PMC 20201215 pmc.key 4802042 CC BY no 0 0 10.1038/ncomms10879 ncomms10879 4802042 26988444 10879 This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ surname:Liu;given-names:Xin surname:Zhang;given-names:Chen-Song surname:Lu;given-names:Chang surname:Lin;given-names:Sheng-Cai surname:Wu;given-names:Jia-Wei surname:Wang;given-names:Zhi-Xin TITLE front 7 2016 0 A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation 0.99966806 protein_type cleaner0 2023-07-11T20:07:53Z MESH: JNK/p38-specific MAPK phosphatases 0.99971896 protein cleaner0 2023-07-11T14:03:57Z PR: JNK1 ABSTRACT abstract 111 Mitogen-activated protein kinases (MAPKs), important in a large array of signalling pathways, are tightly controlled by a cascade of protein kinases and by MAPK phosphatases (MKPs). MAPK signalling efficiency and specificity is modulated by protein–protein interactions between individual MAPKs and the docking motifs in cognate binding partners. Two types of docking interactions have been identified: D-motif-mediated interaction and FXF-docking interaction. Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-Å resolution, providing high-resolution structural insight into the FXF-docking interaction. The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. 0.99968576 protein_type cleaner0 2023-07-11T14:02:48Z MESH: Mitogen-activated protein kinases 0.9995889 protein_type cleaner0 2023-07-11T14:03:00Z MESH: MAPKs 0.9994999 protein_type cleaner0 2023-07-11T14:03:07Z MESH: protein kinases 0.99964887 protein_type cleaner0 2023-07-11T14:03:13Z MESH: MAPK phosphatases 0.99966013 protein_type cleaner0 2023-07-11T14:03:19Z MESH: MKPs 0.99961084 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK 0.99963725 protein_type cleaner0 2023-07-11T14:03:01Z MESH: MAPKs 0.9891355 structure_element cleaner0 2023-07-11T14:06:52Z SO: docking motifs 0.99399155 structure_element cleaner0 2023-07-11T14:07:23Z SO: D-motif 0.99577826 site cleaner0 2023-07-11T19:44:49Z SO: FXF-docking interaction 0.99961877 evidence cleaner0 2023-07-12T14:40:02Z DUMMY: crystal structure 0.9997868 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.99941844 protein_state cleaner0 2023-07-12T14:47:24Z DUMMY: bound to 0.9996116 structure_element cleaner0 2023-07-11T20:18:43Z SO: catalytic domain 0.9998472 protein cleaner0 2023-07-11T14:04:12Z PR: MKP7 0.9984511 site cleaner0 2023-07-11T19:44:50Z SO: FXF-docking interaction 0.94341755 structure_element cleaner0 2023-07-11T19:45:10Z SO: 285FNFL288 segment 0.9998379 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9994817 site cleaner0 2023-07-11T19:44:57Z SO: hydrophobic site 0.9998085 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 protein_type MESH: cleaner0 2023-07-11T14:04:43Z MAPK 0.9996178 structure_element cleaner0 2023-07-11T20:18:49Z SO: helix 0.9984957 structure_element cleaner0 2023-07-11T20:18:52Z SO: αG 0.99952626 experimental_method cleaner0 2023-07-12T14:52:10Z MESH: Biochemical studies 0.999537 protein_state cleaner0 2023-07-12T14:47:28Z DUMMY: highly conserved 0.999419 structure_element cleaner0 2023-07-11T20:18:57Z SO: structural motif 0.99936134 protein_type cleaner0 2023-07-11T20:14:06Z MESH: MKP family 0.99933404 protein_type cleaner0 2023-07-11T20:14:34Z MESH: MKP 0.89675665 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK ABSTRACT abstract 1110 The important MAPK family of signalling proteins is controlled by MAPK phosphatases (MKPs). Here, the authors report the structure of MKP7 bound to JNK1 and characterise the conserved MKP-MAPK interaction. protein_type MESH: cleaner0 2023-07-11T20:14:29Z MAPK family 0.99956846 protein_type cleaner0 2023-07-11T14:03:14Z MESH: MAPK phosphatases 0.99966645 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.99955636 evidence cleaner0 2023-07-12T14:40:06Z DUMMY: structure 0.99985623 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.99945796 protein_state cleaner0 2023-07-12T14:47:31Z DUMMY: bound to 0.99978846 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.9993968 protein_state cleaner0 2023-07-12T14:47:35Z DUMMY: conserved 0.9992163 protein_type cleaner0 2023-07-11T20:14:41Z MESH: MKP protein_type MESH: cleaner0 2023-07-11T14:04:43Z MAPK INTRO paragraph 1317 The mitogen-activated protein kinases (MAPKs) are central components of the signal-transduction pathways, which mediate the cellular response to a variety of extracellular stimuli, ranging from growth factors to environmental stresses. The MAPK signalling pathways are evolutionally highly conserved. The basic assembly of MAPK pathways is a three-tier kinase module that establishes a sequential activation cascade: a MAPK kinase kinase activates a MAPK kinase, which in turn activates a MAPK. The three best-characterized MAPK signalling pathways are mediated by the kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. The ERK pathway is activated by various mitogens and phorbol esters, whereas the JNK and p38 pathways are stimulated mainly by environmental stress and inflammatory cytokines. The MAPKs are activated by MAPK kinases that phosphorylate the MAPKs at conserved threonine and tyrosine residues within their activation loop. After activation, each MAPK phosphorylates a distinct set of protein substrates, which act as the critical effectors that enable cells to mount the appropriate responses to varied stimuli. 0.9996603 protein_type cleaner0 2023-07-11T14:02:49Z MESH: mitogen-activated protein kinases 0.9995908 protein_type cleaner0 2023-07-11T14:03:01Z MESH: MAPKs 0.9994746 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK 0.99851507 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK protein_type MESH: cleaner0 2023-07-11T20:08:19Z kinase 0.95697093 protein_type cleaner0 2023-07-11T14:05:44Z MESH: MAPK kinase kinase 0.99957675 protein_type cleaner0 2023-07-11T14:05:47Z MESH: MAPK kinase 0.9996008 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK 0.99944526 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK 0.9977592 protein_type cleaner0 2023-07-11T14:05:29Z MESH: kinases 0.9959944 protein_type cleaner0 2023-07-11T20:14:47Z MESH: extracellular signal-regulated kinase 0.99826115 protein_type cleaner0 2023-07-11T14:06:43Z MESH: ERK 0.989564 protein_type cleaner0 2023-07-11T14:06:25Z MESH: c-Jun N-terminal kinase 0.98444486 protein_type cleaner0 2023-07-11T14:06:29Z MESH: JNK 0.993874 protein_type cleaner0 2023-07-11T14:06:35Z MESH: p38 0.9856688 protein_type cleaner0 2023-07-11T14:06:42Z MESH: ERK 0.9965958 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.9974468 protein_type cleaner0 2023-07-11T14:06:36Z MESH: p38 protein_type MESH: cleaner0 2023-07-11T20:08:29Z cytokines 0.9995852 protein_type cleaner0 2023-07-11T14:03:01Z MESH: MAPKs 0.99966526 protein_type cleaner0 2023-07-11T20:14:51Z MESH: MAPK kinases 0.9996138 protein_type cleaner0 2023-07-11T14:03:01Z MESH: MAPKs 0.9992895 protein_state cleaner0 2023-07-12T14:48:09Z DUMMY: conserved 0.99927574 residue_name cleaner0 2023-07-11T20:32:43Z SO: threonine 0.9992637 residue_name cleaner0 2023-07-11T20:32:45Z SO: tyrosine 0.99943674 structure_element cleaner0 2023-07-11T20:19:03Z SO: activation loop 0.9995946 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK INTRO paragraph 2487 MAPKs lie at the bottom of conserved three-component phosphorylation cascades and utilize docking interactions to link module components and bind substrates. Two types of docking motifs have been identified in MAPK substrates and cognate proteins: kinase-interacting motif (D-motif) and FXF-motif (also called DEF motif, docking site for ERK FXF). The best-studied docking interactions are those between MAP kinases and ‘D-motifs', which consists of two or more basic residues followed by a short linker and a cluster of hydrophobic residues. The D-motif-docking site (D-site) in MAPKs is situated in a noncatalytic region opposite of the kinase catalytic pocket and is comprised of a highly acidic patch and a hydrophobic groove. D-motifs are found in many MAPK-interacting proteins, including substrates, activating kinases and inactivating phosphatases, as well as scaffolding proteins. A second docking motif for MAPKs consists of two Phe residues separated by one residue (FXF-motif). This motif has been observed in several MAPK substrates. The FXF-motif-binding site of ERK2 has been mapped to a hydrophobic pocket formed between the P+1 site, αG helix and the MAPK insert. However, the generality and mechanism of the FXF-mediated interaction is unclear. 0.999374 protein_type cleaner0 2023-07-11T14:03:01Z MESH: MAPKs 0.7972738 structure_element cleaner0 2023-07-11T14:06:51Z SO: docking motifs 0.9995433 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK 0.99959016 structure_element cleaner0 2023-07-11T20:19:07Z SO: kinase-interacting motif 0.9996815 structure_element cleaner0 2023-07-11T14:07:22Z SO: D-motif 0.9996833 structure_element cleaner0 2023-07-11T14:06:57Z SO: FXF-motif 0.99970543 structure_element cleaner0 2023-07-11T14:07:02Z SO: DEF motif 0.99915403 site cleaner0 2023-07-12T14:33:02Z SO: docking site 0.72890854 protein_type cleaner0 2023-07-11T14:06:43Z MESH: ERK 0.9997389 structure_element cleaner0 2023-07-11T14:07:11Z SO: FXF 0.99966234 protein_type cleaner0 2023-07-11T20:14:55Z MESH: MAP kinases 0.99965477 structure_element cleaner0 2023-07-11T20:19:13Z SO: D-motifs 0.996764 structure_element cleaner0 2023-07-11T20:19:22Z SO: short linker 0.9980342 site cleaner0 2023-07-11T14:08:22Z SO: D-motif-docking site 0.999386 site cleaner0 2023-07-11T14:08:27Z SO: D-site 0.99951327 protein_type cleaner0 2023-07-11T14:03:01Z MESH: MAPKs site SO: cleaner0 2023-07-11T20:20:02Z noncatalytic region 0.98805374 protein_type cleaner0 2023-07-11T20:15:02Z MESH: kinase 0.999212 site cleaner0 2023-07-12T14:33:15Z SO: catalytic pocket 0.99618626 site cleaner0 2023-07-12T14:33:19Z SO: highly acidic patch 0.99940896 site cleaner0 2023-07-12T14:33:24Z SO: hydrophobic groove 0.9996321 structure_element cleaner0 2023-07-11T20:20:08Z SO: D-motifs 0.9996097 protein_type cleaner0 2023-07-11T20:15:06Z MESH: MAPK-interacting proteins 0.98448354 protein_type cleaner0 2023-07-11T14:07:33Z MESH: kinases 0.9988788 protein_type cleaner0 2023-07-11T14:07:31Z MESH: phosphatases 0.9901715 structure_element cleaner0 2023-07-11T20:20:12Z SO: second docking motif 0.99957615 protein_type cleaner0 2023-07-11T14:03:01Z MESH: MAPKs 0.9990539 residue_name cleaner0 2023-07-11T19:59:38Z SO: Phe 0.999645 structure_element cleaner0 2023-07-11T14:06:58Z SO: FXF-motif 0.99953496 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK 0.9996225 site cleaner0 2023-07-11T14:07:52Z SO: FXF-motif-binding site 0.99954295 protein cleaner0 2023-07-11T14:16:33Z PR: ERK2 0.9996133 site cleaner0 2023-07-12T14:33:29Z SO: hydrophobic pocket 0.99959314 site cleaner0 2023-07-12T14:33:32Z SO: P+1 site 0.99970084 structure_element cleaner0 2023-07-11T20:20:16Z SO: αG helix 0.8454261 structure_element cleaner0 2023-07-11T20:20:20Z SO: MAPK insert 0.99787986 structure_element cleaner0 2023-07-11T14:07:12Z SO: FXF INTRO paragraph 3754 The physiological outcome of MAPK signalling depends on both the magnitude and the duration of kinase activation. Downregulation of MAPK activity can be achieved through direct dephosphorylation of the phospho-threonine and/or tyrosine residues by various serine/threonine phosphatases, tyrosine phosphatases and dual-specificity phosphatases (DUSPs) termed MKPs. MKPs constitute a group of DUSPs that are characterized by their ability to dephosphorylate both phosphotyrosine and phosphoserine/phospho-threonine residues within a substrate. Dysregulated expression of MKPs has been associated with pathogenesis of various diseases, and understanding their precise recognition mechanism presents an important challenge and opportunity for drug development. 0.9996532 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK 0.9996407 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK residue_name SO: melaniev@ebi.ac.uk 2023-07-21T14:00:06Z phospho-threonine and/or tyrosine 0.9996766 protein_type cleaner0 2023-07-11T14:08:37Z MESH: serine/threonine phosphatases 0.9996767 protein_type cleaner0 2023-07-11T14:08:40Z MESH: tyrosine phosphatases 0.99968606 protein_type cleaner0 2023-07-11T14:08:45Z MESH: dual-specificity phosphatases 0.99966526 protein_type cleaner0 2023-07-11T14:08:51Z MESH: DUSPs 0.9996773 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.99967456 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.99966264 protein_type cleaner0 2023-07-11T14:08:52Z MESH: DUSPs 0.57390255 residue_name melaniev@ebi.ac.uk 2023-07-21T14:00:06Z SO: phosphotyrosine 0.53731674 residue_name melaniev@ebi.ac.uk 2023-07-21T14:00:06Z SO: phosphoserine residue_name SO: melaniev@ebi.ac.uk 2023-07-21T14:00:06Z phospho-threonine 0.9996741 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs INTRO paragraph 4511 Here, we present the crystal structure of JNK1 in complex with the catalytic domain of MKP7. This structure reveals the molecular mechanism underlying the docking interaction between MKP7 and JNK1. In the JNK1–MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix α5 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction. Biochemical and modelling studies further demonstrate that the molecular interactions mediate this key element for substrate recognition are highly conserved among all MKP-family members. Thus, our study reveals a hitherto unrecognized interaction mode for encoding complex target specificity among MAPK isoforms. 0.9996257 evidence cleaner0 2023-07-12T14:40:11Z DUMMY: crystal structure 0.99982554 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.99903136 protein_state cleaner0 2023-07-12T14:48:29Z DUMMY: in complex with 0.99969137 structure_element cleaner0 2023-07-11T20:20:25Z SO: catalytic domain 0.99986076 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9979746 evidence cleaner0 2023-07-12T14:40:26Z DUMMY: structure 0.9998572 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.99982244 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.9996434 complex_assembly cleaner0 2023-07-11T14:13:00Z GO: JNK1–MKP7 0.9996366 structure_element cleaner0 2023-07-11T20:20:28Z SO: hydrophobic motif 0.9920007 structure_element cleaner0 2023-07-11T20:20:31Z SO: 285FNFL288 0.9997838 structure_element cleaner0 2023-07-11T20:20:34Z SO: helix 0.9869927 structure_element cleaner0 2023-07-11T20:20:38Z SO: α5 0.9998592 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.99969554 structure_element cleaner0 2023-07-11T20:20:41Z SO: catalytic domain 0.99961805 site cleaner0 2023-07-11T14:07:53Z SO: FXF-motif-binding site 0.9998104 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.9645349 site cleaner0 2023-07-12T14:33:42Z SO: FXF-type docking interaction 0.9995483 experimental_method cleaner0 2023-07-12T14:52:15Z MESH: Biochemical and modelling studies 0.99965215 protein_type cleaner0 2023-07-11T20:15:11Z MESH: MKP-family members 0.8800726 protein_type cleaner0 2023-07-11T20:15:14Z MESH: MAPK isoforms RESULTS title_1 5277 Results RESULTS title_2 5285 Interaction of JNK1 with the MKP7 catalytic domain 0.9998319 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.99986243 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9988241 structure_element cleaner0 2023-07-11T20:20:48Z SO: catalytic domain RESULTS paragraph 5336 DUSPs belong to the protein-tyrosine phosphatases (PTPase) superfamily, which is defined by the PTPase-signature motif CXXGXXR. MKPs represent a distinct subfamily within a larger group of DUSPs. In mammalian cells, the MKP subfamily includes 10 distinct catalytically active MKPs. All MKPs contain a highly conserved C-terminal catalytic domain (CD) and an N-terminal kinase-binding domain (KBD). The KBD is homologous to the rhodanese family and contains an intervening cluster of basic amino acids, which has been suggested to be important for interacting with the target MAPKs. On the basis of sequence similarity, substrate specificity and predominant subcellular localization, the MKP family can be further divided into three groups (Fig. 1). Biochemical and structural studies have revealed that the KBD of MKPs is critical for MKP3 docking to ERK2, and MKP5 binding to p38α, although their binding mechanisms are completely different. However, it is unknown if other MAPKs can interact with the KBD of their cognate phosphatases in the same manner as observed for recognition of ERK2 and p38α by their MKPs, or whether they recognize distinct docking motifs of MKPs. 0.99967456 protein_type cleaner0 2023-07-11T14:08:52Z MESH: DUSPs 0.99966884 protein_type cleaner0 2023-07-11T14:13:58Z MESH: protein-tyrosine phosphatases 0.9996518 protein_type cleaner0 2023-07-11T14:14:03Z MESH: PTPase 0.6538119 protein_type cleaner0 2023-07-11T14:14:03Z MESH: PTPase 0.9993011 structure_element cleaner0 2023-07-11T20:20:59Z SO: CXXGXXR 0.9996785 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.99966824 protein_type cleaner0 2023-07-11T14:08:52Z MESH: DUSPs 0.9990175 taxonomy_domain cleaner0 2023-07-11T14:14:09Z DUMMY: mammalian 0.9919881 protein_type cleaner0 2023-07-11T20:15:19Z MESH: MKP subfamily 0.99940425 protein_state cleaner0 2023-07-12T14:48:43Z DUMMY: catalytically active 0.99965084 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.99967396 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.9995341 protein_state cleaner0 2023-07-12T14:48:46Z DUMMY: highly conserved 0.99970675 structure_element cleaner0 2023-07-11T14:16:05Z SO: catalytic domain 0.9998092 structure_element cleaner0 2023-07-11T14:16:09Z SO: CD 0.9996517 structure_element cleaner0 2023-07-11T14:14:16Z SO: kinase-binding domain 0.9997887 structure_element cleaner0 2023-07-11T14:14:21Z SO: KBD 0.99979013 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9995134 