Atomic structure of dual-specificity phosphatase 26, a novel p53 phosphatase.

Dual-specificity phosphatases (DSPs) represent a heterogeneous subclass of the protein tyrosine phosphatase (PTP)-superfamily characterized by the unique ability to dephosphorylate both phospho-tyrosine and phospho-serine/threonine containing substrates (1–5). The first identified member of this family, VH1 is encoded by the Vaccinia virus H1 locus, which is conserved in all viruses of the Poxviridae family (6, 7). Since its identification in 1991 the number of VH1-like DSPs has quickly grown and, to date, it includes 61 members divided into 7 diverse subgroups (5). The human genome encodes 38 different VH1-like DSPs (5) (also referred to as ‘DUSPs’ (5)) that are essential cell signaling enzymes implicated in a multitude of physiological and pathological processes (5). Similar to classical PTPs, DSPs contain a catalytic triad consisting of a Cys, an Arg and an Asp (8). Whereas the catalytic Cys and Arg are part of a phosphate-binding loop (or ‘PTP-signature motif’) that has consensus Cys(X)5Arg(Ser/Thr), the highly conserved Asp residue is located on a separate loop (known as ’general-acid loop’), near the top of the active site, usually 30–40 residues away from the active site motif in the primary sequence (8). DSPs share a similar catalytic mechanism as PTPs, characterized by the formation of a transient enzyme-phospho-substrate intermediate (1, 2). Unlike PTPs, DSPs have broader substrate specificity (9) and can also dephosphorylate non-peptidic substrates. Examples of DPSs specific to non-peptidic substrates include PTEN-like DSPs, that dephosphorylate D3-inositol phospholipids (10), PIR that dephosphorylates mRNA (11) and the glycogen phosphatase laforin (12). The high resolution structure of Vaccinia virus VH1, determined to 1.32 A resolution (13, 14), as well as a wealth of other DSP structures determined over the past twenty years (8), have revealed that the DSP active site consists of a shallow, surface-exposed pocket, usually only ~6 A in depth. This active site is simple and likely not sufficient to discriminate among the thousands of different phospho-substrates present, at any given time, in a living cell (3). Instead, substrate recognition and specificity are likely achieved by a dedicated tertiary/quaternary structural complementarity between the phosphatase and its target phospho-substrate. DUSP26, also known to as MKP-8 (mitogen-activated protein kinase phosphatase-8 (15)), LDP-4 (low molecular-mass DUSP-4 (16)), or SKRP3 (stress-activated protein kinase pathway-regulating phosphatase) is a human DSP of the VH1-superfamily. DUSP26 is mainly expressed in neurons (17), retina (16, 18), heart (15, 17), adrenal gland (18) and skeletal muscle (15, 17, 18), where it localizes primarily to the cell nucleus (15, 16). Several potential substrates of DUSP26 have been identified. DUSP26 can function as a p38-specific phosphatase (15, 19) and an Erk-phosphatase (17), as well as, in PC12 cells, overexpression of DUSP26 was found to down-regulate the PI3K/Akt signaling pathway (18). Furthermore, several direct and indirect lines of evidence connect DUSP26 biology to tumorigenesis. First, DUSP26 inhibits p38-mediated apoptosis, thereby promoting anaplastic thyroid cancer cell growth and survival (19). Second, DUSP26 associates with and dephosphorylates Kap3, a component of the microtubule-directed protein complex KIF3, supporting a role in intracellular transport of β-catenin/N-cadherin (an established KIF3 cargo) and cell-cell adhesion (20). Finally, DUSP26 has been shown to directly bind to and dephosphorylate p53 transactivation domain (TAD) at Ser20 and Ser37, which results in repression of p53 transcriptional activity (21). DUSP26 expression is greatly unregulated in neuroblastoma cell lines, which, unlike many human cancer cells, maintain normal levels of wild type p53. Overexpression of DUSP26 suppresses p53 ‘onco-suppressive’ function in response to genotoxic stress (21). For this reason, DUSP26 is a novel and promising target for the development of small molecule inhibitors for treatment of neuroblastoma and related pediatric malignancies. In this paper, we report a structural and biochemical characterization of human DUSP26. This work sheds light on the organization of a p53 phosphatase whose hyperactivation is linked to neuroblastoma.