bluuebunny/crossref_metadata_2025 huggingface dataset:
The TCR ADAPts to integrin‐mediated cell adhesion
Authors: Peterson Erik J.
Published: January 2004

Summary: Adapter proteins regulate leukocyte signal transduction through recruitment of effector molecules to multiprotein complexes. Recent studies in Adhesion and Degranulation promoting Adapter Protein (ADAP)‐deficient mice have established that the cytoplasmic phosphoprotein ADAP is required for optimal, mature T‐cell proliferation. Furthermore, ADAP plays a key role in T‐cell antigen receptor (TCR)‐mediated ‘inside out’ signaling leading to integrin activation and to enhanced cellular adhesion to integrin ligands. ADAP associates physically with molecules known to play roles in the regulation of TCR‐stimulated actin polymerization. These associations support the hypothesis that ADAP functions in actin cytoskeletal reorganization leading to cellular adhesion and activation.
Protein kinase C‐θ (PKCθ): it's all about location, location, location
Authors: Altman Amnon , Villalba Martin
Published: January 2004

Summary: Much progress has been made in understanding the function of protein kinase C‐θ (PKCθ) in the immune system since this Ca2+‐independent PKC isotype was isolated in 1993 as an enzyme that is highly expressed in T lymphocytes and in muscle cells. Biochemical and genetic approaches revealed that, while dispensable for T‐cell development, PKCθ is required for the activation of mature T cells and for interleukin (IL)‐2 production. This deficiency results from impaired receptor‐induced stimulation of the transcription factors AP‐1 and NF‐κB. PKCθ integrates T‐cell receptor (TCR)/CD28 costimulatory signals, which are essential for productive T‐cell activation and, most likely, for prevention of T‐cell anergy. A unique property of PKCθ is its highly selective recruitment to the central supramolecular activation complex (cSMAC) region of the immunological synapse (IS) in antigen‐stimulated T cells. Our work revealed that this highly selective localization is not entirely dependent on phospholipase C (PLC) activity and diacylglycerol (DAG) production. Instead, a novel signaling pathway that requires functional Vav1, phosphatidylinositol 3‐kinase (PI3‐K), the small GTPase Rac and actin cytoskeleton reorganization regulates the localization and, perhaps, activation of PKCθ. PKCθ also provides a survival signal, which protects T cells from apoptosis. Additional work is required to identify the immediate targets of PKCθ and its immune functions in vivo. This work is likely to validate PKCθ as an attractive drug target.
GTPases and T cell activation
Authors: Cantrell Doreen Ann
Published: January 2004

Summary:  Guanine nucleotide binding proteins rapidly cycle between a guanosine diphosphate (GDP)‐bound and guanosine triphosphate (GTP)‐bound state, and they operate as binary switches that control cell activation in response to environmental cues. GTPases adopt different conformations when binding GTP vs. GDP. The GTP‐bound state is generally considered to be the active conformation that allows GTPases to interact with downstream effectors and thereby initiate downstream signaling pathways, which regulate many important biological processes. Many members of the Ras family of GTPases, notably Ras and Rap1A, and the Rho family GTPases, Cdc42Hs, Rac1, Rac2 and RhoA, are important components of signal transduction pathways used by antigen receptors, costimulatory, cytokine and chemokine receptors to regulate the immune response. This review discusses current knowledge and ideas about the regulation and function of these GTPases in lymphocytes.
The Wiskott–Aldrich syndrome protein: forging the link between actin and cell activation
Authors: Badour Karen , Zhang Jinyi , Siminovitch Katherine A.
Published: January 2004

The Wiskott–Aldrich syndrome protein (WASp) has emerged as a central player in the regulation of actin remodeling in T cells. The unique domain structure of WASp and other WASp family members enables these proteins to associate with a myriad of signaling effectors and to thereby regulate the coupling of T cell antigen receptor (TCR) engagement to both cytoskeletal rearrangement and transcriptional activation. This review focuses on these biochemical properties of WASp and also on the mechanisms whereby WASp interactions with its cognate ligands influence T cell activation. Because of its capacity to shift intracellular location and thereby dictate both the timing and the spatial distribution of actin polymerization following cell stimulation, WASp is well positioned to play major regulatory roles in directing a wide range of cellular processes and signaling pathways. Further dissection of the functional and biochemical properties of WASp therefore represents a promising avenue towards defining the molecular mechanisms that convey TCR stimulatory signals to the actin cytoskeleton and integrate cytoskeletal and other signaling systems so as to evoke a biological response.
Vav1: a key signal transducer downstream of the TCR
Authors: Tybulewicz Victor L. J. , Ardouin Laurence , Prisco Antonella , Reynolds Lucinda F.
Published: January 2004