protein_type cleaner0 2023-07-11T20:15:22Z MESH: rhodanese family 0.9996666 protein_type cleaner0 2023-07-11T14:03:01Z MESH: MAPKs 0.9992954 protein_type cleaner0 2023-07-11T20:14:07Z MESH: MKP family 0.9995157 experimental_method cleaner0 2023-07-12T14:52:20Z MESH: Biochemical and structural studies 0.99979573 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9996674 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.9998385 protein cleaner0 2023-07-11T14:16:26Z PR: MKP3 0.99919564 protein cleaner0 2023-07-11T14:16:33Z PR: ERK2 0.9998448 protein cleaner0 2023-07-11T14:16:41Z PR: MKP5 0.99958605 protein cleaner0 2023-07-11T14:16:52Z PR: p38α 0.9996512 protein_type cleaner0 2023-07-11T14:03:01Z MESH: MAPKs 0.9997993 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.99967206 protein_type cleaner0 2023-07-11T20:15:28Z MESH: phosphatases 0.99907947 protein cleaner0 2023-07-11T14:16:33Z PR: ERK2 0.99940205 protein cleaner0 2023-07-11T14:16:53Z PR: p38α 0.99967 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.99714094 structure_element cleaner0 2023-07-11T14:06:52Z SO: docking motifs 0.99968064 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs RESULTS paragraph 6519 MKP7, the biggest molecule in the MKP family, selectively inactivates JNK and p38 following stress activation. In addition to the CD and KBD, MKP7 has a long C-terminal region that contains both nuclear localization and export sequences by which MKP7 shuttles between the nucleus and the cytoplasm (Fig. 2a). To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). Figure 2b shows the variation of initial rates of the MKP7ΔC304 and MKP7-CD-catalysed reaction with the concentration of phospho-JNK1. Because the concentrations of MKP7 and pJNK1 were comparable in the reaction, the assumption that the free-substrate concentration is equal to the total substrate concentration is not valid. Thus, the kinetic data were analysed using the general initial velocity equation, taking substrate depletion into account: 0.99967444 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9996246 protein_type cleaner0 2023-07-11T20:14:07Z MESH: MKP family 0.8621272 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.9341276 protein_type cleaner0 2023-07-11T14:06:36Z MESH: p38 0.9998054 structure_element cleaner0 2023-07-11T14:16:10Z SO: CD 0.99979025 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9998404 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.999457 structure_element cleaner0 2023-07-11T20:21:18Z SO: C-terminal region 0.9998524 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9995566 structure_element cleaner0 2023-07-11T20:21:21Z SO: N-terminal domain 0.9998579 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9997814 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 ptm MESH: cleaner0 2023-07-11T14:18:30Z dephosphorylation 0.6796221 evidence cleaner0 2023-07-12T14:41:01Z DUMMY: kinetic 0.7138995 experimental_method cleaner0 2023-07-12T14:52:37Z MESH: truncation 0.99983287 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9971969 mutant cleaner0 2023-07-11T14:17:22Z MESH: MKP7ΔC304 0.99902564 residue_range cleaner0 2023-07-12T15:00:47Z DUMMY: 5–303 protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:10Z CD 0.9990709 residue_range cleaner0 2023-07-12T15:00:50Z DUMMY: 156–301 0.99956316 protein_state cleaner0 2023-07-11T20:03:11Z DUMMY: phosphorylated 0.9997745 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 protein_state DUMMY: cleaner0 2023-07-11T14:18:06Z p protein PR: cleaner0 2023-07-11T14:17:56Z JNK1 0.8938303 evidence cleaner0 2023-07-12T14:41:12Z DUMMY: variation of initial rates 0.99916244 mutant cleaner0 2023-07-11T14:17:23Z MESH: MKP7ΔC304 protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:10Z CD 0.99105 protein_state cleaner0 2023-07-12T14:48:56Z DUMMY: phospho 0.9994254 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.99984777 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 protein_state DUMMY: cleaner0 2023-07-11T20:01:02Z p protein PR: cleaner0 2023-07-11T19:46:53Z JNK1 evidence DUMMY: cleaner0 2023-07-12T14:41:37Z kinetic data 0.98305637 evidence cleaner0 2023-07-12T14:41:39Z DUMMY: initial velocity equation RESULTS paragraph 7572 The kcat and Km of the MKP7-CD (0.028 s−1 and 0.26 μM) so determined were nearly identical to those of MKP7ΔC304 (0.029 s−1 and 0.27 μM), indicating that the MKP7-KBD has no effect on enzyme catalysis. 0.9995908 evidence cleaner0 2023-07-12T14:41:43Z DUMMY: kcat 0.99959475 evidence cleaner0 2023-07-12T14:41:46Z DUMMY: Km protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:10Z CD 0.99417454 mutant cleaner0 2023-07-11T14:17:23Z MESH: MKP7ΔC304 0.6997105 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:14:22Z KBD RESULTS paragraph 7789 We next examined the interaction of JNK1 with the CD and KBD of MKP7 by gel filtration analysis. When 3 molar equivalents of CD were mixed with 1 molar equivalent of JNK1, a significant amount of CD co-migrated with JNK1 to earlier fractions, and the excess amount of CD was eluted from the size exclusion column as a monomer, indicating stable complex formation (Fig. 2c). In contrast, no KBD–JNK1 complex was detected when 3 molar equivalents of KBD were mixed with 1 molar equivalent of JNK1. To further confirm the JNK1–MKP7-CD interaction, we performed a pull-down assay using the purified proteins. As shown in Fig. 2d, the CD of MKP7 can be pulled down by JNK1, while the KBD failed to bind to the counterpart protein. Taken together, our data indicate that the CD of MKP7, but not the KBD domain, is responsible for JNK substrate-binding and enzymatic specificity. 0.999777 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.99978906 structure_element cleaner0 2023-07-11T14:16:10Z SO: CD 0.9997913 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9998486 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9995236 experimental_method cleaner0 2023-07-12T14:52:42Z MESH: gel filtration analysis 0.9997826 structure_element cleaner0 2023-07-11T14:16:10Z SO: CD 0.999775 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.9997471 structure_element cleaner0 2023-07-11T14:16:10Z SO: CD 0.99950635 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.99974126 structure_element cleaner0 2023-07-11T14:16:10Z SO: CD 0.99936455 oligomeric_state cleaner0 2023-07-12T15:00:56Z DUMMY: monomer 0.995395 complex_assembly cleaner0 2023-07-11T20:33:13Z GO: KBD–JNK1 0.81012785 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.99950385 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 complex_assembly GO: cleaner0 2023-07-11T19:51:19Z JNK1–MKP7-CD 0.999572 experimental_method cleaner0 2023-07-12T14:52:53Z MESH: pull-down assay 0.9997962 structure_element cleaner0 2023-07-11T14:16:10Z SO: CD 0.999845 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9998209 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.9997838 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.99978584 structure_element cleaner0 2023-07-11T14:16:10Z SO: CD 0.99984205 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9997806 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.99772984 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK RESULTS title_2 8674 Crystal structure of JNK1 in complex with the MKP7-CD 0.99959636 evidence cleaner0 2023-07-12T14:41:51Z DUMMY: Crystal structure 0.99980146 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.99925405 protein_state cleaner0 2023-07-12T14:49:01Z DUMMY: in complex with protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:10Z CD RESULTS paragraph 8728 To understand the molecular basis of JNK1 recognition by MKP7, we determined the crystal structure of unphosphorylated JNK1 in complex with the MKP7-CD (Fig. 3a, Supplementary Fig. 1a and Table 1). In the complex, JNK1 has its characteristic bilobal structure comprising an N-terminal lobe rich in β-sheet and a C-terminal lobe that is mostly α-helical. The overall folding of MKP7-CD is typical of DUSPs, with a central twisted five-stranded β-sheet surrounded by six α-helices. One side of the β-sheet is covered with two α-helices and the other is covered with four α-helices (Fig. 3b). The catalytic domain of MKP7 interacts with JNK1 through a contiguous surface area that is remote from the active site. MKP7-CD is positioned onto the JNK1 molecule so that the active site of the phosphatase faces towards the activation segment. In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). The most striking difference is that helix α0 and loop α0–β1 of VHR are absent in MKP7-CD. Another region that cannot be aligned with VHR is found in loop β3–β4. This loop is shortened by nine residues in MKP7-CD compared with that in VHR. Since helix α0 and the following loop α0–β1 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7. 0.9998361 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.999859 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9996276 evidence cleaner0 2023-07-12T14:41:54Z DUMMY: crystal structure 0.999602 protein_state cleaner0 2023-07-12T14:49:04Z DUMMY: unphosphorylated 0.999816 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.9991393 protein_state cleaner0 2023-07-12T14:49:08Z DUMMY: in complex with 0.93712324 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9997706 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.9554019 structure_element cleaner0 2023-07-11T20:21:29Z SO: N-terminal lobe 0.99935323 structure_element cleaner0 2023-07-11T20:21:32Z SO: β-sheet 0.98967075 structure_element cleaner0 2023-07-11T20:21:36Z SO: C-terminal lobe 0.99887353 structure_element cleaner0 2023-07-11T20:21:39Z SO: α-helical 0.7400553 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.99963737 protein_type cleaner0 2023-07-11T14:08:52Z MESH: DUSPs 0.9988386 structure_element cleaner0 2023-07-11T20:21:43Z SO: twisted five-stranded β-sheet 0.99922544 structure_element cleaner0 2023-07-11T20:21:46Z SO: α-helices 0.99953717 structure_element cleaner0 2023-07-11T20:21:49Z SO: β-sheet 0.9994037 structure_element cleaner0 2023-07-11T20:21:52Z SO: α-helices 0.9919618 structure_element cleaner0 2023-07-11T20:21:55Z SO: α-helices 0.9997327 structure_element cleaner0 2023-07-11T20:20:48Z SO: catalytic domain 0.9998604 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.99982685 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.9996096 site cleaner0 2023-07-12T14:34:20Z SO: active site 0.99024874 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.99980706 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.9995651 site cleaner0 2023-07-12T14:34:24Z SO: active site 0.99965227 protein_type cleaner0 2023-07-11T14:21:28Z MESH: phosphatase 0.9996175 structure_element cleaner0 2023-07-11T20:21:58Z SO: activation segment 0.9996644 experimental_method cleaner0 2023-07-12T14:53:02Z MESH: alignment 0.99963665 evidence cleaner0 2023-07-12T14:41:59Z DUMMY: structure protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9997435 protein cleaner0 2023-07-11T14:22:09Z PR: VHR 0.99946505 protein_type cleaner0 2023-07-11T20:15:35Z MESH: MKP 0.9996985 structure_element cleaner0 2023-07-11T20:20:48Z SO: catalytic domain 0.8229092 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9996706 experimental_method cleaner0 2023-07-12T14:53:05Z MESH: superimposed 0.9995588 evidence cleaner0 2023-07-11T20:09:39Z DUMMY: r.m.s.d. 0.9995039 evidence cleaner0 2023-07-11T20:09:44Z DUMMY: root mean squared deviation 0.99922 structure_element cleaner0 2023-07-11T20:22:05Z SO: helix 0.999746 structure_element cleaner0 2023-07-11T20:22:08Z SO: α0 0.9994394 structure_element cleaner0 2023-07-11T20:22:10Z SO: loop 0.99964577 structure_element cleaner0 2023-07-11T20:22:13Z SO: α0–β1 0.9995042 protein cleaner0 2023-07-11T14:22:09Z PR: VHR 0.6807224 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.99950767 protein cleaner0 2023-07-11T14:22:09Z PR: VHR 0.99970764 structure_element cleaner0 2023-07-11T20:22:17Z SO: loop 0.9996819 structure_element cleaner0 2023-07-11T20:22:19Z SO: β3–β4 0.9990049 structure_element cleaner0 2023-07-11T20:22:22Z SO: loop protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.99971765 protein cleaner0 2023-07-11T14:22:09Z PR: VHR 0.9990748 structure_element cleaner0 2023-07-11T20:22:25Z SO: helix 0.999754 structure_element cleaner0 2023-07-11T20:22:27Z SO: α0 0.9996567 structure_element cleaner0 2023-07-11T20:22:30Z SO: loop 0.99963564 structure_element cleaner0 2023-07-11T20:22:32Z SO: α0–β1 0.95910877 site cleaner0 2023-07-12T14:34:29Z SO: substrate-recognition motif 0.9991061 protein cleaner0 2023-07-11T14:22:08Z PR: VHR 0.9996191 protein_type cleaner0 2023-07-11T14:21:30Z MESH: phosphatases 0.99985707 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 RESULTS paragraph 10297 The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). The MKP7-CD structure near the active site exhibits a typical active conformation as found in VHR and other PTPs. The catalytic residue, Cys244, is located just after strand β5 and optimally positioned for nucleophilic attack. Asp213 in MKP7 also adopts a position similar to that of Asp92 in VHR (Supplementary Fig. 1c), indicating that Asp213 is likely to function as the general acid in MKP7. We also observed the binding of a chloride ion in the active site of MKP7-CD. It is located 3.36 Å from the Cys244 side chain and makes electrostatic interactions with the dipole moment of helix α3 and with several main-chain amide groups. The side chain of strictly conserved Arg250 is oriented towards the negatively charged chloride, similar to the canonical phosphate-coordinating conformation. Thus this chloride ion is a mimic for the phosphate group of the substrate, as revealed by a comparison with the structure of PTP1B in complex with phosphotyrosine (Supplementary Fig. 1d). Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). The difference in the polarity/hydrophobicity of the surface may also point to the origin of the differences in the substrate-recognition mechanism for these two phosphatases (Supplementary Fig. 1e,f). 0.99956113 site cleaner0 2023-07-12T14:34:34Z SO: active site 0.9998584 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9995509 structure_element cleaner0 2023-07-11T20:22:35Z SO: phosphate-binding loop 0.9997042 structure_element cleaner0 2023-07-11T15:41:47Z SO: P-loop residue_name_number DUMMY: cleaner0 2023-07-11T14:23:54Z Cys244 residue_name_number DUMMY: cleaner0 2023-07-11T14:24:06Z Leu245 residue_name_number DUMMY: cleaner0 2023-07-11T14:24:28Z Ala246 0.4037717 residue_name_number cleaner0 2023-07-11T14:24:35Z DUMMY: Gly247 residue_name_number DUMMY: cleaner0 2023-07-11T14:24:57Z Ile248 residue_name_number DUMMY: cleaner0 2023-07-11T14:25:09Z Ser249 0.996447 residue_name_number cleaner0 2023-07-11T14:25:15Z DUMMY: Arg250 0.99989295 residue_name_number cleaner0 2023-07-11T14:25:47Z DUMMY: Asp213 0.9995947 structure_element cleaner0 2023-07-11T20:22:44Z SO: general acid loop 0.96279836 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9995079 evidence cleaner0 2023-07-12T14:42:05Z DUMMY: structure 0.99958664 site cleaner0 2023-07-12T14:34:38Z SO: active site 0.99858224 protein_state cleaner0 2023-07-12T14:49:13Z DUMMY: active conformation 0.99972755 protein cleaner0 2023-07-11T14:22:09Z PR: VHR 0.99964774 protein_type cleaner0 2023-07-11T20:15:43Z MESH: PTPs 0.99247706 site cleaner0 2023-07-12T14:34:43Z SO: catalytic residue 0.99989295 residue_name_number cleaner0 2023-07-11T14:24:11Z DUMMY: Cys244 0.9970216 structure_element cleaner0 2023-07-11T20:22:47Z SO: strand 0.99965894 structure_element cleaner0 2023-07-11T20:22:50Z SO: β5 0.99989736 residue_name_number cleaner0 2023-07-11T14:25:48Z DUMMY: Asp213 0.9998536 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9998957 residue_name_number cleaner0 2023-07-11T14:25:25Z DUMMY: Asp92 0.99980706 protein cleaner0 2023-07-11T14:22:09Z PR: VHR 0.9998938 residue_name_number cleaner0 2023-07-11T14:25:48Z DUMMY: Asp213 0.99986017 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9991714 chemical cleaner0 2023-07-12T15:01:27Z CHEBI: chloride 0.9996083 site cleaner0 2023-07-12T14:34:50Z SO: active site 0.9866303 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9998913 residue_name_number cleaner0 2023-07-11T14:24:12Z DUMMY: Cys244 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z electrostatic interactions 0.9997094 structure_element cleaner0 2023-07-11T20:22:55Z SO: helix 0.99973077 structure_element cleaner0 2023-07-11T20:22:58Z SO: α3 0.9994043 protein_state cleaner0 2023-07-12T14:49:18Z DUMMY: strictly conserved 0.99989843 residue_name_number cleaner0 2023-07-11T14:25:16Z DUMMY: Arg250 0.99835026 chemical cleaner0 2023-07-12T15:01:32Z CHEBI: chloride 0.99666464 structure_element cleaner0 2023-07-12T14:35:20Z SO: phosphate-coordinating conformation 0.9994386 chemical cleaner0 2023-07-12T15:01:36Z CHEBI: chloride 0.99971646 chemical cleaner0 2023-07-12T15:01:46Z CHEBI: phosphate 0.99766505 evidence cleaner0 2023-07-12T14:42:09Z DUMMY: structure 0.9998487 protein cleaner0 2023-07-11T15:49:21Z PR: PTP1B 0.99920243 protein_state cleaner0 2023-07-12T14:49:22Z DUMMY: in complex with 0.9996356 residue_name melaniev@ebi.ac.uk 2023-07-21T14:00:06Z SO: phosphotyrosine 0.99006784 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9998066 protein cleaner0 2023-07-11T14:22:09Z PR: VHR 0.99970084 structure_element cleaner0 2023-07-11T15:41:47Z SO: P-loop 0.9998542 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9996877 protein cleaner0 2023-07-11T14:22:09Z PR: VHR 0.56615037 residue_name_number cleaner0 2023-07-11T14:28:39Z DUMMY: Cys124 residue_name_number DUMMY: cleaner0 2023-07-11T14:29:03Z Arg125 0.7130929 residue_name_number cleaner0 2023-07-11T14:30:49Z DUMMY: Glu126 0.71502244 