Vav1 is a 95‐kDa protein expressed in all hemopoietic cells that becomes rapidly tyrosine phosphorylated following T cell antigen receptor (TCR) stimulation. Vav1 contains multiple domains characteristic of signal transducing proteins, including a Dbl homology domain, a hallmark of a guanine nucleotide exchange factor (GEF) for Rho‐family GTPases. Indeed Vav1 is a GEF for Rac1, Rac2 and RhoG, and it is activated following tyrosine phosphorylation. Generation of mice deficient in Vav1 has shown that it plays an important role in selection events within the thymus, including both positive and negative selection, consistent with Vav1 transducing TCR signals required to drive these processes. Furthermore, Vav1‐deficient T cells are defective in TCR‐induced proliferation and cytokine synthesis.

Analysis of TCR signaling pathways in Vav1‐deficient T cells and thymocytes has shown that Vav1 is required to transduce signals to the activation of a calcium flux, extracellular signal‐regulated kinase (ERK) and the nuclear factor κB (NF‐κB) transcription factor. Vav1 has also been shown to control the activation of phospholipase Cγ1 (PLCγ1) via both phosphoinositide‐3‐kinase (PI3K)‐dependent and ‐independent pathways. Finally, Vav1 has been shown to transduce TCR signals to some but not all cytoskeleton‐dependent pathways. In particular, Vav1 is required for efficient TCR‐induced conjugate formation with antigen presenting cells (APCs), activation of the integrin leukocyte function‐associated antigen‐1 (LFA‐1) and cell polarization.
Molecular modifiers of T cell antigen receptor triggering threshold: the mechanism of CD28 costimulatory receptor
Authors: Acuto Oreste , Mise‐Omata Setsuko , Mangino Giorgio , Michel Frédérique
Published: January 2004

CD28 was thought to represent a prototypic membrane receptor responsible for delivering the classically defined ‘second signal’ needed to avoid T cell paralysis when recognizing antigen presented by appropriate antigen presenting cells (APCs). Almost two decades after its molecular identification, the mechanism by which this ‘second receptor’ facilitates clonal expansion and differentiation upon antigen encounter is still not fully elucidated. There may be at least two reasons for this partially gray picture: the use of nonphysiological experimental conditions to study it and the fact that the action of CD28 may be partly masked by the presence of additional T cell surface receptors that also provide some costimulatory signals, although not equivalent to the one delivered through CD28. Thus, instead of aging, the study of CD28 is still a topical subject. What is appearing through work of recent years is that far from being purely qualitative, the CD28 signal provides a key quantitative contribution to potently boost the T cell antigen receptor (TCR) signal. In other words, CD28 is in part a signaling ‘sosia’ of the TCR. Also, it is clear now that CD28 operates via multiple molecular effects. Still, what we do not understand is the ‘qualitative’ part of this signal, perhaps due to lack of identification of unique signaling components and/or pathways activated by CD28 only. Here we review a series of recent findings pointing towards novel avenues to better understand the molecular basis of CD28 function.
Investigation of metal binding and activation of Escherichia coli glyoxalase I: kinetic, thermodynamic and mutagenesis studies
Authors: CLUGSTON Susan L. , YAJIMA Rieko , HONEK John F.
Published: January 2004

GlxI (glyoxalase I) isomerizes the hemithioacetal formed between glutathione and methylglyoxal. Unlike other GlxI enzymes, Escherichia coli GlxI exhibits no activity with Zn2+ but maximal activation with Ni2+. To elucidate further the metal site in E. coli GlxI, several approaches were undertaken. Kinetic studies indicate that the catalytic metal ion affects the kcat without significantly affecting the Km for the substrate. Inductively coupled plasma analysis and isothermal titration calorimetry confirmed one metal ion bound to the enzyme, including Zn2+, which produces an inactive enzyme. Isothermal titration calorimetry was utilized to determine the relative binding affinity of GlxI for various bivalent metals. Each metal ion examined bound very tightly to GlxI with an association constant (Ka)>107 M−1, with the exception of Mn2+ (Ka of the order of 106 M−1). One of the ligands to the catalytic metal, His5, was altered to glutamine, a side chain found in the Zn2+-active Homo sapiens GlxI. The affinity of the mutant protein for all bivalent metals was drastically decreased. However, low levels of activity were now observed for Zn2+-bound GlxI. Although this residue has a marked effect on metal binding and activation, it is not the sole factor determining the differential metal activation between the human and E. coli GlxI enzymes.
A membrane proximal region of the integrin alpha5 subunit is important for its interaction with nischarin
Authors: ALAHARI Suresh K. , NASRALLAH Hani
Published: January 2004