residue_name_number cleaner0 2023-07-11T14:30:22Z DUMMY: Gly127 0.98875487 residue_name_number cleaner0 2023-07-11T14:30:29Z DUMMY: Tyr128 0.99159086 residue_name_number cleaner0 2023-07-11T14:30:38Z DUMMY: Gly129 0.99976474 residue_name_number cleaner0 2023-07-11T14:30:43Z DUMMY: Arg130 0.999539 protein_type cleaner0 2023-07-11T14:30:55Z MESH: phosphatases RESULTS paragraph 11945 In the complex, MKP7-CD and JNK1 form extensive protein–protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e). As a result, the buried solvent-accessible surface area is ∼1,315 Å. In the C-terminal domain, JNK1 has an insertion after the helix αG. This insertion consists of two helices (α1L14 and α2L14) that are common to all members of the MAPK family. The interactive surface in JNK1, formed by the helices αG and α2L14, displays a hydrophobic region, centred at Trp234 (Fig. 3d). The MKP7-docking region includes two helices, α4 and α5, and the general acid loop. The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. The second interactive area involves the α4 helix of MKP7 and charged/polar residues of JNK1 (Fig. 3e). The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9997383 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.9009522 structure_element cleaner0 2023-07-11T20:23:02Z SO: C-terminal helices protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9996624 structure_element cleaner0 2023-07-11T20:23:08Z SO: C-lobe 0.99978286 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.99970305 structure_element cleaner0 2023-07-11T20:23:11Z SO: C-terminal domain 0.9997002 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.99934155 structure_element cleaner0 2023-07-11T20:23:14Z SO: helix 0.99979514 structure_element cleaner0 2023-07-11T20:23:17Z SO: αG 0.9945833 structure_element cleaner0 2023-07-11T20:23:25Z SO: helices 0.99979657 structure_element cleaner0 2023-07-11T20:23:28Z SO: α1L14 0.999795 structure_element cleaner0 2023-07-11T20:23:30Z SO: α2L14 0.99658537 protein_type cleaner0 2023-07-11T20:15:48Z MESH: MAPK family 0.99957514 site cleaner0 2023-07-12T14:35:31Z SO: interactive surface 0.9997404 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.9991308 structure_element cleaner0 2023-07-11T20:24:00Z SO: helices 0.9998018 structure_element cleaner0 2023-07-11T20:24:08Z SO: αG 0.99978906 structure_element cleaner0 2023-07-11T20:24:11Z SO: α2L14 0.9994271 site cleaner0 2023-07-12T14:35:37Z SO: hydrophobic region 0.99990344 residue_name_number cleaner0 2023-07-11T15:42:39Z DUMMY: Trp234 0.9992646 site cleaner0 2023-07-12T14:35:43Z SO: MKP7-docking region 0.9909559 structure_element cleaner0 2023-07-11T20:23:37Z SO: helices 0.9998104 structure_element cleaner0 2023-07-11T20:23:40Z SO: α4 0.9998042 structure_element cleaner0 2023-07-11T20:23:42Z SO: α5 0.9997103 structure_element cleaner0 2023-07-11T20:23:44Z SO: general acid loop 0.99990416 residue_name_number cleaner0 2023-07-11T15:42:47Z DUMMY: Phe285 0.9998337 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.99970245 structure_element cleaner0 2023-07-11T20:23:51Z SO: α5-helix 0.99957085 site cleaner0 2023-07-12T14:35:48Z SO: hydrophobic pocket 0.99971503 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.99990094 residue_name_number cleaner0 2023-07-11T15:42:56Z DUMMY: Ile197 0.9999 residue_name_number cleaner0 2023-07-11T15:43:02Z DUMMY: Leu198 0.9999014 residue_name_number cleaner0 2023-07-11T15:43:08Z DUMMY: Ile231 0.99990284 residue_name_number cleaner0 2023-07-11T15:42:41Z DUMMY: Trp234 0.99990404 residue_name_number cleaner0 2023-07-11T15:43:16Z DUMMY: Val256 0.99990463 residue_name_number cleaner0 2023-07-11T15:43:21Z DUMMY: Tyr259 0.9999045 residue_name_number cleaner0 2023-07-11T15:43:26Z DUMMY: Val260 0.9999076 residue_name_number cleaner0 2023-07-11T15:43:33Z DUMMY: His230 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z hydrogen bonds 0.9999056 residue_name_number cleaner0 2023-07-11T15:43:38Z DUMMY: Ser282 0.99990463 residue_name_number cleaner0 2023-07-11T15:43:44Z DUMMY: Asn286 0.9997948 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.99990773 residue_name_number cleaner0 2023-07-11T15:43:34Z DUMMY: His230 0.9999039 residue_name_number cleaner0 2023-07-11T15:43:52Z DUMMY: Thr255 0.9994832 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.9999082 residue_name_number cleaner0 2023-07-11T15:44:00Z DUMMY: Phe215 0.99971133 structure_element cleaner0 2023-07-11T20:24:14Z SO: general acid loop 0.99982077 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z hydrogen-bonded 0.9999063 residue_name_number cleaner0 2023-07-11T15:44:07Z DUMMY: Gln253 0.9997098 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 site SO: cleaner0 2023-07-12T14:36:35Z second interactive area 0.9997213 structure_element cleaner0 2023-07-11T20:24:17Z SO: α4 helix 0.9998319 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.99964833 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.99990463 residue_name_number cleaner0 2023-07-11T15:44:17Z DUMMY: Asp268 0.99982387 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z salt bridge 0.99990475 residue_name_number cleaner0 2023-07-11T15:44:33Z DUMMY: Arg263 0.99966395 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.9999026 residue_name_number cleaner0 2023-07-11T15:44:38Z DUMMY: Lys275 0.9998275 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z hydrogen bond bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z salt bridge 0.9999002 residue_name_number cleaner0 2023-07-11T15:44:22Z DUMMY: Thr228 0.99990034 residue_name_number cleaner0 2023-07-11T15:44:27Z DUMMY: Asp229 0.9996506 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 RESULTS title_2 13355 Mutational analysis of the JNK1–MKP7 docking interface 0.9995778 experimental_method cleaner0 2023-07-12T14:53:09Z MESH: Mutational analysis site SO: cleaner0 2023-07-11T15:45:03Z JNK1–MKP7 docking interface RESULTS paragraph 13412 To assess the importance of the aforementioned interactions, we generated a series of point mutations on the MKP7-CD and examined their effect on the MKP7-catalysed JNK1 dephosphorylation (Fig. 4a). When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold. In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP. Mutation of Leu288 markedly reduced its solubility when expressed in Escherichia coli, resulting in the insoluble aggregation of the mutant protein. Gel filtration analysis further confirmed the key role of Phe285 in the MKP7–JNK1 interaction: no F285D–JNK1 complex was detected when 3 molar equivalents of MKP7-CD (F285D) were mixed with 1 molar equivalent of JNK1 (Fig. 4b). Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a). We also generated a series of point mutations in the JNK1 and assessed the effect on JNK1 binding using the GST pull-down assay (Fig. 4c). Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK. To determine whether the deficiencies in their abilities to bind partner proteins or carry out catalytic function are owing to misfolding of the purified mutant proteins, we also examined the folding properties of the JNK1 and MKP7 mutants with circular dichroism. The spectra of these mutants are similar to the wild-type proteins, indicating that these mutants fold as well as the wild-type proteins (Fig. 4d,e). Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme–substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1. experimental_method MESH: cleaner0 2023-07-12T14:53:26Z point mutations protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 0.58665764 structure_element cleaner0 2023-07-11T14:16:11Z SO: CD 0.99985516 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9996363 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T13:57:01Z dephosphorylation 0.99989676 residue_name_number cleaner0 2023-07-11T15:42:48Z DUMMY: Phe285 0.99989974 residue_name_number cleaner0 2023-07-11T15:47:33Z DUMMY: Phe287 0.9997677 structure_element cleaner0 2023-07-11T20:24:24Z SO: α5 protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 0.4852582 structure_element cleaner0 2023-07-11T14:16:11Z SO: CD 0.99880517 experimental_method cleaner0 2023-07-12T14:53:31Z MESH: replaced 0.9990822 residue_name cleaner0 2023-07-11T15:45:28Z SO: Asp 0.9991892 residue_name cleaner0 2023-07-11T15:45:31Z SO: Ala 0.99865085 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T13:57:12Z dephosphorylation 0.9996031 experimental_method cleaner0 2023-07-12T14:53:34Z MESH: replacement 0.99989915 residue_name_number cleaner0 2023-07-11T15:44:01Z DUMMY: Phe215 0.999897 residue_name_number cleaner0 2023-07-11T15:44:17Z DUMMY: Asp268 0.9998969 residue_name_number cleaner0 2023-07-11T15:44:39Z DUMMY: Lys275 0.99989736 residue_name_number cleaner0 2023-07-11T15:43:39Z DUMMY: Ser282 0.99989784 residue_name_number cleaner0 2023-07-11T15:43:45Z DUMMY: Asn286 0.99989796 residue_name_number cleaner0 2023-07-11T15:45:52Z DUMMY: Leu292 0.99912673 residue_name cleaner0 2023-07-11T15:45:33Z SO: Ala 0.999175 residue_name cleaner0 2023-07-11T15:45:36Z SO: Asp 0.99946874 protein_type cleaner0 2023-07-11T20:15:51Z MESH: MKP 0.99959475 experimental_method cleaner0 2023-07-12T14:53:50Z MESH: Mutation 0.99990356 residue_name_number cleaner0 2023-07-11T15:46:00Z DUMMY: Leu288 species MESH: cleaner0 2023-07-12T14:54:13Z Escherichia coli 0.9973037 protein_state cleaner0 2023-07-11T15:48:13Z DUMMY: mutant 0.99871475 experimental_method cleaner0 2023-07-12T14:54:41Z MESH: Gel filtration analysis 0.99989915 residue_name_number cleaner0 2023-07-11T15:42:48Z DUMMY: Phe285 0.9884856 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.7853465 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 complex_assembly GO: cleaner0 2023-07-12T14:42:42Z F285D–JNK1 protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9996942 mutant cleaner0 2023-07-11T15:53:12Z MESH: F285D 0.999648 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 0.99958867 experimental_method cleaner0 2023-07-12T14:54:46Z MESH: mutation 0.99990153 residue_name_number cleaner0 2023-07-11T15:47:32Z DUMMY: Phe287 protein_state DUMMY: cleaner0 2023-07-11T20:02:20Z p protein PR: cleaner0 2023-07-11T19:47:26Z JNK1 0.9948709 evidence cleaner0 2023-07-12T14:42:18Z DUMMY: affinity protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.99906033 protein cleaner0 2023-07-11T14:03:58Z PR: JNK1 experimental_method MESH: cleaner0 2023-07-12T14:55:11Z point mutations 0.99979895 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.99973184 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.9995672 experimental_method cleaner0 2023-07-12T14:55:19Z MESH: GST pull-down assay 0.99960667 experimental_method cleaner0 2023-07-12T14:55:23Z MESH: Substitution 0.9998933 residue_name_number cleaner0 2023-07-11T15:44:28Z DUMMY: Asp229 0.9998952 residue_name_number cleaner0 2023-07-11T15:42:41Z DUMMY: Trp234 0.99989414 residue_name_number cleaner0 2023-07-11T15:43:53Z DUMMY: Thr255 0.9998956 residue_name_number cleaner0 2023-07-11T15:43:17Z DUMMY: Val256 0.9998952 residue_name_number cleaner0 2023-07-11T15:43:22Z DUMMY: Tyr259 0.99989724 residue_name_number cleaner0 2023-07-11T15:43:27Z DUMMY: Val260 0.9995673 evidence cleaner0 2023-07-12T14:42:49Z DUMMY: binding affinity protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 0.5284776 structure_element cleaner0 2023-07-11T14:16:11Z SO: CD 0.99918693 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK protein_state DUMMY: cleaner0 2023-07-11T15:48:15Z mutant 0.9997172 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.99974996 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.99952084 experimental_method cleaner0 2023-07-12T14:46:19Z MESH: circular dichroism 0.9995652 evidence cleaner0 2023-07-12T14:42:52Z DUMMY: spectra protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.99955815 protein_state cleaner0 2023-07-11T15:48:09Z DUMMY: wild-type protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.9995454 protein_state cleaner0 2023-07-11T15:48:08Z DUMMY: wild-type 0.9967828 evidence cleaner0 2023-07-12T14:42:55Z DUMMY: crystallographic model bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z hydrophobic contacts 0.999858 protein cleaner0 2023-07-11T14:04:13Z PR: MKP7 0.9994093 structure_element cleaner0 2023-07-11T20:20:49Z SO: catalytic domain 0.999542 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.9999 residue_name_number cleaner0 2023-07-11T15:42:48Z DUMMY: Phe285 protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 0.7495031 structure_element cleaner0 2023-07-11T14:16:11Z SO: CD 0.9997335 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 RESULTS paragraph 15610 It has previously been reported that several cytosolic and inducible nuclear MKPs undergo catalytic activation upon interaction with the MAPK substrates. This allosteric activation of MKP3 has been well-documented in vitro using pNPP, a small-molecule phosphotyrosine analogue of its normal substrate. We then assayed pNPPase activities of MKP7ΔC304 and MKP7-CD in the presence of JNK1. Incubation of MKP7 with JNK1 did not markedly stimulate the phosphatase activity, which is consistent with previous results that MKP7 solely possesses the intrinsic activity (Supplementary Fig. 2b). The small pNPP molecule binds directly at the enzyme active site and can be used to probe the reaction mechanism of protein phosphatases. We therefore examined the effects of the MKP7-CD mutants on their pNPPase activities. As shown in Fig. 4f, all the mutants, except F287D/A, showed little or no activity change compared with the wild-type MKP7-CD. In the JNK1/MKP7-CD complex structure, Phe287 of MKP7 does not make contacts with JNK1 substrate. It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). 0.99966466 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.99960107 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK 0.99984586 protein cleaner0 2023-07-11T14:16:27Z PR: MKP3 0.99979967 chemical cleaner0 2023-07-12T15:01:52Z CHEBI: pNPP 0.9996563 residue_name melaniev@ebi.ac.uk 2023-07-21T14:00:06Z SO: phosphotyrosine 0.9685103 protein_type cleaner0 2023-07-11T20:16:18Z MESH: pNPPase 0.9973181 mutant cleaner0 2023-07-11T14:17:23Z MESH: MKP7ΔC304 protein PR: cleaner0 2023-07-11T14:04:13Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9957655 protein_state cleaner0 2023-07-12T14:49:29Z DUMMY: presence of 0.9997247 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.98657924 experimental_method cleaner0 2023-07-12T14:55:33Z MESH: Incubation 0.99985695 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9997564 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.99723995 protein_type cleaner0 2023-07-11T20:16:22Z MESH: phosphatase 0.9998536 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9998017 chemical cleaner0 2023-07-12T15:01:55Z CHEBI: pNPP 0.9995545 site cleaner0 2023-07-12T14:36:41Z SO: active site 0.99950284 protein_type cleaner0 2023-07-11T20:16:25Z MESH: protein phosphatases protein PR: cleaner0 2023-07-11T14:04:14Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9039243 protein_state cleaner0 2023-07-11T20:04:10Z DUMMY: mutants 0.99861705 protein_type cleaner0 2023-07-11T20:16:29Z MESH: pNPPase protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.9993043 mutant cleaner0 2023-07-11T20:02:34Z MESH: F287D/A 0.9995575 protein_state cleaner0 2023-07-11T15:48:09Z DUMMY: wild-type protein PR: cleaner0 2023-07-11T14:04:14Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.99967325 complex_assembly cleaner0 2023-07-11T20:33:18Z GO: JNK1/MKP7-CD 0.9995315 evidence cleaner0 2023-07-12T14:43:01Z DUMMY: structure 0.999907 residue_name_number cleaner0 2023-07-11T15:47:33Z DUMMY: Phe287 0.9998541 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9997867 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.9994991 site cleaner0 2023-07-12T14:36:47Z SO: pocket 0.9997178 structure_element cleaner0 2023-07-11T15:41:48Z SO: P-loop 0.99967957 structure_element cleaner0 2023-07-11T20:24:31Z SO: general acid loop bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z hydrophobic contacts 0.9999043 residue_name_number cleaner0 2023-07-11T14:25:16Z DUMMY: Arg250 0.99990535 residue_name_number cleaner0 2023-07-11T20:31:42Z DUMMY: Glu217 0.99990547 residue_name_number cleaner0 2023-07-11T20:31:44Z DUMMY: Ile219 0.9999083 residue_name_number cleaner0 2023-07-11T15:47:33Z DUMMY: Phe287 0.999853 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.99966335 protein_type cleaner0 2023-07-11T20:16:32Z MESH: PTPs 0.99990714 residue_name_number cleaner0 2023-07-11T15:49:12Z DUMMY: Gln266 0.9997998 protein cleaner0 2023-07-11T15:49:20Z PR: PTP1B 0.9814004 protein cleaner0 2023-07-11T14:22:09Z PR: VHR 0.9999057 residue_name_number cleaner0 2023-07-11T15:49:26Z DUMMY: Phe166 0.99110174 protein cleaner0 2023-07-11T14:22:09Z PR: VHR 0.9995959 site cleaner0 2023-07-12T14:36:52Z SO: active-site residues 0.9998474 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 RESULTS paragraph 17123 Kinase-associated phosphatase (KAP), a member of the DUSP family, plays a crucial role in cell cycle regulation by dephosphorylating the pThr160 residue of CDK2 (cyclin-dependent kinase 2). The crystal structure of the CDK2/KAP complex has been determined at 3.0 Å (Fig. 5a). The interface between these two proteins consists of three discontinuous contact regions. Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the αG helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b). There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2. Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c). The substitution of the two hydrophobic residues with charged/polar residues (F285I/N286K) seriously disrupts the hydrophobic interaction required for MKP7 binding on JNK1 (Fig. 4a). In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK. These data indicated that a unique hydrophobic pocket formed between the MAPK insert and αG helix plays a major role in the substrate recognition by MKPs. 0.8595056 protein cleaner0 2023-07-11T15:50:57Z PR: Kinase-associated phosphatase 0.8032938 protein cleaner0 2023-07-11T15:50:48Z PR: KAP 0.9996263 protein_type cleaner0 2023-07-11T20:16:37Z MESH: DUSP family 0.9998877 ptm cleaner0 2023-07-11T20:30:37Z MESH: pThr160 0.99959224 protein cleaner0 2023-07-11T15:51:11Z PR: CDK2 0.84290755 protein cleaner0 2023-07-11T15:51:07Z PR: cyclin-dependent kinase 2 0.9995709 evidence cleaner0 2023-07-12T14:43:16Z DUMMY: crystal structure 0.9996457 complex_assembly cleaner0 2023-07-11T20:33:22Z GO: CDK2/KAP 0.99838305 site cleaner0 2023-07-12T14:36:57Z SO: interface 0.9886779 protein cleaner0 2023-07-11T15:50:49Z PR: KAP 0.9997129 protein cleaner0 2023-07-11T15:51:12Z PR: CDK2 0.9996252 site cleaner0 2023-07-12T14:37:17Z SO: recognition site 0.99971896 protein cleaner0 2023-07-11T15:51:12Z PR: CDK2 0.9995949 structure_element cleaner0 2023-07-11T20:24:34Z SO: αG helix 0.99938977 structure_element cleaner0 2023-07-11T20:24:37Z SO: L14 loop structure_element SO: cleaner0 2023-07-11T20:25:04Z N-terminal helical region 0.9977049 protein cleaner0 2023-07-11T15:50:49Z PR: KAP bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z hydrogen bond 0.9999007 residue_name_number cleaner0 2023-07-11T20:31:50Z DUMMY: Ile183 0.9984602 protein cleaner0 2023-07-11T15:50:49Z PR: KAP 0.9999026 residue_name_number cleaner0 2023-07-11T20:31:55Z DUMMY: Glu208 0.9996748 protein cleaner0 2023-07-11T15:51:12Z PR: CDK2 0.99989843 residue_name_number cleaner0 2023-07-11T20:32:01Z DUMMY: Lys184 0.9994535 protein cleaner0 2023-07-11T15:50:49Z PR: KAP 0.9998981 residue_name_number cleaner0 2023-07-11T20:32:04Z DUMMY: Asp235 0.9997507 protein cleaner0 2023-07-11T15:51:12Z PR: CDK2 0.99959075 experimental_method cleaner0 2023-07-12T14:56:07Z MESH: Structural analysis 0.99918985 experimental_method cleaner0 2023-07-12T14:56:09Z MESH: sequence alignment protein PR: cleaner0 2023-07-11T14:04:14Z MKP7 0.62528604 structure_element cleaner0 2023-07-11T14:16:11Z SO: CD 0.9986525 protein cleaner0 2023-07-11T15:50:49Z PR: KAP 0.9995457 site cleaner0 2023-07-12T14:37:26Z SO: substrate-binding region 0.99974567 structure_element cleaner0 2023-07-11T20:25:09Z SO: FNFL 0.99516207 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9981623 structure_element cleaner0 2023-07-11T20:25:12Z SO: IKQY 0.99752086 protein cleaner0 2023-07-11T15:50:49Z PR: KAP 0.9995154 experimental_method cleaner0 2023-07-12T14:56:14Z MESH: substitution 0.9996402 mutant cleaner0 2023-07-11T20:30:46Z MESH: F285I 0.99964404 mutant cleaner0 2023-07-11T20:30:49Z MESH: N286K bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z hydrophobic interaction 0.9998348 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.99979585 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.9999008 residue_name_number cleaner0 2023-07-11T15:43:34Z DUMMY: His230 0.9998977 residue_name_number cleaner0 2023-07-11T15:43:17Z DUMMY: Val256 0.9997652 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.9998976 residue_name_number cleaner0 2023-07-11T20:32:07Z DUMMY: Glu208 0.9998958 residue_name_number cleaner0 2023-07-11T20:32:10Z DUMMY: Asp235 0.9998272 protein cleaner0 2023-07-11T15:51:12Z PR: CDK2 0.96913147 protein cleaner0 2023-07-11T15:51:12Z PR: CDK2 0.99889547 site cleaner0 2023-07-12T14:37:30Z SO: interactive surface 0.9995783 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.99939847 site cleaner0 2023-07-12T14:37:35Z SO: hydrophobic pocket 0.9705114 structure_element cleaner0 2023-07-11T20:25:17Z SO: MAPK insert 0.99964076 structure_element cleaner0 2023-07-11T20:25:19Z SO: αG helix 0.999629 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs RESULTS title_2 18683 F-site interaction is crucial for JNK1 inactivation in vivo 0.9995083 site cleaner0 2023-07-12T14:37:38Z SO: F-site 0.9998393 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 RESULTS paragraph 18743 JNK is activated following cellular exposure to a number of acute stimuli such as anisomycin, H2O2, ultraviolet light, sorbitol, DNA-damaging agents and several strong apoptosis inducers (etoposide, cisplatin and taxol). To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. As shown in Fig. 6a–c, immunobloting showed similar expression levels for the different MKP7 constructs in all the cells. Overexpressed full-length MKP7, MKP7ΔC304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells. Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. We next tested in vivo interactions between JNK1 mutants and full-length MKP7 by coimmunoprecipitation experiments under unstimulated conditions. When co-expressed in HEK293T cells, wild-type (HA)-JNK1 was readily precipitated with (Myc)-MKP7 (Fig. 6d), indicating that MKP7 binds dephosphorylated JNK1 protein in vivo. In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1–MKP7 interaction (Fig. 6d and Supplementary Fig. 3a). 0.9604673 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.9985983 chemical cleaner0 2023-07-12T15:02:01Z CHEBI: anisomycin 0.99502426 chemical cleaner0 2023-07-12T15:02:05Z CHEBI: H2O2 0.98596144 chemical cleaner0 2023-07-11T15:51:53Z CHEBI: sorbitol 0.9941076 chemical cleaner0 2023-07-11T15:51:59Z CHEBI: etoposide 0.9951277 chemical cleaner0 2023-07-11T15:52:04Z CHEBI: cisplatin 0.9956655 chemical cleaner0 2023-07-11T15:52:09Z CHEBI: taxol 0.9998487 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.6975378 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.9971235 protein_state cleaner0 2023-07-12T14:49:37Z DUMMY: HA-tagged 0.99959403 protein_state cleaner0 2023-07-11T15:54:25Z DUMMY: full-length 0.9998418 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.99450654 mutant cleaner0 2023-07-11T14:17:23Z MESH: MKP7ΔC304 protein PR: cleaner0 2023-07-11T14:04:14Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9996773 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.94985515 chemical cleaner0 2023-07-11T15:52:00Z CHEBI: etoposide 0.99964476 experimental_method cleaner0 2023-07-12T14:56:29Z MESH: immunobloting 0.9997931 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.99441683 experimental_method cleaner0 2023-07-12T14:56:32Z MESH: Overexpressed 0.9995976 protein_state cleaner0 2023-07-11T15:54:25Z DUMMY: full-length 0.9998485 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9981046 mutant cleaner0 2023-07-11T14:17:23Z MESH: MKP7ΔC304 protein PR: cleaner0 2023-07-11T14:04:14Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9932893 protein_state cleaner0 2023-07-11T20:03:10Z DUMMY: phosphorylated 0.9973918 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.99957913 structure_element cleaner0 2023-07-11T14:07:23Z SO: D-motif 0.99302083 protein_state cleaner0 2023-07-11T20:04:10Z DUMMY: mutants 0.9996798 mutant cleaner0 2023-07-11T15:52:49Z MESH: R56A 0.9894162 mutant cleaner0 2023-07-11T15:52:54Z MESH: R57A 0.9996897 mutant cleaner0 2023-07-11T15:52:59Z MESH: V63A 0.99854505 mutant cleaner0 2023-07-11T15:53:04Z MESH: I65A 0.9985159 protein_state cleaner0 2023-07-11T20:03:16Z DUMMY: dephosphorylated 0.9928854 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.9995721 protein_state cleaner0 2023-07-11T15:54:34Z DUMMY: wild type 0.9998369 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:14:22Z KBD 0.69305307 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.99383813 protein_state cleaner0 2023-07-11T20:03:11Z DUMMY: phosphorylated 0.995398 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.9998392 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.99957305 structure_element cleaner0 2023-07-11T14:06:58Z SO: FXF-motif 0.99624014 protein_state cleaner0 2023-07-11T20:04:10Z DUMMY: mutants 0.99967384 mutant cleaner0 2023-07-11T15:53:11Z MESH: F285D 0.9996712 mutant cleaner0 2023-07-11T15:53:17Z MESH: F287D 0.99967074 mutant cleaner0 2023-07-11T15:53:45Z MESH: L288D 0.999846 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9996885 mutant cleaner0 2023-07-11T15:53:33Z MESH: D268A 0.99969137 mutant cleaner0 2023-07-11T15:53:39Z MESH: N286A 0.99719185 protein_state cleaner0 2023-07-11T20:04:10Z DUMMY: mutants 0.9376806 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.9992361 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.999608 protein_state cleaner0 2023-07-11T15:54:24Z DUMMY: full-length 0.99984133 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.99945056 experimental_method cleaner0 2023-07-12T14:56:41Z MESH: coimmunoprecipitation experiments 0.9995473 experimental_method cleaner0 2023-07-12T14:56:44Z MESH: co-expressed 0.9995973 protein_state cleaner0 2023-07-11T15:48:09Z DUMMY: wild-type 0.9994735 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.9990482 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9998165 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9996216 protein_state cleaner0 2023-07-11T20:03:16Z DUMMY: dephosphorylated 0.99959725 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.9960538 experimental_method cleaner0 2023-07-12T14:56:47Z MESH: in vitro pull-down 0.9902006 protein_state cleaner0 2023-07-11T20:04:10Z DUMMY: mutants 0.99968827 mutant cleaner0 2023-07-11T15:53:51Z MESH: D229A 0.99967825 mutant cleaner0 2023-07-11T15:53:59Z MESH: W234D 0.9996753 mutant cleaner0 2023-07-11T15:54:04Z MESH: Y259D 0.9998559 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9996773 mutant cleaner0 2023-07-11T20:31:09Z MESH: I231D 0.99910635 protein_state cleaner0 2023-07-11T15:48:15Z DUMMY: mutant 0.98990566 complex_assembly cleaner0 2023-07-11T14:13:02Z GO: JNK1–MKP7 RESULTS paragraph 20599 Activation of the JNK signalling pathway is frequently associated with apoptotic cell death, and inhibition of JNK can prevent apoptotic death of multiple cells. To examine whether the inhibition of JNK activity by MKP7 would provide protections against the apoptosis, we analysed the rate of apoptosis in ultraviolet-irradiated cells transfected with MKP7 (wild type or mutants) by flow cytometry. The results showed similar apoptotic rates between cells transfected with blank vector or with MKP7 (wild type or mutants) under unstimulated conditions (Supplementary Fig. 3b), while ultraviolet-irradiation significantly increased apoptotic rate in cells transfected with blank vector (Fig. 6e). Expressions of wild-type MKP7, MKP7ΔC304 and MKP7-CD significantly decreased the proportion of apoptotic cells after ultraviolet treatment. Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did. In contrast, cells transfected with the MKP7 FXF-motif mutants (F285D, F287D and L288D) showed little protective effect after ultraviolet treatment and similar levels of apoptosis rates were detected to cells transfected with control vectors (Fig. 6e,f). Taken together, our results suggested that FXF-motif-mediated, rather than KBD-mediated, interaction is essential for MKP7 to block ultraviolet-induced apoptosis. 0.966076 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.98420215 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.9612107 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.99984944 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.99985313 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9995705 protein_state cleaner0 2023-07-11T15:54:35Z DUMMY: wild type 0.9624378 protein_state cleaner0 2023-07-11T20:04:10Z DUMMY: mutants 0.9994309 experimental_method cleaner0 2023-07-12T14:56:52Z MESH: flow cytometry 0.9998561 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.99956214 protein_state cleaner0 2023-07-11T15:54:35Z DUMMY: wild type 0.9825302 protein_state cleaner0 2023-07-11T20:04:09Z DUMMY: mutants 0.55191964 experimental_method cleaner0 2023-07-12T14:56:57Z MESH: Expressions 0.9995586 protein_state cleaner0 2023-07-11T15:48:09Z DUMMY: wild-type 0.9998543 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9996325 mutant cleaner0 2023-07-11T14:17:23Z MESH: MKP7ΔC304 protein PR: cleaner0 2023-07-11T14:04:14Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.79603076 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:14:22Z KBD 0.99613756 protein_state cleaner0 2023-07-11T20:04:10Z DUMMY: mutants 0.9996768 mutant cleaner0 2023-07-11T15:52:50Z MESH: R56A 0.78404593 mutant cleaner0 2023-07-11T15:52:54Z MESH: R57A 0.9996897 mutant cleaner0 2023-07-11T15:53:00Z MESH: V63A 0.9893594 mutant cleaner0 2023-07-11T15:53:05Z MESH: I65A 0.99985623 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9995667 protein_state cleaner0 2023-07-11T15:54:35Z DUMMY: wild type 0.99985516 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9996304 structure_element cleaner0 2023-07-11T14:06:58Z SO: FXF-motif 0.99723876 protein_state cleaner0 2023-07-11T20:04:10Z DUMMY: mutants 0.99966097 mutant cleaner0 2023-07-11T15:53:12Z MESH: F285D 0.9996561 mutant cleaner0 2023-07-11T15:53:18Z MESH: F287D 0.9996691 mutant cleaner0 2023-07-11T15:53:46Z MESH: L288D 0.99962074 structure_element cleaner0 2023-07-11T14:06:58Z SO: FXF-motif 0.99956053 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9998573 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 RESULTS title_2 22013 A similar docking mechanism for JNK1 recognition by MKP5 0.9998292 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.9998512 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 RESULTS paragraph 22070 MKP5 belongs to the same subfamily as MKP7. MKP5 is unique among the MKPs in possessing an additional domain of unknown function at the N-terminus (Fig. 7a). The KBD of MKP5 interacts with the D-site of p38α to mediate the enzyme–substrate interaction. Deletion of the KBD in MKP5 leads to a 280-fold increase in Km for p38α substrate. In contrast to p38α substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b). The substrate specificity constant kcat /Km value for MKP5-CD was calculated as 1.0 × 105 M−1 s−1, which is very close to that of MKP7-CD (1.07 × 105 M−1 s−1). The crystal structure of human MKP5-CD has been determined. Comparisons between catalytic domains structures of MKP5 and MKP7 reveal that the overall folds of the two proteins are highly similar, with only a few regions exhibiting small deviations (r.m.s.d. of 0.79 Å; Fig. 7c). 0.99984145 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.999816 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.999853 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.9996393 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.9997284 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9998511 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.99933696 site cleaner0 2023-07-11T14:08:28Z SO: D-site 0.9997428 protein cleaner0 2023-07-11T14:16:53Z PR: p38α 0.995196 experimental_method cleaner0 2023-07-12T14:57:09Z MESH: Deletion of 0.99958664 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9998568 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.9995534 evidence cleaner0 2023-07-12T14:43:22Z DUMMY: Km 0.99974126 protein cleaner0 2023-07-11T14:16:53Z PR: p38α 0.99971825 protein cleaner0 2023-07-11T14:16:53Z PR: p38α 0.94834405 experimental_method cleaner0 2023-07-12T14:57:11Z MESH: deletion of 0.9998579 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.7168404 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9996953 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.9997081 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.99985504 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.99968207 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.9992036 evidence cleaner0 2023-07-12T14:43:26Z DUMMY: substrate specificity constant 0.9992015 evidence cleaner0 2023-07-12T14:43:29Z DUMMY: kcat /Km 0.9990727 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD protein PR: cleaner0 2023-07-11T14:04:14Z MKP7 0.5896785 structure_element cleaner0 2023-07-11T14:16:11Z SO: CD 0.99961245 evidence cleaner0 2023-07-12T14:43:32Z DUMMY: crystal structure 0.99919623 species cleaner0 2023-07-11T20:32:27Z MESH: human 0.9881953 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9996147 structure_element cleaner0 2023-07-11T20:25:29Z SO: catalytic domains 0.99957293 evidence cleaner0 2023-07-12T14:43:36Z DUMMY: structures 0.99985886 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.9998543 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9995599 evidence cleaner0 2023-07-12T14:43:39Z DUMMY: r.m.s.d. RESULTS paragraph 23094 Given the distinct interaction mode revealed by the crystal structure of JNK1–MKP7-CD, one obvious question is whether this is a general mechanism used by all members of the JNK-specific MKPs. To address this issue, we first examined the docking ability of JNK1 to the KBD and CD of MKP5 using gel filtration analysis and pull-down assays. It can be seen from gel filtration experiments that JNK1 can forms a stable heterodimer with MKP5-CD in solution, but no detectable interaction was found with the KBD domain (Fig. 7d). Pull-down assays also confirmed the protein–protein interactions observed above. The catalytic domain of MKP5, but not its KBD, was able to pull-down a detectable amount of JNK1 (Fig. 7e), implicating a different substrate-recognition mechanisms for p38 and JNK MAPKs. To further test our hypothesis, we generated forms of MKP5-CD bearing mutations corresponding to the changes we made on MKP7-CD on the basis of sequence and structural alignment and examined their effects on the phosphatase activity. As shown in Fig. 7f, the T432A and L449F MKP5 mutant showed little or no difference in phosphatase activity, whereas the other mutants showed reduced specific activities of MKP5. As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). In addition, there were no significant differences in the CD spectra between wild-type and mutant proteins, indicating that the overall structures of these mutants did not change significantly from that of wild-type MKP5 protein (Supplementary Fig. 4a). Taken together, our results suggest that MKP5 binds JNK1 in a docking mode similar to that in the JNK1–MKP7 complex, and the detailed interaction model can be generated using molecular dynamics simulation based on the structure of JNK1–MKP7-CD complex (Supplementary Fig. 4b,c). In this model, the MKP5-CD adopts a conformation nearly identical to that in its unbound form, suggesting that the conformation of the catalytic domain undergoes little change, if any at all, upon JNK1 binding. In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1–MKP7-CD complex (Supplementary Fig. 4d). Despite the strong similarities between JNK1–MKP5-CD and JNK1–MKP7-CD, however, there are differences. The JNK1–MKP7-CD interaction is better and more extensive. Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1. In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD). 0.9996052 evidence cleaner0 2023-07-12T14:43:43Z DUMMY: crystal structure 0.99971896 complex_assembly cleaner0 2023-07-11T15:55:37Z GO: JNK1–MKP7-CD 0.9985678 protein_type cleaner0 2023-07-11T19:54:00Z MESH: JNK-specific MKPs 0.9996025 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.99980146 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9997985 structure_element cleaner0 2023-07-11T14:16:11Z SO: CD 0.9998498 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.9994723 experimental_method cleaner0 2023-07-12T14:57:21Z MESH: gel filtration analysis 0.9995785 experimental_method cleaner0 2023-07-12T14:57:24Z MESH: pull-down assays 0.99940306 experimental_method cleaner0 2023-07-12T14:57:27Z MESH: gel filtration experiments 0.99954104 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.9941077 protein_state cleaner0 2023-07-12T14:49:42Z DUMMY: stable 0.99865675 oligomeric_state cleaner0 2023-07-12T15:01:02Z DUMMY: heterodimer 0.99686253 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9997793 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9995878 experimental_method cleaner0 2023-07-12T14:57:30Z MESH: Pull-down assays 0.9997058 structure_element cleaner0 2023-07-11T20:20:49Z SO: catalytic domain 0.9998578 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.99981016 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9997764 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.99910957 protein_type cleaner0 2023-07-11T14:06:36Z MESH: p38 0.99906963 protein_type cleaner0 2023-07-11T14:06:30Z MESH: JNK 0.9905059 protein_type cleaner0 2023-07-11T14:03:02Z MESH: MAPKs 0.99416006 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9255612 experimental_method cleaner0 2023-07-12T14:57:37Z MESH: mutations 0.9993222 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9994841 experimental_method cleaner0 2023-07-12T14:57:41Z MESH: sequence and structural alignment 0.99898106 protein_type cleaner0 2023-07-11T20:16:43Z MESH: phosphatase 0.9996252 mutant cleaner0 2023-07-11T20:31:14Z MESH: T432A 0.9996165 mutant cleaner0 2023-07-11T20:31:17Z MESH: L449F 0.99975866 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.999532 protein_state cleaner0 2023-07-11T15:48:15Z DUMMY: mutant protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.9998572 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.99984705 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.99950796 mutant cleaner0 2023-07-11T20:31:21Z MESH: F451D/A 0.9959241 protein_type cleaner0 2023-07-11T20:16:46Z MESH: pNPPase 0.999556 protein_state cleaner0 2023-07-11T15:48:09Z DUMMY: wild-type 0.9991085 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9973254 experimental_method cleaner0 2023-07-12T14:57:44Z MESH: point mutations 0.99965835 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.9994711 evidence cleaner0 2023-07-12T14:43:48Z DUMMY: binding affinity 0.99926347 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.99963176 protein cleaner0 2023-07-11T14:03:59Z PR: JNK1 0.9987402 evidence cleaner0 2023-07-12T14:43:51Z DUMMY: CD spectra 0.9995606 protein_state cleaner0 2023-07-11T15:48:09Z DUMMY: wild-type protein_state DUMMY: cleaner0 2023-07-11T15:48:15Z mutant 0.8988361 evidence cleaner0 2023-07-12T14:43:55Z DUMMY: structures protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.9995599 protein_state cleaner0 2023-07-11T15:48:09Z DUMMY: wild-type 0.99984586 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.99984026 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.99974054 protein cleaner0 2023-07-11T14:04:00Z PR: JNK1 0.99969023 complex_assembly cleaner0 2023-07-11T14:13:02Z GO: JNK1–MKP7 0.99949646 experimental_method cleaner0 2023-07-12T14:57:48Z MESH: molecular dynamics simulation 0.998095 evidence cleaner0 2023-07-12T14:43:57Z DUMMY: structure 0.9997139 complex_assembly cleaner0 2023-07-11T19:51:19Z GO: JNK1–MKP7-CD 0.99576855 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9996253 protein_state cleaner0 2023-07-12T14:49:50Z DUMMY: unbound 0.99968714 structure_element cleaner0 2023-07-11T20:20:49Z SO: catalytic domain 0.9995883 protein cleaner0 2023-07-11T14:04:00Z PR: JNK1 0.999897 residue_name_number cleaner0 2023-07-11T20:32:17Z DUMMY: Leu449 0.99985313 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.999897 residue_name_number cleaner0 2023-07-11T15:42:48Z DUMMY: Phe285 0.9998419 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.99959517 site cleaner0 2023-07-12T14:37:46Z SO: hydrophobic pocket 0.99971 protein cleaner0 2023-07-11T14:04:00Z PR: JNK1 residue_name_number DUMMY: cleaner0 2023-07-11T15:42:48Z Phe285 complex_assembly GO: cleaner0 2023-07-11T19:51:19Z JNK1–MKP7-CD protein PR: cleaner0 2023-07-11T14:04:00Z JNK1 protein PR: cleaner0 2023-07-11T14:16:42Z MKP5 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD complex_assembly GO: cleaner0 2023-07-11T19:51:19Z JNK1–MKP7-CD complex_assembly GO: cleaner0 2023-07-11T19:51:19Z JNK1–MKP7-CD residue_name_number DUMMY: cleaner0 2023-07-11T15:44:17Z Asp268 protein PR: cleaner0 2023-07-11T14:04:14Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z salt bridge protein PR: cleaner0 2023-07-11T14:04:00Z JNK1 residue_name_number DUMMY: cleaner0 2023-07-11T15:44:34Z Arg263 residue_name_number DUMMY: cleaner0 2023-07-11T20:05:29Z Thr432 protein PR: cleaner0 2023-07-11T14:16:42Z MKP5 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD protein PR: cleaner0 2023-07-11T14:04:00Z JNK1 protein PR: cleaner0 2023-07-11T14:04:14Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD residue_name_number DUMMY: cleaner0 2023-07-11T15:44:01Z Phe215 residue_name_number DUMMY: cleaner0 2023-07-11T20:04:45Z Leu267 residue_name_number DUMMY: cleaner0 2023-07-11T15:46:01Z Leu288 residue_name_number DUMMY: cleaner0 2023-07-11T20:04:57Z Asn379 residue_name_number DUMMY: cleaner0 2023-07-11T20:05:07Z Met431 residue_name_number DUMMY: cleaner0 2023-07-11T20:05:18Z Met452 protein PR: cleaner0 2023-07-11T14:16:42Z MKP5 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z hydrophobic interactions protein PR: cleaner0 2023-07-11T14:16:42Z MKP5 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD protein PR: cleaner0 2023-07-11T14:04:00Z JNK1 protein PR: cleaner0 2023-07-11T14:04:00Z JNK1 protein PR: cleaner0 2023-07-11T14:04:14Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD protein PR: cleaner0 2023-07-11T14:16:42Z MKP5 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD protein PR: cleaner0 2023-07-11T14:16:42Z MKP5 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD protein_state DUMMY: cleaner0 2023-07-11T20:05:56Z p protein PR: cleaner0 2023-07-11T20:06:05Z JNK1 protein PR: cleaner0 2023-07-11T14:04:14Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD DISCUSS title_1 26278 Discussion DISCUSS paragraph 26289 The MAPKs p38, ERK and JNK, are central to evolutionarily conserved signalling pathways that are present in all eukaryotic cells. Each MAPK cascade is activated in response to a diverse array of extracellular signals and culminates in the dual-phosphorylation of a threonine and a tyrosine residue in the MAPK-activation loop. The propagation of MAPK signals is attenuated through the actions of the MKPs. Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the ‘kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL). Crystal structures of ERK2 bound with the D-motif sequences derived from MKP3 and HePTP have been reported. These structures revealed that linear docking motifs in interacting proteins bind to a common docking site on MAPKs outside the kinase active site. The particular amino acids and their spacing within D-motif sequences and amino acid composition of the docking sites on MAPKs appear to determine the specificity of D-motifs for individual MAPKs. 0.9995801 protein_type cleaner0 2023-07-11T14:03:02Z MESH: MAPKs 0.9994368 protein_type cleaner0 2023-07-11T14:06:36Z MESH: p38 0.99916995 protein_type cleaner0 2023-07-11T14:06:43Z MESH: ERK 0.99858177 protein_type cleaner0 2023-07-11T14:06:31Z MESH: JNK 0.99923587 taxonomy_domain cleaner0 2023-07-11T20:32:36Z DUMMY: eukaryotic 0.99952877 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK 0.97636795 ptm cleaner0 2023-07-12T15:04:02Z MESH: dual-phosphorylation 0.9991327 residue_name cleaner0 2023-07-11T20:32:50Z SO: threonine 0.99905163 residue_name cleaner0 2023-07-11T20:33:01Z SO: tyrosine 0.9992236 structure_element cleaner0 2023-07-11T19:48:38Z SO: MAPK-activation loop 0.9995788 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK 0.99965894 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.99959964 protein_type cleaner0 2023-07-11T14:03:02Z MESH: MAPKs 0.9996265 protein_type cleaner0 2023-07-11T20:16:52Z MESH: phosphatases 0.9995668 structure_element cleaner0 2023-07-11T19:48:41Z SO: kinase-interaction motif 0.9996681 structure_element cleaner0 2023-07-11T14:07:23Z SO: D-motif 0.99964 protein_type cleaner0 2023-07-11T14:08:52Z MESH: DUSPs 0.99963486 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.9996373 protein_type cleaner0 2023-07-11T14:08:41Z MESH: tyrosine phosphatases 0.9723622 protein cleaner0 2023-07-11T19:48:17Z PR: HePTP 0.73130083 protein cleaner0 2023-07-11T19:48:22Z PR: STEP 0.998229 protein cleaner0 2023-07-11T19:48:27Z PR: PTP-SL 0.9996085 evidence cleaner0 2023-07-12T14:44:03Z DUMMY: Crystal structures 0.9965006 protein cleaner0 2023-07-11T14:16:33Z PR: ERK2 0.99931073 protein_state cleaner0 2023-07-12T14:49:55Z DUMMY: bound with 0.9996767 structure_element cleaner0 2023-07-11T14:07:23Z SO: D-motif 0.9997563 protein cleaner0 2023-07-11T14:16:27Z PR: MKP3 0.9996892 protein cleaner0 2023-07-11T19:48:17Z PR: HePTP 0.998662 evidence cleaner0 2023-07-12T14:44:06Z DUMMY: structures 0.84627134 structure_element cleaner0 2023-07-11T19:48:06Z SO: linear docking motifs 0.9994099 site cleaner0 2023-07-12T14:37:56Z SO: docking site 0.9995753 protein_type cleaner0 2023-07-11T14:03:02Z MESH: MAPKs 0.9915377 protein_type cleaner0 2023-07-11T20:06:36Z MESH: kinase 0.980252 site cleaner0 2023-07-12T14:37:58Z SO: active site 0.9995621 structure_element cleaner0 2023-07-11T14:07:23Z SO: D-motif 0.9995978 site cleaner0 2023-07-12T14:38:01Z SO: docking sites 0.99960834 protein_type cleaner0 2023-07-11T14:03:02Z MESH: MAPKs 0.99966985 structure_element cleaner0 2023-07-11T20:25:35Z SO: D-motifs 0.9996258 protein_type cleaner0 2023-07-11T14:03:02Z MESH: MAPKs DISCUSS paragraph 27390 Recently, the crystal structure of a complex between the KBD of MKP5 and p38α has been obtained. This complex has revealed a distinct interaction mode for MKP5. The KBD of MKP5 binds to p38α in the opposite polypeptide direction compared with how the D-motif of MKP3 binds to ERK2. In contrast to the canonical D-motif-binding mode, separate helices, α2 and α3′, in the KBD of MKP5 engage the p38α-docking site. Further structural and biochemical studies indicate that KBD of MKP7 may interact with p38α in a similar manner to that of MKP5. In contrast to MKP5, removal of the KBD domain from MKP7 does not drastically affect enzyme catalysis, and the kinetic parameters of MKP7-CD for p38α substrate are very similar to those for JNK1 substrate. Taken together, these results suggest that MKP7 utilizes a bipartite recognition mechanism to achieve the efficiency and fidelity of p38α signalling. The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. 0.9996283 evidence cleaner0 2023-07-12T14:44:09Z DUMMY: crystal structure 0.99979156 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9998586 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.9997658 protein cleaner0 2023-07-11T14:16:53Z PR: p38α 0.9998615 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.9997876 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.99985623 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.99980146 protein cleaner0 2023-07-11T14:16:53Z PR: p38α 0.9996622 structure_element cleaner0 2023-07-11T14:07:23Z SO: D-motif 0.9998518 protein cleaner0 2023-07-11T14:16:27Z PR: MKP3 0.99966013 protein cleaner0 2023-07-11T14:16:33Z PR: ERK2 0.94805497 site cleaner0 2023-07-11T19:48:51Z SO: D-motif-binding mode 0.9082678 structure_element cleaner0 2023-07-11T20:25:43Z SO: helices 0.9998097 structure_element cleaner0 2023-07-11T20:25:46Z SO: α2 0.99963236 structure_element cleaner0 2023-07-11T20:25:48Z SO: α3′ 0.99978834 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.99986005 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 site SO: cleaner0 2023-07-11T19:49:31Z p38α-docking site 0.9994786 experimental_method cleaner0 2023-07-12T14:57:53Z MESH: structural and biochemical studies 0.99978584 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9998585 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.99980944 protein cleaner0 2023-07-11T14:16:53Z PR: p38α 0.99985754 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.9998597 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.9972884 experimental_method cleaner0 2023-07-12T14:57:59Z MESH: removal of 0.9997774 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9998616 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9995437 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9997954 protein cleaner0 2023-07-11T14:16:53Z PR: p38α 0.99980885 protein cleaner0 2023-07-11T14:04:00Z PR: JNK1 0.99985254 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9997168 protein cleaner0 2023-07-11T14:16:53Z PR: p38α 0.9990908 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.92762035 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.99951345 site cleaner0 2023-07-11T14:08:28Z SO: D-site 0.99981517 protein cleaner0 2023-07-11T14:16:53Z PR: p38α 0.9996432 site cleaner0 2023-07-12T14:38:06Z SO: catalytic pocket 0.999741 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9997526 protein cleaner0 2023-07-11T14:16:53Z PR: p38α 0.99972093 structure_element cleaner0 2023-07-11T20:26:06Z SO: activation loop 0.9998603 protein cleaner0 2023-07-11T14:04:14Z PR: MKP7 0.9996278 site cleaner0 2023-07-12T14:38:09Z SO: active site 0.9998229 protein cleaner0 2023-07-11T14:16:53Z PR: p38α DISCUSS paragraph 28756 In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs. This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix αG. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d). MKP3 is highly specific in dephosphorylating and inactivating ERK2, and the phosphatase activity of the MKP3-catalysed pNPP reaction can be markedly increased in the presence of ERK2 (refs). Sequence alignment of all MKPs reveals a high degree of conservation of residues surrounding the interacting region observed in JNK1–MKP7-CD complex (Supplementary Fig. 5). Therefore, it is tempting to speculate that the catalytic domain of MKP3 may bind to ERK2 in a manner analogous to the way by which MKP7-CD binds to JNK1. A comprehensive examination of the molecular basis of the specific ERK2 recognition by MKP3 is underway. The ongoing work demonstrates that although the overall interaction modes are similar between the JNK1–MKP7-CD and ERK2–MKP3-CD complexes, the ERK2–MKP3-CD interaction is less extensive and helix α4 from MKP3-CD does not interact directly with ERK2. The FXF-motif-mediated interaction is critical for both pERK2 inactivation and ERK2-induced MKP3 activation (manuscript in preparation). 