In a previous study [Alahari, Lee and Juliano (2000) J. Cell Biol. 151, 1141–1154], we have identified a novel protein, nischarin, that specifically interacts with the cytoplasmic tail of the α5 integrin subunit. Overexpression of this protein profoundly affects cell migration. To examine the nischarin–α5 interaction in detail, and to find the minimal region required for the interaction, several mutants of nischarin and α5 were created. The results obtained for the yeast two-hybrid system indicate that a 99-aminoacid region of nischarin (from residues 464 to 562) is indispensable for the interaction. Also, we demonstrate that the membrane proximal region (from residues 1017 to 1030) of the α5 cytoplasmic tail is essential for the interaction. To characterize more directly the properties of the interaction between nischarin and α5, we performed surface-plasmon resonance studies in which peptides were immobilized on the surface of a sensor chip, and the recombinant nischarin protein fragments were injected. Consistent with the two-hybrid results, recombinant nischarin binds well to immobilized α5 peptides. In addition, mutational analysis revealed that residues Tyr1018 and Lys1022 are crucial for α5–nischarin interactions. These results provide evidence that nischarin is capable of directly and selectively binding to a portion of the α5 cytoplasmic domain. Further studies demonstrated that the minimal α5 binding region of nischarin does not affect cell migration.
Characterization of human brain nicotinamide 5′-mononucleotide adenylyltransferase-2 and expression in human pancreas
Authors: YALOWITZ Joel A. , XIAO Suhong , BIJU Mangatt P. , ANTONY Aśok C. , CUMMINGS Oscar W. , DEEG Mark A. , JAYARAM Hiremagalur N.
Published: January 2004

NMNAT (nicotinamide 5´-mononucleotide adenylyltransferase; EC 2.7.7.1) catalyses the transfer of the adenylyl group from ATP to NMN to form NAD. We have cloned a novel human NMNAT cDNA, designated hNMNAT-2, from human brain. The cDNA contains a 924 bp open reading frame that encodes a 307 amino acid peptide that was expressed as a histidine-patch-containing thioredoxin fusion protein. Expressed hNMNAT-2 shared only 35% amino acid sequence homology with the human NMNAT enzyme (hNMNAT-1), but possessed enzymic activity comparable with hNMNAT-1. Using human genomic databases, hNMNAT-2 was localized to chromosome 1q25 within a 171 kb gene, whereas hNMNAT-1 is on chromosome 1p32–35. Northern blot analysis revealed highly restricted expression of hNMNAT-2 to brain, heart and muscle tissues, which contrasts with the wide tissue expression of hNMNAT-1; different regions of the brain exhibited differential expression of hNMNAT-2. Substitution mutations of either of two invariant residues, His-24 or Trp-92, abolished enzyme activity. Anti-peptide antibody to a unique epitope within hNMNAT-2 was produced, and immunohistochemical analysis of sections of normal adult human pancreas revealed that hNMNAT-2 protein was markedly expressed in the islets of Langerhans. However, the pancreatic exocrine cells exhibited weak expression of hNMNAT-2 protein. Sections of pancreas from insulinoma patients showed strong expression of hNMNAT-2 protein in the insulin-producing tumour cells, whereas acinar cells exhibited relatively low expression of hNMNAT-2 protein. These data suggest that the unique tissue-expression patterns of hNMNAT-2 reflect distinct functions for the isoforms in the regulation of NAD metabolism.
The regulatory domain of the inositol 1,4,5-trisphosphate receptor is necessary to keep the channel domain closed: possible physiological significance of specific cleavage by caspase 3
Authors: NAKAYAMA Tomohiro , HATTORI Mitsuharu , UCHIDA Keiko , NAKAMURA Takeshi , TATEISHI Yoko , BANNAI Hiroko , IWAI Miwako , MICHIKAWA Takayuki , INOUE Takafumi , MIKOSHIBA Katsuhiko
Published: January 2004