0.99955744 site cleaner0 2023-07-11T14:08:28Z SO: D-site 0.99954635 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK 0.99971265 protein cleaner0 2023-07-11T14:16:33Z PR: ERK2 0.99957544 site cleaner0 2023-07-12T14:38:17Z SO: second binding site 0.99919015 protein_type cleaner0 2023-07-11T20:17:00Z MESH: phosphatases 0.99955225 site cleaner0 2023-07-11T14:07:53Z SO: FXF-motif-binding site 0.99956304 site cleaner0 2023-07-12T14:38:21Z SO: F-site 0.99965453 protein_state cleaner0 2023-07-12T14:50:01Z DUMMY: active 0.9998123 protein cleaner0 2023-07-11T14:16:33Z PR: ERK2 0.746325 structure_element cleaner0 2023-07-11T14:07:23Z SO: D-motif 0.9995926 protein_type cleaner0 2023-07-11T14:03:02Z MESH: MAPKs 0.9996002 site cleaner0 2023-07-11T19:44:58Z SO: hydrophobic site 0.8181511 evidence cleaner0 2023-07-12T14:44:20Z DUMMY: changes in deuterium exchange profiles 0.8763486 structure_element cleaner0 2023-07-11T20:26:10Z SO: MAPK insertion 0.99932504 structure_element cleaner0 2023-07-11T20:26:13Z SO: helix 0.9997248 structure_element cleaner0 2023-07-11T20:26:16Z SO: αG 0.99982846 protein cleaner0 2023-07-11T14:04:00Z PR: JNK1 protein PR: cleaner0 2023-07-11T14:04:14Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.99978536 protein cleaner0 2023-07-11T14:16:33Z PR: ERK2 0.99960566 site cleaner0 2023-07-12T14:38:25Z SO: DEF-site 0.9997335 protein cleaner0 2023-07-11T14:16:34Z PR: ERK2 0.9998536 protein cleaner0 2023-07-11T14:16:27Z PR: MKP3 0.99983525 protein cleaner0 2023-07-11T14:16:34Z PR: ERK2 0.9998573 protein cleaner0 2023-07-11T14:16:27Z PR: MKP3 0.9996147 chemical cleaner0 2023-07-12T15:02:14Z CHEBI: pNPP 0.999213 protein_state cleaner0 2023-07-12T14:50:14Z DUMMY: presence of 0.9997465 protein cleaner0 2023-07-11T14:16:34Z PR: ERK2 0.9995199 experimental_method cleaner0 2023-07-12T14:58:02Z MESH: Sequence alignment 0.99966586 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.99782705 site cleaner0 2023-07-12T14:38:30Z SO: interacting region 0.9997303 complex_assembly cleaner0 2023-07-11T19:51:18Z GO: JNK1–MKP7-CD 0.9996654 structure_element cleaner0 2023-07-11T20:20:49Z SO: catalytic domain 0.9998479 protein cleaner0 2023-07-11T14:16:27Z PR: MKP3 0.9998105 protein cleaner0 2023-07-11T14:16:34Z PR: ERK2 protein PR: cleaner0 2023-07-11T14:04:14Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:11Z CD 0.9997949 protein cleaner0 2023-07-11T14:04:00Z PR: JNK1 0.9998253 protein cleaner0 2023-07-11T14:16:34Z PR: ERK2 0.9998517 protein cleaner0 2023-07-11T14:16:27Z PR: MKP3 0.9997066 complex_assembly cleaner0 2023-07-11T19:51:19Z GO: JNK1–MKP7-CD 0.9997077 complex_assembly cleaner0 2023-07-11T19:51:53Z GO: ERK2–MKP3-CD 0.9992817 complex_assembly cleaner0 2023-07-11T19:51:54Z GO: ERK2–MKP3-CD 0.99969053 structure_element cleaner0 2023-07-11T20:26:21Z SO: helix 0.9997199 structure_element cleaner0 2023-07-11T20:26:23Z SO: α4 protein PR: cleaner0 2023-07-11T14:16:27Z MKP3 0.947457 structure_element cleaner0 2023-07-11T14:16:11Z SO: CD 0.9998349 protein cleaner0 2023-07-11T14:16:34Z PR: ERK2 structure_element SO: cleaner0 2023-07-11T14:06:58Z FXF-motif protein_state DUMMY: cleaner0 2023-07-11T20:07:12Z p protein PR: cleaner0 2023-07-11T19:54:59Z ERK2 0.9995442 protein cleaner0 2023-07-11T14:16:34Z PR: ERK2 0.9998511 protein cleaner0 2023-07-11T14:16:27Z PR: MKP3 DISCUSS paragraph 30429 In summary, we have resolved the structure of JNK1 in complex with the catalytic domain of MKP7. This structure reveals an FXF-docking interaction mode between MAPK and MKP. Results from biochemical characterization of the Phe285 and Phe287 MKP7 mutants combined with structural information support the conclusion that the two Phe residues serve different roles in the catalytic reaction. Phe285 is essential for JNK1 substrate binding, whereas Phe287 plays a role for the precise alignment of active-site residues, which are important for transition-state stabilization. This newly identified FXF-type motif is present in all MKPs, except that the residue at the first position in MKP5 is an equivalent hydrophobic leucine residue (see also Fig. 7f,g), suggesting that these two Phe residues would play a similar role in facilitating substrate recognition and catalysis, respectively. An important feature of MKP–JNK1 interactions is that MKP7 or MKP5 only interact with the F-site of JNK1. One possible explanation is that JNK1 needs to use the D-site to interact with JIP-1, a scaffold protein for JNK signalling. The N-terminal JNK-binding domain of JIP-1 interacts with the D-site on JNK and this interaction is required for JIP-1-mediated enhancement of JNK activation. In addition, JIP-1 can also associate with MKP7 via the C-terminal region of MKP7 (ref.). When MKP7 is bound to JIP-1, it reduces JNK activation, leading to reduced phosphorylation of the JNK target c-Jun. Thus, our biochemical and structural data allow us to present a model for the JNK1–JIP-1–MKP7 ternary complex and provide an important insight into the assembly and function of JNK signalling modules (Supplementary Fig. 6). 0.99756986 evidence cleaner0 2023-07-12T14:44:22Z DUMMY: structure 0.9997992 protein cleaner0 2023-07-11T14:04:00Z PR: JNK1 0.99904174 protein_state cleaner0 2023-07-12T14:50:31Z DUMMY: in complex with 0.9996628 structure_element cleaner0 2023-07-11T20:20:49Z SO: catalytic domain 0.99985576 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.9973513 evidence cleaner0 2023-07-12T14:44:25Z DUMMY: structure 0.99884284 site cleaner0 2023-07-12T14:38:36Z SO: FXF-docking interaction mode 0.999608 protein_type cleaner0 2023-07-11T14:04:43Z MESH: MAPK 0.9996295 protein_type cleaner0 2023-07-11T20:17:04Z MESH: MKP 0.99131435 experimental_method cleaner0 2023-07-12T14:58:13Z MESH: biochemical characterization 0.9998313 residue_name_number cleaner0 2023-07-11T15:42:48Z DUMMY: Phe285 0.99976903 residue_name_number cleaner0 2023-07-11T15:47:33Z DUMMY: Phe287 0.99977034 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.9915985 protein_state cleaner0 2023-07-11T20:04:10Z DUMMY: mutants 0.99624157 evidence cleaner0 2023-07-12T14:44:33Z DUMMY: structural information 0.9991603 residue_name cleaner0 2023-07-11T19:56:01Z SO: Phe 0.99989176 residue_name_number cleaner0 2023-07-11T15:42:48Z DUMMY: Phe285 0.99978596 protein cleaner0 2023-07-11T14:04:00Z PR: JNK1 0.9998894 residue_name_number cleaner0 2023-07-11T15:47:33Z DUMMY: Phe287 0.99960524 site cleaner0 2023-07-12T14:38:41Z SO: active-site residues 0.99946177 structure_element cleaner0 2023-07-11T20:26:29Z SO: FXF-type motif 0.9996111 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.99986124 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.9991714 residue_name cleaner0 2023-07-11T19:55:59Z SO: leucine 0.9991885 residue_name cleaner0 2023-07-11T20:33:07Z SO: Phe 0.9982412 protein_type cleaner0 2023-07-11T20:33:47Z MESH: MKP 0.95433605 protein cleaner0 2023-07-11T14:04:00Z PR: JNK1 0.99985945 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.99986255 protein cleaner0 2023-07-11T14:16:42Z PR: MKP5 0.9994425 site cleaner0 2023-07-12T14:38:50Z SO: F-site 0.9997942 protein cleaner0 2023-07-11T14:04:00Z PR: JNK1 0.9997756 protein cleaner0 2023-07-11T14:04:00Z PR: JNK1 0.99938655 site cleaner0 2023-07-11T14:08:28Z SO: D-site 0.9997222 protein cleaner0 2023-07-11T19:55:47Z PR: JIP-1 0.9912742 protein_type cleaner0 2023-07-11T14:06:31Z MESH: JNK 0.99856734 structure_element cleaner0 2023-07-11T20:26:33Z SO: JNK-binding domain 0.99969196 protein cleaner0 2023-07-11T19:55:47Z PR: JIP-1 0.99909693 site cleaner0 2023-07-11T14:08:28Z SO: D-site 0.6355919 protein_type cleaner0 2023-07-11T14:06:31Z MESH: JNK 0.9996818 protein cleaner0 2023-07-11T19:55:47Z PR: JIP-1 0.91881114 protein_type cleaner0 2023-07-11T14:06:31Z MESH: JNK 0.99969906 protein cleaner0 2023-07-11T19:55:47Z PR: JIP-1 0.99985313 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.9994958 structure_element cleaner0 2023-07-11T20:26:36Z SO: C-terminal region 0.9998529 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.9998578 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.9994645 protein_state cleaner0 2023-07-12T14:50:35Z DUMMY: bound to 0.99965245 protein cleaner0 2023-07-11T19:55:47Z PR: JIP-1 0.52655923 protein_type cleaner0 2023-07-11T14:06:31Z MESH: JNK protein_type MESH: cleaner0 2023-07-11T14:06:31Z JNK 0.9402229 protein_type cleaner0 2023-07-11T20:18:37Z MESH: c-Jun 0.9990047 evidence cleaner0 2023-07-12T14:44:44Z DUMMY: biochemical and structural data 0.99968415 complex_assembly cleaner0 2023-07-11T19:55:38Z GO: JNK1–JIP-1–MKP7 0.9929068 protein_type cleaner0 2023-07-11T14:06:31Z MESH: JNK METHODS title_1 32141 Methods METHODS title_2 32149 Protein preparation METHODS paragraph 32169 The cDNAs of human MKP7 and MKP5 were kindly provided by Dr Mathijs Baens (University of Leuven) and Dr Eisuke Nishida (Kyoto University), respectively. The cDNAs of human ASK1, MKK4, MKK7 and JNK1 were kindly provided by Dr Zhenguo Wu (Hong Kong University of Science and Technology). The catalytic domains of MKP7 (MKP7-CD, residues 156–301) and MKP5 (MKP5-CD, 320–467) and the full-length MKP5 were cloned into the pET15b vector, resulting in the N-terminal His-fusion proteins. The KBD domains of MKP7 (MKP7-KBD, 5–138) and MKP5 (MKP5-KBD, 139–287), and the C-terminal truncation of MKP7 (MKP7ΔC304, 5–303) were cloned into pET21b vector for generation of C-terminal His-tagged proteins. The human full-length JNK1, MKK4, MKK7 and the kinase domain of ASK1 (659–951) were cloned into pGEX4T-2, pET15b and/or pET21b vectors to produce a GST- or His-tagged protein. Mutations of MKP7-CD, MKP5-CD and JNK1 were generated by overlap PCR procedure. All constructs were verified by DNA sequencing. All proteins, overexpressed in BL21(DE3) cells at 20 °C, were first purified over Ni-NTA (Qiagen) or GS4B (GE Healthcare) columns, and then by ion exchange and gel filtration chromatography (Source-15Q/15S and Superdex-200, GE Healthcare) at 4 °C. The double-phosphorylated JNK1 (phospho-JNK1) was generated by mixing JNK1 with upstream kinases MKK4, MKK7 and ASK1 in buffer containing 10 mM MgCl2 and 2 mM ATP, and further purified by gel filtration chromatography (Superdex-200, GE Healthcare) at 4 °C (ref.). Proteins were stored at −80 °C, and stocks for phosphatase assays were supplemented with glycerol to a final concentration of 20% (v/v). Protein concentrations were determined spectrophotometrically using theoretical molar extinction coefficients at 280 nm (ref.). species MESH: cleaner0 2023-07-11T20:32:27Z human species MESH: cleaner0 2023-07-11T20:32:27Z human species MESH: cleaner0 2023-07-11T20:32:27Z human METHODS title_2 33975 Crystallography METHODS paragraph 33991 The mixture of unphosphorylated JNK1 and MKP7-CD at 1:1 molar ratio was subjected to crystallization trials. Crystals were grown by the vapor-diffusion technique in hanging drops, and the drops were prepared by mixing equal volumes of protein with reservoir solution containing 0.1 M HEPES, pH 7.0, 14% PEG3350, 0.2 M MgCl2, 6% 1,6-Hexanediol and 0.005 M EDTA at 21 °C. Crystals were cryo-protected in reservoir solutions supplemented with 10% glycerol and then flash frozen in liquid nitrogen. The diffraction data sets were collected at beamline 17U at Shanghai Synchrotron Radiation Facility and processed with the HKL2000 package. The crystals belong to space group P1 and comprise eight molecules per asymmetric unit (four complexes). Structure was solved by molecular replacement using Phaser with JNK1 (PDB 1UKH) and MKP5-CD (PDB 1ZZW) as the search models. Standard refinement was performed with programs PHENIX and Coot. The crystal structure of unphosphorylated JNK1 in complex with the catalytic domain of MKP7 was refined to 2.4 Å resolution. Initial structural refinement was performed with NCS restraints, and after several rounds the restraints were removed from the calculations. The final Rwork and Rfree were 21.7 and 23.9%, respectively. The crystallographic asymmetric unit contains four JNK1–MKP7-CD complexes. The four complexes are nearly identical with an r.m.s.d.<1 Å for any complex pair in the asymmetric unit. Ramachandran analysis was carried out using PROCHECK. Additional density at the active site of MKP7-CD was attributed to a chloride ion incorporated as a crystallizing agent, similar to those observed in the structures of MKP3-CD and MKP5-CD (refs). The data collection and refinement statistics are summarized in Table 1. All structural representations in this paper were prepared with PYMOL (http://www.pymol.org). structure_element SO: cleaner0 2023-07-11T20:20:49Z catalytic domain METHODS title_2 35863 Phosphatase assays METHODS paragraph 35882 The activities of MKP7 and MKP5 was assayed using phospho-JNK1 as substrate in the coupled enzyme system containing 50 mM MOPS, pH 7.0, 100 mM NaCl, 0.1 mM EDTA, 50 μM MESG and 0.1 mg ml−1 PNPase. This coupled assay uses PNPase and its chromogenic substrate MESG to monitor the production of inorganic phosphate. The reactions were initiated by addition of 0.4 μM MKP7-CD and MKP7ΔC304 (or 0.1 μM MKP5 full-length and 0.15 μM MKP5-CD) for substrate phospho-JNK1. All experiments were carried out at 25 °C in 1.8 ml reaction mixtures, and the continuous absorbance changes were recorded with a PerkinElmer LAMBDA 45 spectrophotometer equipped with a magnetic stirrer in the cuvette holder. The quantification of inorganic phosphate produced was monitored at 360 nm with the extinction coefficient of 11,200 M−1 cm−1 (ref.). The initial rates were determined from the linear slope of the progress curves obtained. The activity of MKP7-CD or MKP5-CD mutants were assayed using pNPP or phospho-JNK1 as substrate. The phospho-JNK1 assay was performed as the same procedure mentioned above, and in the presence of wild type (as a control) or indicated mutants, and equal concentrations of phospho-JNK1. The pNPP assay was performed in the reaction mixture containg 50 mM MOPS, pH 7.0, 100 mM NaCl, 0.1 mM EDTA and 20 mM pNPP. The amount of the product p-nitrophenol was determined from the absorbance at 405 nm using a molar extinction coefficient of 18,000 M−1 cm−1 (ref.). METHODS title_2 37409 Assays for protein–protein interaction METHODS paragraph 37450 The interactions of JNK1 with the CD and KBD domains of MKP7 and MKP5 were examined by gel filtration analyses using a Superdex-200 10/300 column on an ÄKTA FPLC (GE Healthcare). The column was equilibrated with a buffer containing 10 mM HEPES, pH 7.5, 150 mM NaCl and 2 mM dithiothreitol, and calibrated with molecular mass standards. Samples of individual proteins and indicated mixtures (500 μl each) were loaded to the Superdex-200 column and then eluted at a flow rate of 0.5 ml min−1. Fractions of 0.5 ml each were collected, and aliquots of relevant fractions were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed Coomassie Blue staining. METHODS paragraph 38133 The interactions between various JNK1 mutants and MKP7-CD or MKP5-CD were assessed by GST-mediated pull-down assays at 4 °C. First, 0.5 ml GST-JNK1 proteins (6 μM) were loaded to 0.2 ml GS4B resin. The excess unbound JNK1 or other contaminants were removed by washing the column 5 times, each with 1.0 ml buffer containing 25 mM Tris-HCl, pH 8.0, 150 mM NaCl and 2 mM dithiothreitol. Then, 0.5 ml MKP7-CD or MKP5-CD (20 μM) was allowed to flow through the JNK1-bound column. After extensive washing, the bound proteins were eluted with 0.5 ml reduced glutathione (10 mM). The interactions of JNK1 with the CD and KBD domains of MKP7 and MKP5 were also examined by GST-mediated pull-down assays. The GST protein alone was used as a control. Aliquots of all eluates were subjected to SDS-PAGE, and proteins were visualized by Coomassie Blue staining. The uncropped gels are shown in Supplementary Fig. 7. METHODS title_2 39063 Building JNK and MKP5 interaction model METHODS paragraph 39103 The model of catalytic domain of MKP5 bound to JNK was constructed by superimposition of previous deposited structures of MKP5-CD (PDB 1ZZW) to the corresponding domains in the crystal structure of JNK1–MKP7-CD. The fractured loops in deposited structures were computationally generated using Modeller. The program CHARMM22 (ref.) was then used to add hydrogen atoms, N- and C-terminal patches to the model. The model was then subjected to restrained energy minimization to optimize bonds and remove any nonbonded steric clashes. Refinement of the modelled complex was performed using NAMD2.9 package at 1 atm pressure and 300 K. The generated complex structure was solvated and neutralized in a box with TIP3P water at a minimum of 13 Å between the model and the wall of the box. The simulation was first set up with 1 fs time step under periodic boundary conditions. The particle mesh Ewald method was applied to model the electrostatics and the van der Waals interactions cutoff was set at 12 Å. The system was restrained for 5 ps minimization and 5 ps simulation, and followed by removing all the restraints and performing a minimization of 10 ps and an equilibration of 10 ns. Simulations were viewed using VMD. METHODS title_2 40342 Circular dichroism spectra METHODS paragraph 40369 The experiments were performed on a Chirascan-plus circular dichroism Spectrometer (Applied Photophysics, Surrey, UK) using 0.1 mm quartz cuvette. The protein sample were analysed at a concentration of 0.5 mg ml−1. Data were collected over a wavelength range from 260 to 190 nm with 1 nm intervals at room temperature, three scans were averaged, and the baseline spetrum of solution buffer containing 10 mM HEPES (pH 7.5) and 150 mM NaCl was subtracted. METHODS title_2 40839 Cell culture and transfections METHODS paragraph 40870 pcDNA3.3-Myc-MKP7, pCMV5-3HA-JNK1 and pBOBI-HA-MKP7 were generated with standard molecular techniques. Mutants with amino acid substitution and truncation constructs were generated through PCR-based site-directed mutagenesis method using Pfu polymerase (Stratagene). The authenticities of all constructs were confirmed by sequencing (Invitrogen, China). HEK293T and HeLa cells (ATCC) were maintained in DMEM supplemented with 10% fetal bovine serum, 100 IU penicillin, 100 mg ml−1 streptomycin at 37 °C in a humidified incubator containing 5% CO2. Polyethylenimine (Polysciences, #23966) at a final concentration of 10 μM was used to transfect HEK293T cells. Total DNA for each plate was adjusted to the same amount by adding relevant blank vector. Lentiviruses for infection were packaged in HEK293T cells after transfection using Lipofectamine 2000 (Invitrogen, 11668-027). At 30 h post transfection, medium was collected for further infection. METHODS title_2 41833 Coimmunoprecipitation and immunoblotting METHODS paragraph 41874 Cells were lysed in a lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 2 mM Na3VO4, 25 mM NaF, 1 mM phenylmethanesulfonyl fluoride, 1 μg ml−1 leupeptin and 1 μg ml−1 aprotinin. Cell lysates were incubated with respective antibodies overnight at 4 °C. Protein aggregates resulting from the overnight incubation were removed by centrifugation, and protein A/G beads (Santa-Cruz Biotechnology, Dallas, TX, USA) were then added into the lysates and incubated for another 3 h. After spinning and washing for three times with the lysis buffer, the beads were mixed with 2 × SDS sample buffer, boiled and subjected to 15% SDS/PAGE. The samples were transferred to PVDF membranes (Millipore), and immunoblotted with indicated antibodies. Levels of total proteins and the levels of phosphorylation of proteins were analysed on separate gels. The uncropped blots are shown in Supplementary Fig. 7. METHODS title_2 42830 Antibodies and drugs METHODS paragraph 42851 Antibodies used in this study: mouse anti-HA (1:100 for immunoprecipitation; F-7) and anti-JNK1 (1:1,000 for immunoblotting; F-3) antibodies were purchased from Santa-Cruz Biotechnology. Anti-c-Myc Agarose Affinity Gel antibody produced in rabbit (1:200 for immunoprecipitation; A7470) was purchased from Sigma. Rabbit anti-HA-tag (1:1,000 for immunoblotting; #3724), anti-phospho-JNK-T183/Y185 (1:1,000 for immunoblotting; #4668) and mouse anti-Myc-tag (1:1,000 for immunoblotting; #2276) antibodies were purchased from Cell Signaling Technology. Etoposide (E1383) was purchased from Sigma. METHODS title_2 43443 Apoptosis assay METHODS paragraph 43459 HeLa cells were infected with lentiviruses expressing MKP7 or its mutants. At 36 h post infection, cells were irradiated with 25 J m−2 ultraviolet light and collected at 6 h after irradiation. Cells were then stained with the Annexin-V-APC/PI double-staining solution (BD Biosciences) and analysed with a flow cytometer (BD LSRFortessa). The percentages of apoptotic cells were quantified with FlowJo 7.6.1 software. METHODS title_1 43886 Additional information METHODS paragraph 43909 Accession codes: The coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 4YR8 for the JNK1–MKP7-CD structure. METHODS paragraph 44069 How to cite this article: Liu, X. et al. A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation. Nat. Commun. 7:10879 doi: 10.1038/ncomms10879 (2016). SUPPL title_1 44276 Supplementary Material 1150 1160 surname:Avruch;given-names:J. 17229475 REF Biochim. Biophys. 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Z.-X.W., J.-W.W. and S.-C.L. conceived and designed the experiments, and wrote the manuscript. ncomms10879-f1.jpg f1 FIG fig_title_caption 50739 Domain structures of ten human MKPs and the atypical VHR. 0.9988306 evidence cleaner0 2023-07-12T14:44:49Z DUMMY: structures 0.999137 species cleaner0 2023-07-11T20:32:28Z MESH: human 0.9996724 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.9510513 protein cleaner0 2023-07-11T14:22:09Z PR: VHR ncomms10879-f1.jpg f1 FIG fig_caption 50797 On the basis of sequence similarity, protein structure, substrate specificity and subcellular localization, the ten members of MKP family can be divided into three groups. The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs. The second subfamily contains MKP3, MKP4 and MKPX, which are cytoplasmic ERK-specific MKPs. The third subfamily comprises MKP5, MKP7 and hVH5, which were located in both nucleus and cytoplasm, and selectively inactivate JNK and p38. All MKPs contain both the CD and KBD domains, whereas VHR, an atypical MKP, only contains a highly conserved catalytic domain. In addition to the CD and KBD, MKP7 contains a unique long C-terminal region that contains NES, NLS and PEST motifs, which has no effect on the binding ability and phosphatase activity of MKP7 toward MAPKs. NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines. 0.6954108 evidence cleaner0 2023-07-12T14:44:52Z DUMMY: structure 0.9993874 protein_type cleaner0 2023-07-11T20:14:08Z MESH: MKP family 0.99983966 protein cleaner0 2023-07-11T19:57:04Z PR: MKP1 0.9998387 protein cleaner0 2023-07-11T19:57:06Z PR: MKP2 0.99979764 protein cleaner0 2023-07-11T19:57:09Z PR: PAC1 0.9997702 protein cleaner0 2023-07-11T19:57:11Z PR: hVH3 0.99476105 protein_state cleaner0 2023-07-12T14:50:43Z DUMMY: inducible protein_type MESH: cleaner0 2023-07-12T14:50:59Z nuclear phosphatases 0.9887932 protein_type cleaner0 2023-07-11T14:06:43Z MESH: ERK 0.69141513 protein_type cleaner0 2023-07-11T14:06:31Z MESH: JNK 0.91759735 protein_type cleaner0 2023-07-11T14:06:37Z MESH: p38 0.99923766 protein_type cleaner0 2023-07-11T14:03:02Z MESH: MAPKs 0.99983656 protein cleaner0 2023-07-11T14:16:27Z PR: MKP3 0.9998363 protein cleaner0 2023-07-11T19:57:16Z PR: MKP4 0.9997894 protein cleaner0 2023-07-11T19:57:18Z PR: MKPX 0.9996675 protein_type cleaner0 2023-07-11T18:52:27Z MESH: ERK-specific MKPs 0.99985766 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 0.9998541 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.99983954 protein cleaner0 2023-07-11T19:57:27Z PR: hVH5 0.81356543 protein_type cleaner0 2023-07-11T14:06:31Z MESH: JNK 0.81705546 protein_type cleaner0 2023-07-11T14:06:37Z MESH: p38 0.99963415 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs 0.9998186 structure_element cleaner0 2023-07-11T14:16:12Z SO: CD 0.9998085 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.99979275 protein cleaner0 2023-07-11T14:22:09Z PR: VHR 0.9995327 protein_type cleaner0 2023-07-11T20:17:18Z MESH: MKP 0.9995488 protein_state cleaner0 2023-07-12T14:51:29Z DUMMY: highly conserved 0.9996837 structure_element cleaner0 2023-07-11T20:20:49Z SO: catalytic domain 0.99982965 structure_element cleaner0 2023-07-11T14:16:12Z SO: CD 0.99981815 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9998565 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.9995472 structure_element cleaner0 2023-07-11T20:26:41Z SO: C-terminal region 0.9997634 structure_element cleaner0 2023-07-11T19:57:33Z SO: NES 0.99974984 structure_element cleaner0 2023-07-11T19:57:36Z SO: NLS 0.9996542 structure_element cleaner0 2023-07-11T20:26:46Z SO: PEST motifs 0.9998586 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.9992887 protein_type cleaner0 2023-07-11T14:03:02Z MESH: MAPKs 0.9963917 structure_element cleaner0 2023-07-11T19:57:40Z SO: NES structure_element SO: cleaner0 2023-07-11T19:57:55Z nuclear export signal 0.9986312 structure_element cleaner0 2023-07-11T19:57:38Z SO: NLS 0.90655786 structure_element cleaner0 2023-07-11T19:57:57Z SO: nuclear localization signal 0.99957305 structure_element cleaner0 2023-07-11T19:57:59Z SO: PEST 0.94908106 structure_element cleaner0 2023-07-11T19:58:01Z SO: C-terminal sequence rich 0.9986249 residue_name cleaner0 2023-07-11T18:52:32Z SO: prolines 0.99889416 residue_name cleaner0 2023-07-11T18:52:34Z SO: glutamates 0.9987664 residue_name cleaner0 2023-07-11T18:52:37Z SO: serines 0.99859756 residue_name cleaner0 2023-07-11T18:52:39Z SO: threonines ncomms10879-f2.jpg f2 FIG fig_title_caption 51826 MKP7-CD is crucial for JNK1 binding and enzyme catalysis. 0.9993863 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.9998307 protein cleaner0 2023-07-11T14:04:01Z PR: JNK1 ncomms10879-f2.jpg f2 FIG fig_caption 51884 (a) Domain organization of human MKP7 and JNK1. The KBD and CD of MKP7 are shown in green and blue, and the N-lobe and C-lobe of JNK1 are coloured in lemon and yellow, respectively. The key structural elements are indicated. The colour scheme is the same in the following figures unless indicated otherwise. (b) Plots of initial velocity of the MKP7-catalysed reaction versus phospho-JNK1 concentration. The solid lines are best-fitting results according to equation (1). Each experiment was performed in replicate for at least three times. The error bars represent s.e.m. (c) Gel filtration analysis for interaction of JNK1 with MKP7-CD and MKP7-KBD. (d) GST-mediated pull-down assay for interaction of JNK1 with MKP7-CD and MKP7-KBD. The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. The protein amounts of MKP7 used are shown at the bottom. 0.9990736 species cleaner0 2023-07-11T20:32:28Z MESH: human 0.9998344 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.99976367 protein cleaner0 2023-07-11T14:04:01Z PR: JNK1 0.9998006 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.9997886 structure_element cleaner0 2023-07-11T14:16:12Z SO: CD 0.99983644 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.99769574 structure_element cleaner0 2023-07-11T20:26:51Z SO: N-lobe 0.9928906 structure_element cleaner0 2023-07-11T20:26:54Z SO: C-lobe 0.99968266 protein cleaner0 2023-07-11T14:04:01Z PR: JNK1 evidence DUMMY: cleaner0 2023-07-12T14:45:13Z Plots of initial velocity 0.9997818 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.7825839 ptm cleaner0 2023-07-12T15:02:31Z MESH: phospho 0.9747847 protein cleaner0 2023-07-11T14:04:01Z PR: JNK1 0.99949336 experimental_method cleaner0 2023-07-12T14:58:18Z MESH: Gel filtration analysis 0.9996594 protein cleaner0 2023-07-11T14:04:01Z PR: JNK1 0.99578506 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD protein PR: cleaner0 2023-07-11T14:04:15Z MKP7 structure_element SO: cleaner0 2023-07-11T14:14:22Z KBD 0.9995559 experimental_method cleaner0 2023-07-12T14:58:21Z MESH: GST-mediated pull-down assay 0.9995567 protein cleaner0 2023-07-11T14:04:01Z PR: JNK1 0.9985934 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD protein PR: cleaner0 2023-07-11T14:04:15Z MKP7 structure_element SO: cleaner0 2023-07-11T14:14:22Z KBD 0.9938221 evidence cleaner0 2023-07-12T14:45:20Z DUMMY: affinities 0.984475 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD protein PR: cleaner0 2023-07-11T14:04:15Z MKP7 structure_element SO: cleaner0 2023-07-11T14:14:22Z KBD 0.9995722 protein cleaner0 2023-07-11T14:04:01Z PR: JNK1 0.999506 evidence cleaner0 2023-07-12T14:45:23Z DUMMY: affinity 0.9986137 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.99982244 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.9996997 protein cleaner0 2023-07-11T14:04:01Z PR: JNK1 0.9995669 experimental_method cleaner0 2023-07-12T14:58:25Z MESH: GST pull-down assays 0.9998379 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 ncomms10879-f3.jpg f3 FIG fig_title_caption 52894 Structure of JNK1 in complex with MKP7-CD. 0.99948704 evidence cleaner0 2023-07-12T14:45:27Z DUMMY: Structure 0.9998179 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.9992642 protein_state cleaner0 2023-07-12T14:51:34Z DUMMY: in complex with protein PR: cleaner0 2023-07-11T14:04:15Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD ncomms10879-f3.jpg f3 FIG fig_caption 52937 (a) Ribbon diagram of JNK1–MKP7-CD complex in two views related by a 45° rotation around a vertical axis. (b) Structure of MKP7-CD with its active site highlight in cyan. The 2Fo−Fc omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). The JNK1 is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red). (e) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. MKP7-CD is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red). Blue dashed lines represent polar interactions. (f) The 2Fo−Fc omit map (contoured at 1.5σ) clearly shows electron density for the 285FNFL288 segment of MKP7-CD. 0.99973774 complex_assembly cleaner0 2023-07-11T19:51:19Z GO: JNK1–MKP7-CD 0.95589375 evidence cleaner0 2023-07-12T14:45:30Z DUMMY: Structure 0.9310647 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.9996388 site cleaner0 2023-07-12T14:38:56Z SO: active site 0.9995763 evidence cleaner0 2023-07-12T14:45:33Z DUMMY: 2Fo−Fc omit map 0.99972206 structure_element cleaner0 2023-07-11T15:41:48Z SO: P-loop protein PR: cleaner0 2023-07-11T14:04:15Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.9885592 evidence cleaner0 2023-07-12T14:45:36Z DUMMY: Structure 0.99786264 protein cleaner0 2023-07-11T14:22:09Z PR: VHR 0.99964476 site cleaner0 2023-07-12T14:38:59Z SO: active site 0.9995858 site cleaner0 2023-07-12T14:39:02Z SO: JNK1–MKP7 interface 0.9985259 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 protein PR: cleaner0 2023-07-11T14:04:15Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.9991696 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.9993895 site cleaner0 2023-07-12T14:39:07Z SO: Interaction networks 0.9997509 structure_element cleaner0 2023-07-11T20:26:59Z SO: helices 0.9997986 structure_element cleaner0 2023-07-11T20:27:02Z SO: α4 0.9997985 structure_element cleaner0 2023-07-11T20:27:04Z SO: α5 protein PR: cleaner0 2023-07-11T14:04:15Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.9997863 structure_element cleaner0 2023-07-11T20:27:07Z SO: αG 0.9997346 structure_element cleaner0 2023-07-11T20:27:09Z SO: α2L14 0.99909556 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 protein PR: cleaner0 2023-07-11T14:04:15Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z polar interactions 0.9995697 evidence cleaner0 2023-07-12T14:45:39Z DUMMY: 2Fo−Fc omit map 0.99958444 evidence cleaner0 2023-07-12T14:45:42Z DUMMY: electron density structure_element SO: cleaner0 2023-07-11T19:45:11Z 285FNFL288 segment protein PR: cleaner0 2023-07-11T14:04:15Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD ncomms10879-f4.jpg f4 FIG fig_title_caption 53913 Mutational analysis on interactions between MKP7-CD and JNK1. 0.99958706 experimental_method cleaner0 2023-07-12T14:58:42Z MESH: Mutational analysis 0.9992169 protein cleaner0 2023-07-11T14:04:15Z PR: MKP7 0.58408344 structure_element cleaner0 2023-07-11T14:16:12Z SO: CD 0.99970716 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 ncomms10879-f4.jpg f4 FIG fig_caption 53975 (a) Effects of mutations in MKP7-CD on the JNK1 dephosphorylation (mean±s.e.m., n=3). Residues involved in hydrophobic and hydrophilic contacts are coloured in red and blue, respectively. (b) Gel filtration analysis for interaction of JNK1 with MKP7-CD mutant F285D. Mutant F285D and JNK1 were eluted as monomers, with the molecular masses of ∼17 and 44 kDa, respectively. However, in contrast to the wild-type MKP7-CD, mutant F285D did not co-migrate with JNK1. (c) Pull-down assays of MKP7-CD by GST-tagged JNK1 mutants. The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. The protein amounts of MKP7-CD used are shown at the bottom. (d) Circular dichroism spectra for MKP7-CD wild type and mutants. Measurements were averaged for three scans. (e) Circular dichroism spectra for JNK1 wild type and mutants. Measurements were averaged for three scans. (f) Effects of mutations in MKP7-CD on the pNPP hydrolysis reaction (mean±s.e.m., n=3). protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.99977106 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T14:01:33Z dephosphorylation bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z hydrophobic and hydrophilic contacts 0.9995289 experimental_method cleaner0 2023-07-12T14:58:45Z MESH: Gel filtration analysis 0.9996489 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.9994962 protein_state cleaner0 2023-07-11T15:48:15Z DUMMY: mutant 0.99963117 mutant cleaner0 2023-07-11T15:53:12Z MESH: F285D 0.9994444 protein_state cleaner0 2023-07-11T15:48:15Z DUMMY: Mutant 0.99965394 mutant cleaner0 2023-07-11T15:53:12Z MESH: F285D 0.9996574 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.9994036 oligomeric_state cleaner0 2023-07-12T15:01:07Z DUMMY: monomers 0.9995597 protein_state cleaner0 2023-07-11T15:48:09Z DUMMY: wild-type protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.99948275 protein_state cleaner0 2023-07-11T15:48:15Z DUMMY: mutant 0.99964154 mutant cleaner0 2023-07-11T15:53:12Z MESH: F285D 0.9996581 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.99957776 experimental_method cleaner0 2023-07-12T14:58:48Z MESH: Pull-down assays protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.9913747 protein_state cleaner0 2023-07-12T14:51:39Z DUMMY: GST-tagged 0.9995147 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.96717983 evidence cleaner0 2023-07-12T14:45:47Z DUMMY: affinities protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.9995888 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.9993345 evidence cleaner0 2023-07-12T14:45:51Z DUMMY: affinity 0.9995756 protein_state cleaner0 2023-07-11T15:48:09Z DUMMY: wild-type 0.99977297 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.99935895 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.99956286 experimental_method cleaner0 2023-07-12T14:58:51Z MESH: GST pull-down assays protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD experimental_method MESH: cleaner0 2023-07-12T14:46:18Z Circular dichroism evidence DUMMY: cleaner0 2023-07-12T14:46:27Z spectra protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.9995651 protein_state cleaner0 2023-07-11T15:54:35Z DUMMY: wild type protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants experimental_method MESH: cleaner0 2023-07-12T14:46:19Z Circular dichroism evidence DUMMY: cleaner0 2023-07-12T14:46:44Z spectra 0.99968576 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.99957126 protein_state cleaner0 2023-07-11T15:54:35Z DUMMY: wild type protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.59046876 experimental_method cleaner0 2023-07-12T14:58:55Z MESH: mutations protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.99979097 chemical cleaner0 2023-07-12T15:02:37Z CHEBI: pNPP ncomms10879-f5.jpg f5 FIG fig_title_caption 55099 Comparison of CDK2-KAP and JNK1–MKP7-CD. 0.8294056 complex_assembly cleaner0 2023-07-11T20:33:53Z GO: CDK2-KAP 0.9901239 complex_assembly cleaner0 2023-07-11T19:51:19Z GO: JNK1–MKP7-CD ncomms10879-f5.jpg f5 FIG fig_caption 55142 (a) Superposition of the complex structures of CDK2-KAP (PDB 1FQ1) and JNK1–MKP7-CD. The N-lobe and C-lobe of CDK2 are coloured in grey and pink, respectively, and KAP is coloured in green. The interactions between these two proteins consist of three discontinuous contact regions, centred at the multiple hydrogen bonds between the pThr160 of CDK2 and the active site of KAP (region I). Interestingly, the recognition of CDK2 by KAP is augmented by a similar interface as that observed in the complex of JNK1 and MKP7-CD (region II). (b) Interactions networks at the auxiliary region II mainly involving helix α7 from KAP and the αG helix and following L14 loop of CDK2. The orientation of the panel is almost identical to those of Fig. 3d. The CDK2 is shown in surface representation coloured according to the electrostatic potential (positive, blue; negative, red). Residues of KAP and CDK2 are highlighted as green and red sticks, respectively. Blue dashed lines represent polar interactions. One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. Residues of MKP7-CD involved in JNK1 recognition are indicated by cyan asterisks, and the conserved FXF-motif is highlighted in cyan. The secondary structure assignments of MKP7-CD and KAP are shown above and below each sequence. (d) Sequence alignment of the F-site regions on MAPKs. Residues of JNK1 involved in recognition of MKP7 are indicated by orange asterisks, and those forming the F-site are highlighted in yellow. 