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is an intracellular Ca2+ channel protein that plays crucial roles in generating complex Ca2+ signalling patterns. IP3R1 consists of three domains: a ligand-binding domain, a regulatory domain and a channel domain. In order to investigate the function of these domains in its gating machinery and the physiological significance of specific cleavage by caspase 3 that is observed in cells undergoing apoptosis, we utilized various IP3R1 constructs tagged with green fluorescent protein (GFP). Expression of GFP-tagged full-length IP3R1 or IP3R1 lacking the ligand-binding domain in HeLa and COS-7 cells had little effect on cells’ responsiveness to an IP3-generating agonist ATP and Ca2+ leak induced by thapsigargin. On the other hand, in cells expressing the caspase-3-cleaved form (GFP–IP3R1-casp) or the channel domain alone (GFP–IP3R1-ES), both ATP and thapsigargin failed to induce increase of cytosolic Ca2+ concentration. Interestingly, store-operated (−like) Ca2+ entry was normally observed in these cells, irrespective of thapsigargin pre-treatment. These findings indicate that the Ca2+ stores of cells expressing GFP–IP3R1-casp or GFP–IP3R1-ES are nearly empty in the resting state and that these proteins continuously leak Ca2+. We therefore propose that the channel domain of IP3R1 tends to remain open and that the large regulatory domain of IP3R1 is necessary to keep the channel domain closed. Thus cleavage of IP3R1 by caspase 3 may contribute to the increased cytosolic Ca2+ concentration often observed in cells undergoing apoptosis. Finally, GFP–IP3R1-casp or GFP–IP3R1-ES can be used as a novel tool to deplete intracellular Ca2+ stores.
Parasite-specific inserts in the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase of Plasmodium falciparum modulate catalytic activities and domain interactions
Authors: BIRKHOLTZ Lyn-Marie , WRENGER Carsten , JOUBERT Fourie , WELLS Gordon A. , WALTER Rolf D. , LOUW Abraham I.
Published: January 2004

Polyamine biosynthesis of the malaria parasite, Plasmodium falciparum, is regulated by a single, hinge-linked bifunctional PfAdoMetDC/ODC [P. falciparum AdoMetDC (S-adenosylmethionine decarboxylase)/ODC (ornithine decarboxylase)] with a molecular mass of 330 kDa. The bifunctional nature of AdoMetDC/ODC is unique to Plasmodia and is shared by at least three species. The PfAdoMetDC/ODC contains four parasite-specific regions ranging in size from 39 to 274 residues. The significance of the parasite-specific inserts for activity and protein–protein interactions of the bifunctional protein was investigated by a single- and multiple-deletion strategy. Deletion of these inserts in the bifunctional protein diminished the corresponding enzyme activity and in some instances also decreased the activity of the neighbouring, non-mutated domain. Intermolecular interactions between AdoMetDC and ODC appear to be vital for optimal ODC activity. Similar results have been reported for the bifunctional P. falciparum dihydrofolate reductase-thymidylate synthase [Yuvaniyama, Chitnumsub, Kamchonwongpaisan, Vanichtanankul, Sirawaraporn, Taylor, Walkinshaw and Yuthavong (2003) Nat. Struct. Biol. 10, 357–365]. Co-incubation of the monofunctional, heterotetrameric ≈150 kDa AdoMetDC domain with the monofunctional, homodimeric ODC domain (≈180 kDa) produced an active hybrid complex of 330 kDa. The hinge region is required for bifunctional complex formation and only indirectly for enzyme activities. Deletion of the smallest, most structured and conserved insert in the ODC domain had the biggest impact on the activities of both decarboxylases, homodimeric ODC arrangement and hybrid complex formation. The remaining large inserts are predicted to be non-globular regions located on the surface of these proteins. The large insert in AdoMetDC in contrast is not implicated in hybrid complex formation even though distinct interactions between this insert and the two domains are inferred from the effect of its removal on both catalytic activities. Interference with essential protein–protein interactions mediated by parasite-specific regions therefore appears to be a viable strategy to aid the design of selective inhibitors of polyamine metabolism of P. falciparum.
Analysis of the interaction between piD261/Bud32, an evolutionarily conserved protein kinase of Saccharomyces cerevisiae, and the Grx4 glutaredoxin
Authors: LOPREIATO Raffaele , FACCHIN Sonia , SARTORI Geppo , ARRIGONI Giorgio , CASONATO Stefano , RUZZENE Maria , PINNA Lorenzo A. , CARIGNANI Giovanna
Published: January 2004

The Saccharomyces cerevisiae piD261/Bud32 protein and its structural homologues, which are present along the Archaea–Eukarya lineage, constitute a novel protein kinase family (the piD261 family) distantly related in sequence to the eukaryotic protein kinase superfamily. It has been demonstrated that the yeast protein displays Ser/Thr phosphotransferase activity in vitro and contains all the invariant residues of the family. This novel protein kinase appears to play an important cellular role as deletion in yeast of the gene encoding piD261/Bud32 results in the alteration of fundamental processes such as cell growth and sporulation. In this work we show that the phosphotransferase activity of Bud32 is relevant to its functionality in vivo, but is not the unique role of the protein, since mutants which have lost catalytic activity but not native conformation can partially complement the disruption of the gene encoding piD261/Bud32. A two-hybrid approach has led to the identification of several proteins interacting with Bud32; in particular a glutaredoxin (Grx4), a putative glycoprotease (Ykr038/Kae1) and proteins of the Imd (inosine monophosphate dehydrogenase) family seem most plausible interactors. We further demonstrate that Grx4 directly interacts with Bud32 and that it is phosphorylated in vitro by Bud32 at Ser-134. The functional significance of the interaction between Bud32 and the putative protease Ykr038/Kae1 is supported by its evolutionary conservation.
Analysis of Stat3 (signal transducer and activator of transcription 3) dimerization by fluorescence resonance energy transfer in living cells
Authors: KRETZSCHMAR Antje K. , DINGER Michaela C. , HENZE Christian , BROCKE-HEIDRICH Katja , HORN Friedemann
Published: January 2004

Signal transducer and activator of transcription 3 (Stat3) dimerization is commonly thought to be triggered by its tyrosine phosphorylation in response to interleukin-6 (IL-6) or other cytokines. Accumulating evidence from in vitro studies, however, suggests that cytoplasmic Stat3 may be associated with high-molecular-mass protein complexes and/or dimerize prior to its activation. To directly study Stat3 dimerization and subcellular localization upon cytokine stimulation, we used live-cell fluorescence spectroscopy and imaging microscopy combined with fluorescence resonance energy transfer (FRET). Stat3 fusion proteins with spectral variants of green fluorescent protein (GFP), cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) were constructed and expressed in human hepatoma cells (HepG2) and human embryonic kidney cells (HEK-293). Like wild-type Stat3, the fusion proteins redistributed from a preferentially cytoplasmic to nuclear localization upon IL-6 stimulation and supported IL-6-dependent target gene expression. FRET studies in cells co-expressing Stat3–CFP and Stat3–YFP demonstrated that Stat3 dimers exist in the absence of tyrosine phosphorylation. IL-6 induced a 2-fold increase of this basal FRET signal, indicating that tyrosine phosphorylation either increases the dimer/monomer ratio of Stat3 or induces a conformational change of the dimer yielding a higher FRET efficiency. Studies using a mutated Stat3 with a non-functional src-homology 2 (SH2) domain showed that the SH2 domain is essential for dimer formation of phosphorylated as well as non-phosphorylated Stat3. Furthermore, our data show that visualization of normalized FRET signals allow insights into the spatiotemporal dynamics of Stat3 signal transduction.
Interleukin-8 binds to syndecan-2 on human endothelial cells
Authors: HALDEN Yvonne , REK Angelika , ATZENHOFER Werner , SZILAK Laszlo , WABNIG Astrid , KUNGL Andreas J.
Published: January 2004

Application of reverse transcription–PCR to total RNA prepared from TNF-α (tumour necrosis factor-α)-stimulated HUVECs (human umbilical vein endothelial cells) revealed that the syndecan-2 mRNA was up-regulated by this inflammatory stimulus. By immunoprecipitation using an anti-syndecan-2 antibody on TNF-α-stimulated HUVEC lysates, inflammation-induced interleukin-8 was found to be an interaction partner of this HS (heparan sulphate) proteoglycan, but not of any other syndecan on these cells. The glycosylated [Syn2ect(+HS)] and non-glycosylated [Syn2ect(−HS)] forms of Syn2ect (the syndecan-2 ectodomain) were purified from a stably transfected human cell line and from a bacterial expression system respectively. By CD spectroscopy, Syn2ect was found to adopt an all-β secondary structure. The dissociation constant of Syn2ect(+HS) with respect to interleukin-8 binding was determined by isothermal fluorescence titrations to be 23 nM. Despite its lack of HS chains, Syn2ect(−HS) exhibited significant binding to the chemokine, with a Kd of >1 µM. Thus, in addition to glycosaminoglycan binding, protein–protein contacts might also contribute to the chemokine–proteoglycan interaction.
Saccharomyces cerevisiae ubiquitin-like protein Rub1 conjugates to cullin proteins Rtt101 and Cul3 in vivo
Authors: LAPLAZA Jose M. , BOSTICK Magnolia , SCHOLES Derek T. , CURCIO M. Joan , CALLIS Judy
Published: January 2004