0.99969125 experimental_method cleaner0 2023-07-12T14:58:59Z MESH: Superposition 0.99635834 evidence cleaner0 2023-07-12T14:46:51Z DUMMY: structures 0.9996756 complex_assembly cleaner0 2023-07-11T20:33:59Z GO: CDK2-KAP 0.9996969 complex_assembly cleaner0 2023-07-11T19:51:19Z GO: JNK1–MKP7-CD 0.9981871 structure_element cleaner0 2023-07-11T20:27:15Z SO: N-lobe 0.996067 structure_element cleaner0 2023-07-11T20:27:18Z SO: C-lobe 0.9996972 protein cleaner0 2023-07-11T15:51:12Z PR: CDK2 0.9975011 protein cleaner0 2023-07-11T15:50:50Z PR: KAP 0.8763778 site cleaner0 2023-07-12T14:39:12Z SO: contact regions bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z hydrogen bonds 0.99980813 ptm cleaner0 2023-07-11T20:12:29Z MESH: pThr160 0.99976426 protein cleaner0 2023-07-11T15:51:12Z PR: CDK2 0.9995154 site cleaner0 2023-07-12T14:39:16Z SO: active site 0.9991155 protein cleaner0 2023-07-11T15:50:50Z PR: KAP 0.75867003 structure_element cleaner0 2023-07-11T20:27:21Z SO: region I 0.99981576 protein cleaner0 2023-07-11T15:51:12Z PR: CDK2 0.9995796 protein cleaner0 2023-07-11T15:50:50Z PR: KAP 0.9992055 site cleaner0 2023-07-12T14:39:23Z SO: interface 0.99970776 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD structure_element SO: cleaner0 2023-07-11T19:58:56Z region II 0.99902946 structure_element cleaner0 2023-07-11T20:27:25Z SO: auxiliary region II 0.99974304 structure_element cleaner0 2023-07-11T20:27:28Z SO: helix 0.9998036 structure_element cleaner0 2023-07-11T20:27:30Z SO: α7 0.9989945 protein cleaner0 2023-07-11T15:50:50Z PR: KAP 0.9997145 structure_element cleaner0 2023-07-11T20:27:33Z SO: αG helix 0.9996296 structure_element cleaner0 2023-07-11T20:27:36Z SO: L14 loop 0.99973935 protein cleaner0 2023-07-11T15:51:12Z PR: CDK2 0.9997421 protein cleaner0 2023-07-11T15:51:12Z PR: CDK2 0.9988533 protein cleaner0 2023-07-11T15:50:50Z PR: KAP 0.9997558 protein cleaner0 2023-07-11T15:51:12Z PR: CDK2 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:06Z polar interactions 0.99966705 structure_element cleaner0 2023-07-11T20:27:40Z SO: helix 0.9997894 structure_element cleaner0 2023-07-11T20:27:43Z SO: α6 0.9979475 protein cleaner0 2023-07-11T15:50:50Z PR: KAP 0.99966335 structure_element cleaner0 2023-07-11T20:27:46Z SO: helix 0.99980253 structure_element cleaner0 2023-07-11T20:27:48Z SO: α4 protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.9987024 protein_state cleaner0 2023-07-12T14:51:45Z DUMMY: stable 0.99910814 oligomeric_state cleaner0 2023-07-12T15:01:11Z DUMMY: heterodimer 0.9997851 protein cleaner0 2023-07-11T15:51:12Z PR: CDK2 0.999603 protein cleaner0 2023-07-11T15:50:50Z PR: KAP 0.9995645 experimental_method cleaner0 2023-07-12T14:59:03Z MESH: Sequence alignment 0.9743006 site cleaner0 2023-07-12T14:39:27Z SO: JNK-interacting regions 0.9995679 protein_type cleaner0 2023-07-11T14:03:20Z MESH: MKPs protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.9996482 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.9995695 protein_state cleaner0 2023-07-12T14:51:48Z DUMMY: conserved 0.9996901 structure_element cleaner0 2023-07-11T14:06:58Z SO: FXF-motif protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.99891937 protein cleaner0 2023-07-11T15:50:50Z PR: KAP 0.99956197 experimental_method cleaner0 2023-07-12T14:59:06Z MESH: Sequence alignment structure_element SO: cleaner0 2023-07-11T20:28:31Z F-site regions 0.9995608 protein_type cleaner0 2023-07-11T14:03:02Z MESH: MAPKs 0.9998103 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.99984515 protein cleaner0 2023-07-11T14:04:16Z PR: MKP7 0.99884516 site cleaner0 2023-07-12T14:39:31Z SO: F-site ncomms10879-f6.jpg f6 FIG fig_title_caption 56864 FXF-motif is critical for controlling the phosphorylation of JNK and ultraviolet-induced apoptosis. 0.99970436 structure_element cleaner0 2023-07-11T14:06:58Z SO: FXF-motif 0.9987393 ptm cleaner0 2023-07-12T15:04:07Z MESH: phosphorylation 0.8759518 protein_type cleaner0 2023-07-11T14:06:31Z MESH: JNK ncomms10879-f6.jpg f6 FIG fig_caption 56964 (a–c) FXF-motif is essential for the dephosphorylation of JNK by MKP7. HEK293T cells were infected with lentiviruses expressing MKP7 and its mutants (1.0 μg). After 36 h infection, cells were untreated in a, stimulated with 30 μM etoposide for 3 h in b or irradiated with 25 J m−2 ultraviolet light at 30 min before lysis in c. Whole-cell extracts were then immunoblotted with antibody indicated. Shown is a typical immunoblot for phosphorylated JNK from three independent experiments. (d) F-site is required for JNK1 to interact with MKP7. HEK293T cells were co-transfected with MKP7 full-length (1.0 μg) and JNK1 (wild type or mutants as indicated, 1.0 μg). At 16 h post transfection, cells were lysed. Whole-cell extracts were then immunoprecipitated with antibody against Myc for MKP7; immunobloting was carried out with antibodies indicated. IP, immunoprecipitation; TCL, total cell lysate. Shown is a typical result from three independent experiments. (e) Effect of MKP7 (wild type or mutants) expression on ultraviolet-induced apoptosis. HeLa cells were infected with lentiviruses expressing MKP7 full-length and its mutants. At 36 h post infection, cells were irradiated with 25 J m−2 ultraviolet light and collected at 6 h after irradiation. Cells were then subjected to flow cytometry analysis. Apoptotic cells were determined by Annexin-V-APC/PI staining. The results using Annexin-V stain for membrane phosphatidylserine eversion, combined with propidium iodide (PI) uptake to evaluate cells whose membranes had been compromised. Staining with both Annexin-V and PI indicate apoptosis (upper right quadrant). The values shown in the lower left, and upper right quadrants of each panel represent the percentage of viable, and apoptotic cells, respectively. All results are representative of three independent experiments. (f) Statistical analysis of apoptotic cells (mean±s.e.m., n=3), *P<0.05, ***P<0.001 (ANOVA followed by Tukey's test). NS, not significant. 0.99860597 structure_element cleaner0 2023-07-11T14:06:58Z SO: FXF-motif 0.9997149 protein_type cleaner0 2023-07-11T14:06:31Z MESH: JNK 0.9998548 protein cleaner0 2023-07-11T14:04:16Z PR: MKP7 0.99673367 taxonomy_domain cleaner0 2023-07-12T14:59:33Z DUMMY: lentiviruses 0.99986315 protein cleaner0 2023-07-11T14:04:16Z PR: MKP7 protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.75931406 chemical cleaner0 2023-07-11T15:52:00Z CHEBI: etoposide 0.99608105 protein_state cleaner0 2023-07-11T20:03:12Z DUMMY: phosphorylated 0.9987192 protein_type cleaner0 2023-07-11T14:06:31Z MESH: JNK 0.8712673 site cleaner0 2023-07-12T14:39:37Z SO: F-site 0.9998406 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.99986076 protein cleaner0 2023-07-11T14:04:16Z PR: MKP7 0.9993498 experimental_method cleaner0 2023-07-12T14:59:46Z MESH: co-transfected 0.9998604 protein cleaner0 2023-07-11T14:04:16Z PR: MKP7 0.99951607 protein_state cleaner0 2023-07-11T15:54:25Z DUMMY: full-length 0.9998323 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.9995818 protein_state cleaner0 2023-07-11T15:54:35Z DUMMY: wild type protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.9974833 experimental_method cleaner0 2023-07-12T14:59:49Z MESH: immunoprecipitated 0.9998566 protein cleaner0 2023-07-11T14:04:16Z PR: MKP7 0.99774736 experimental_method cleaner0 2023-07-12T14:59:53Z MESH: IP 0.99863476 experimental_method cleaner0 2023-07-12T14:59:56Z MESH: immunoprecipitation 0.9998597 protein cleaner0 2023-07-11T14:04:16Z PR: MKP7 0.999576 protein_state cleaner0 2023-07-11T15:54:35Z DUMMY: wild type protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.9986125 taxonomy_domain cleaner0 2023-07-12T15:00:10Z DUMMY: lentiviruses 0.9998605 protein cleaner0 2023-07-11T14:04:16Z PR: MKP7 0.9995164 protein_state cleaner0 2023-07-11T15:54:25Z DUMMY: full-length protein_state DUMMY: cleaner0 2023-07-11T20:04:10Z mutants 0.99808496 experimental_method cleaner0 2023-07-12T15:00:15Z MESH: flow cytometry 0.9895574 chemical cleaner0 2023-07-12T15:03:07Z CHEBI: Annexin-V-APC 0.9218436 chemical cleaner0 2023-07-12T15:03:10Z CHEBI: PI 0.99781734 chemical cleaner0 2023-07-12T15:03:28Z CHEBI: Annexin-V 0.9977014 chemical cleaner0 2023-07-12T15:03:12Z CHEBI: propidium iodide 0.9991844 chemical cleaner0 2023-07-12T15:03:16Z CHEBI: PI 0.9990835 chemical cleaner0 2023-07-12T15:03:31Z CHEBI: Annexin-V 0.9988003 chemical cleaner0 2023-07-12T15:03:49Z CHEBI: PI evidence DUMMY: cleaner0 2023-07-11T20:13:34Z *P evidence DUMMY: cleaner0 2023-07-11T20:13:41Z ***P 0.99305326 experimental_method cleaner0 2023-07-12T15:00:17Z MESH: ANOVA experimental_method MESH: cleaner0 2023-07-11T20:13:19Z Tukey's test ncomms10879-f7.jpg f7 FIG fig_title_caption 58975 MKP5-CD is crucial for JNK1 binding and enzyme catalysis. 0.99952304 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.99982834 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 ncomms10879-f7.jpg f7 FIG fig_caption 59033 (a) Domain organization of human MKP5. The KBD and CD of MKP5 are shown in brown and grey, respectively. (b) Plots of initial velocity of the MKP5-catalysed reaction versus phospho-JNK1 concentration. The solid lines are best-fitting results according to the Michaelis–Menten equation with Km and kcat values indicated. Each experiment was performed in replicate for at least three times. The error bars represent s.e.m. (c) Structural comparison of the JNK-interacting residues on MKP5-CD (PDB 1ZZW) and MKP7-CD. The corresponding residues on MKP5 are depicted as orange sticks, and MKP5 residues numbers are in parentheses. (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. The panels are arranged the same as in Fig. 2d. (f) Effects of mutations in MKP5-CD on the JNK1 dephosphorylation (mean±s.e.m., n=3). (g) Effects of mutations in MKP5-CD on the pNPP hydrolysis reaction (mean±s.e.m., n=3). (h) Pull-down assays of MKP5-CD by GST-tagged JNK1 mutants. The panels are arranged the same as in Fig. 4c. 0.99896634 species cleaner0 2023-07-11T20:32:28Z MESH: human 0.9998555 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 0.9997814 structure_element cleaner0 2023-07-11T14:14:22Z SO: KBD 0.999783 structure_element cleaner0 2023-07-11T14:16:12Z SO: CD 0.9998547 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 evidence DUMMY: cleaner0 2023-07-12T14:47:11Z Plots of initial velocity 0.9998386 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 0.61602974 protein_state cleaner0 2023-07-12T14:51:53Z DUMMY: phospho 0.99366367 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.99945515 evidence cleaner0 2023-07-12T14:47:14Z DUMMY: Km 0.9994019 evidence cleaner0 2023-07-12T14:47:17Z DUMMY: kcat 0.999575 experimental_method cleaner0 2023-07-12T15:00:23Z MESH: Structural comparison 0.9991039 site cleaner0 2023-07-12T14:39:54Z SO: JNK-interacting residues 0.99977833 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 0.62858474 structure_element cleaner0 2023-07-11T14:16:12Z SO: CD protein PR: cleaner0 2023-07-11T14:04:16Z MKP7 0.46788436 structure_element cleaner0 2023-07-11T14:16:12Z SO: CD 0.99983907 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 0.9998492 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 0.9995819 experimental_method cleaner0 2023-07-12T15:00:26Z MESH: Gel filtration analysis 0.9997434 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.99965286 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.9639164 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 structure_element SO: cleaner0 2023-07-11T14:14:22Z KBD 0.9995555 experimental_method cleaner0 2023-07-12T15:00:30Z MESH: GST-mediated pull-down assays 0.99949336 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.9991891 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 0.49584374 structure_element cleaner0 2023-07-11T14:16:12Z SO: CD protein PR: cleaner0 2023-07-11T14:16:43Z MKP5 structure_element SO: cleaner0 2023-07-11T14:14:22Z KBD 0.6695175 experimental_method cleaner0 2023-07-12T15:00:33Z MESH: mutations 0.9997912 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 0.6887164 structure_element cleaner0 2023-07-11T14:16:12Z SO: CD 0.9993088 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.8962566 experimental_method cleaner0 2023-07-12T15:00:36Z MESH: mutations 0.9997826 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 0.8319109 structure_element cleaner0 2023-07-11T14:16:12Z SO: CD 0.9997385 chemical cleaner0 2023-07-12T15:03:54Z CHEBI: pNPP 0.9995843 experimental_method cleaner0 2023-07-12T15:00:39Z MESH: Pull-down assays 0.9994916 protein cleaner0 2023-07-11T14:16:43Z PR: MKP5 structure_element SO: cleaner0 2023-07-11T14:16:12Z CD 0.99779916 protein_state cleaner0 2023-07-12T14:51:56Z DUMMY: GST-tagged 0.99959534 protein cleaner0 2023-07-11T14:04:02Z PR: JNK1 0.99732274 protein_state cleaner0 2023-07-11T20:04:10Z DUMMY: mutants t1.xml t1 TABLE table_title_caption 60157 Data collection and refinement statistics. t1.xml t1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"> </th><th align="center" valign="top" charoff="50"><bold>JNK1–MKP7-CD</bold></th></tr></thead><tbody valign="top"><tr><td colspan="2" align="left" valign="top" charoff="50"><italic>Data collection</italic><xref ref-type="fn" rid="t1-fn1">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> Space group</td><td align="center" valign="top" charoff="50"><italic>P</italic>1</td></tr><tr><td colspan="2" align="left" valign="top" charoff="50"> Cell dimensions</td></tr><tr><td align="left" valign="top" charoff="50">  <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td align="center" valign="top" charoff="50">58.1, 74.8, 134.8</td></tr><tr><td align="left" valign="top" charoff="50">  α, β, γ (°)</td><td align="center" valign="top" charoff="50">76.9, 84.3, 67.4</td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="char" valign="top" char="(" charoff="50">40.00–2.40 (2.49–2.40)<xref ref-type="fn" rid="t1-fn2">†</xref></td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic>_merge</td><td align="char" valign="top" char="(" charoff="50">6.4 (60.9)</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>I</italic>/σ<italic>I</italic></td><td align="char" valign="top" char="(" charoff="50">14.1 (1.9)</td></tr><tr><td align="left" valign="top" charoff="50"> Completeness (%)</td><td align="char" valign="top" char="(" charoff="50">99.6 (99.9)</td></tr><tr><td align="left" valign="top" charoff="50"> Redundancy</td><td align="char" valign="top" char="(" charoff="50">3.5 (3.4)</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="2" align="left" valign="top" charoff="50"><italic>Refinement</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="char" valign="top" char="(" charoff="50">38.02–2.40 (2.43–2.40)</td></tr><tr><td align="left" valign="top" charoff="50"> No. of reflections</td><td align="center" valign="top" charoff="50">66889</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic>_work/<italic>R</italic>_free</td><td align="center" valign="top" charoff="50">21.7/23.9</td></tr><tr><td colspan="2" align="left" valign="top" charoff="50"> No. atoms</td></tr><tr><td align="left" valign="top" charoff="50">  Protein</td><td align="center" valign="top" charoff="50">14637</td></tr><tr><td align="left" valign="top" charoff="50">  Ligand/ion</td><td align="center" valign="top" charoff="50">4</td></tr><tr><td align="left" valign="top" charoff="50">  Water</td><td align="center" valign="top" charoff="50">457</td></tr><tr><td colspan="2" align="left" valign="top" charoff="50"> <italic>B</italic>-factors</td></tr><tr><td align="left" valign="top" charoff="50">  Protein</td><td align="center" valign="top" charoff="50">47.04</td></tr><tr><td align="left" valign="top" charoff="50">  Ligand/ion</td><td align="center" valign="top" charoff="50">30</td></tr><tr><td align="left" valign="top" charoff="50">  Water</td><td align="center" valign="top" charoff="50">37.28</td></tr><tr><td colspan="2" align="left" valign="top" charoff="50"> R.m.s.d.</td></tr><tr><td align="left" valign="top" charoff="50">  Bond lengths (Å)</td><td align="center" valign="top" charoff="50">0.015</td></tr><tr><td align="left" valign="top" charoff="50">  Bond angles (°)</td><td align="center" valign="top" charoff="50">1.564</td></tr></tbody></table> 60200   JNK1–MKP7-CD Data collection*  Space group P1  Cell dimensions   a, b, c (Å) 58.1, 74.8, 134.8   α, β, γ (°) 76.9, 84.3, 67.4  Resolution (Å) 40.00–2.40 (2.49–2.40)†  Rmerge 6.4 (60.9)  I/σI 14.1 (1.9)  Completeness (%) 99.6 (99.9)  Redundancy 3.5 (3.4)     Refinement  Resolution (Å) 38.02–2.40 (2.43–2.40)  No. of reflections 66889  Rwork/Rfree 21.7/23.9  No. atoms   Protein 14637   Ligand/ion 4   Water 457  B-factors   Protein 47.04   Ligand/ion 30   Water 37.28  R.m.s.d.   Bond lengths (Å) 0.015   Bond angles (°) 1.564 t1.xml t1 TABLE table_footnote 60875 *The data set was collected from a single crystal. t1.xml t1 TABLE table_footnote 60926 †Values in parentheses are for the highest resolution shell.