In Saccharomyces cerevisiae, the ubiquitin-like protein Rub1p (related to ubiquitin 1 protein) covalently attaches to the cullin protein Cdc53p (cell division cycle 53 protein), a subunit of a class of ubiquitin E3 ligases named SCF (Skp1–Cdc53–F-box protein) complex. We identified Rtt101p (regulator of Ty transposition 101 protein, where Ty stands for transposon of yeast), initially found during a screen for proteins to confer retrotransposition suppression, and Cul3p (cullin 3 protein), a protein encoded by the previously uncharacterized open reading frame YGR003w, as two new in vivo targets for Rub1p conjugation. These proteins show significant identity with Cdc53p and, therefore, are cullin proteins. Modification of Cul3p is eliminated by deletion of the Rub1p pathway through disruption of either RUB1 or its activating enzyme ENR2/ULA1. The same disruptions in the Rub pathway decreased the percentage of total Rtt101p that is modified from approx. 60 to 30%. This suggests that Rtt101p has an additional RUB1- and ENR2-independent modification. All modified forms of Rtt101p and Cul3p were lost when a single lysine residue in a conserved region near the C-terminus was replaced by an arginine residue. These results suggest that this lysine residue is the site of Rub1p-dependent and -independent modifications in Rtt101p and of Rub1p-dependent modification in Cul3p. An rtt101Δ strain was hypersensitive to thiabendazole, isopropyl (N-3-chlorophenyl) carbamate and methyl methanesulphonate, but rub1Δ strains were not. Whereas rtt101Δ strains exhibited a 14-fold increase in Ty1 transposition, isogenic rub1Δ strains did not show statistically significant increases. Rtt101K791Rp, which cannot be modified, complemented for Rtt101p function in a transposition assay. Altogether, these results suggest that neither the RUB1-dependent nor the RUB1-independent form of Rtt101p is required for Rtt101p function. The identification of additional Rub1p targets in S. cerevisiae suggests an expanded role for Rub in this organism.
Arf-1 (ADP-ribosylation factor-1) is involved in the activation of a mammalian Na+-selective current
Authors: STRAUBE Sebastian , PAREKH Anant B.
Published: January 2004

Stimulation of mammalian cells often results in an increase in the intracellular Na+ concentration, brought about by Na+ influx into the cell via Na+-permeable ion channels. In some cell types, particularly renal epithelia and mast cells, non-hydrolysable analogues of GTP, such as GTP[S] (guanosine 5´-[γ-thio]triphosphate), activate a non-voltage-activated Na+-selective current. We have carried out whole-cell patch–clamp experiments to examine how GTP[S] activates the Na+ current in a rat mast cell line. The ability of GTP[S] to activate Na+ influx was prevented by including GTP in the pipette solution, indicating the involvement of small G-proteins. Brefeldin A and Arf-1-(2–17), inhibitors of Arf-1 (ADP-ribosylation factor-1) proteins, suppressed the activation of Na+ entry by GTP[S]. However, non-active succinylated Arf-1-(2–17) or an N-terminal myristoylated peptide directed towards Arf-5 were ineffective. Arf proteins modulate the cytoskeleton, and disruption of the cytoskeleton with cytochalasin D or its stabilization with phalloidin impaired the development of the Na+ current. Disaggregation of microtubules was without effect. Dialysis with cAMP or inhibition of cAMP phosphodiesterase with caffeine both decreased the extent of Na+ entry, and this was not prevented by pre-treatment with broad-spectrum protein kinase inhibitors. Collectively, our results suggest that the mechanism of activation of a mammalian non-voltage-activated Na+-selective current requires an Arf small G-protein, most probably Arf-1.
Accumulation of manganese superoxide dismutase under metal-depleted conditions: proposed role for zinc ions in cellular redox balance
Authors: OTSU Kaoru , IKEDA Yoshitaka , FUJII Junichi
Published: January 2004

A diet low in copper results in increased levels of MnSOD (manganese superoxide dismutase), a critical antioxidative enzyme conferring protection against oxidative stress, in rat liver mitochondria. The mechanism for this was investigated using cultured HepG2 cells, a human hepatocellular carcinoma-derived line. MnSOD activity increased 5–7-fold during incubation in a medium supplemented with metal-depleted fetal bovine serum, with a corresponding elevation of its mRNA levels. Metal depletion also decreased CuZnSOD and glutathione peroxidase levels to approx. 70–80% of baseline. When zinc ions were added to the medium at micromolar levels, MnSOD accumulation was suppressed; however, copper ions had essentially no effect on MnSOD expression. Since the intracellular redox status was shifted to a more oxidized state by metal depletion, we examined the DNA-binding activity of NF-κB (nuclear factor-κB), an oxidative stress-sensitive transactivating factor that plays a primary role in MnSOD induction. A gel shift assay indicated that the DNA-binding activity of NF-κB was increased in cells maintained in metal-depleted culture, suggesting the involvement of the transactivating function of NF-κB in this induction. This was further supported by the observation that curcumin suppressed both the DNA-binding activity of NF-κB and the induction of MnSOD mRNA in cells cultivated under metal-depleted conditions. These results suggest that the level of zinc, rather than copper, is a critical regulatory factor in MnSOD expression. It is possible that a deficiency of zinc in the low-copper diet may be primarily involved in MnSOD induction.
Fatty acids enhance membrane permeabilization by pro-apoptotic Bax
Authors: EPAND Raquel F. , MARTINOU Jean-Claude , MONTESSUIT Sylvie , EPAND Richard M.
Published: January 2004

Fatty acids are known promoters of apoptosis. In the present study, the direct role of fatty acids with regard to their ability to cause membrane permeabilization by Bax was explored. Addition of fatty acids to liposomes in the presence of cations greatly enhanced the permeabilizing activity of Bax, a pro-apoptotic Bcl-2 protein. This provides a putative mechanism for the role of fatty acids in apoptosis. It is not a result of detergent-like properties of fatty acids, since a different micelle-forming amphiphile, dilysocardiolipin, was strongly inhibitory. We also demonstrate that there is a synergistic effect on Bax-induced permeabilization between Ca2+ and Mg2+, both on the binding of Bax to liposomes as well as on the induction of the leakage of liposomal contents. Micromolar concentrations of Ca2+ added externally or submicromolar concentrations of free Ca2+ present in the medium were sufficient to promote Bax-induced permeabilization synergistically with externally added Mg2+. These results indicate that Bax can induce leakage from liposomes at ion concentrations resembling those found physiologically. The synergistic effects of Ca2+ and Mg2+ were observed with liposomes with different lipid compositions. Thus the action of Bax is strongly modulated by the presence of bivalent cations that can act synergistically, as well as by micelle-forming lipid components that can be either stimulatory or inhibitory.
Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures
Authors: BOZZO Gale G. , RAGHOTHAMA Kashchandra G. , PLAXTON William C.
Published: January 2004

An intracellular acid phosphatase (IAP) from Pi-starved (−Pi) tomato (Lycopersicon esculentum) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (λmax=546 nm) and was insensitive to l-tartrate. PAGE, periodic acid–Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (α-subunit) and 57 kDa (β-subunit) polypeptides. However, the nine N-terminal amino acids of the α- and β-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to Pi-deprivation was correlated with similar increases in the amount of antigenic IAP α- and β-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg2+, but was potently inhibited by Zn2+, Cu2+, Fe3+, molybdate, vanadate, fluoride and Pi. Although IAP demonstrated broad substrate selectivity, its specificity constant (Vmax/Km) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg2+ or molybdate. This IAP is proposed to scavenge Pi from intracellular phosphate esters in −Pi tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of −Pi tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278–6286].
Identification of sequences required for the import of human protoporphyrinogen oxidase to mitochondria
Authors: MORGAN Rhian R. , ERRINGTON Rachel , ELDER George H.
Published: January 2004

Protoporphyrinogen oxidase (PPOX; EC 1.3.3.4), the penultimate enzyme of haem biosynthesis, is a nucleus-encoded flavoprotein strongly associated with the outer surface of the inner mitochondrial membrane. It is attached to this membrane by an unknown mechanism that appears not to involve a membrane-spanning domain. The pathway for its import to mitochondria and insertion into the inner membrane has not been established. We have fused human PPOXs containing N-terminal deletions, C-terminal deletions or missense mutations to yellow fluorescent protein (YFP) and have used these constructs to investigate the mitochondrial import of PPOX in human cells. We show that all the information required for efficient import is contained within the first 250 amino acid residues of human PPOX and that targeting to mitochondria is prevented by fusion of YFP to the N-terminus. Deletion of between 151 and 175 residues from the N-terminus is required to abolish import, whereas shorter deletions impair its efficiency. Fully efficient targeting appears to require both a major targeting signal, the whole or part of which is contained between residues 151 and 175, and which may be involved in anchoring to the inner mitochondrial membrane, together with interaction between this region and a sequence(s) within the first 150 residues. These features suggest that the mechanism for import of human PPOX to mitochondria differs from those identified for the translocation of nucleus-encoded, membrane-spanning, inner membrane proteins. In addition, a missense mutation outside this region (Val335→Gly) prevented targeting to mitochondria and delayed the appearance of YFP fluorescence. This mutation appeared to prevent import by a direct effect on protein folding rather than by altering a sequence required for targeting. It may lead to sequestration of the PPOX–YFP construct in an unfolded conformation, followed by proteolytic degradation, possibly through enhanced binding to a cytosolic chaperone protein.
Functional characterization of alpha-glucan,water dikinase, the starch phosphorylating enzyme
Authors: MIKKELSEN René , BAUNSGAARD Lone , BLENNOW Andreas
Published: January 2004

GWD (α-glucan,water dikinase) is the enzyme that catalyses the phosphorylation of starch by a dikinase-type reaction in which the β-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glycosyl residue of amylopectin. GWD shows similarity in both sequence and reaction mechanism to bacterial PPS (pyruvate,water dikinase) and PPDK (pyruvate,phosphate dikinase). Amino acid sequence alignments identified a conserved histidine residue located in the putative phosphohistidine domain of potato GWD. Site-directed mutagenesis of this histidine residue resulted in an inactive enzyme and loss of autophosphorylation. Native GWD is a homodimer and shows a strict requirement for the presence of α-1,6 branch points in its polyglucan substrate, and exhibits a sharp 20-fold increase in activity when the degree of polymerization is increased from 27.8 to 29.5. In spite of the high variability in the degree of starch phosphorylation, GWD proteins are ubiquitous in plants. The overall reaction mechanism of GWD is similar to that of PPS and PPDK, but the GWD family appears to have arisen after divergence of the plant kingdom. The nucleotide-binding domain of GWD exhibits a closer phylogenetic relationship to prokaryotic PPSs than to PPDKs.
Evidence for specific tetraspanin homodimers: inhibition of palmitoylation makes cysteine residues available for cross-linking
Authors: KOVALENKO Oleg V. , YANG Xiuwei , KOLESNIKOVA Tatiana V. , HEMLER Martin E.
Published: January 2004

It is a well-established fact that tetraspanin proteins, a large family of integral membrane proteins involved in cell motility, fusion and signalling, associate extensively with one another and with other transmembrane and membrane-proximal proteins. In this study, we present results strongly suggesting that tetraspanin homodimers are fundamental units within larger tetraspanin complexes. Evidence for constitutive CD9 homodimers was obtained using several cell lines, utilizing the following four methods: (1) spontaneous cross-linking via intermolecular disulphide bonds, (2) use of a cysteine-reactive covalent cross-linking agent, (3) use of an amino-reactive covalent cross-linking agent, and (4) covalent cross-linking via direct intermolecular disulphide bridging between unpalmitoylated membrane-proximal cysteine residues. In the last case, incubation of cells with the palmitoylation inhibitor 2-bromopalmitate exposed membrane-proximal cysteine residues, thus effectively promoting ‘zero-length’ cross-linking to stabilize homodimers. Similar to CD9, other tetraspanins (CD81 and CD151) also showed a tendency to homodimerize. Tetraspanin homodimers were assembled from newly synthesized proteins in the Golgi, as evidenced by cycloheximide and Brefeldin A inhibition studies. Importantly, tetraspanin homodimers appeared on the cell surface and participated in typical ‘tetraspanin web’ interactions with other proteins. Whereas homodimers were the predominant cross-linked species, we also observed some higher-order complexes (trimers, tetramers or higher) and a much lower level of cross-linking between different tetraspanins (CD81–CD9, CD9–CD151, CD81–CD151). In conclusion, our results strongly suggest that tetraspanin homodimers, formed in the Golgi and present at the cell surface, serve as building blocks for the assembly of larger, multicomponent tetraspanin protein complexes.
Rho GTPases have diverse effects on the organization of the actin filament system
Authors: ASPENSTRÖM Pontus , FRANSSON Åsa , SARAS Jan
Published: January 2004

The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1–Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1–Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases.
Calcium/calmodulin-dependent protein kinase IIdelta associates with the ryanodine receptor complex and regulates channel function in rabbit heart
Authors: CURRIE Susan , LOUGHREY Christopher M. , CRAIG Margaret-Anne , SMITH Godfrey L.
Published: January 2004

Cardiac ryanodine receptors (RyR2s) play a critical role in excitation–contraction coupling by providing a pathway for the release of Ca2+ from the sarcoplasmic reticulum into the cytosol. RyR2s exist as macromolecular complexes that are regulated via binding of Ca2+ and protein phosphorylation/dephosphorylation. The present study examined the association of endogenous CaMKII (calcium/calmodulin-dependent protein kinase II) with the RyR2 complex and whether this enzyme could modulate RyR2 function in isolated rabbit ventricular myocardium. Endogenous phosphorylation of RyR2 was verified using phosphorylation site-specific antibodies. Co-immunoprecipitation studies established that RyR2 was physically associated with CaMKIIδ. Quantitative assessment of RyR2 protein was performed by [3H]ryanodine binding to RyR2 immunoprecipitates. Parallel kinase assays allowed the endogenous CaMKII activity associated with these immunoprecipitates to be expressed relative to the amount of RyR2. The activity of RyR2 in isolated cardiac myocytes was measured in two ways: (i) RyR2-mediated Ca2+ release (Ca2+ sparks) using confocal microscopy and (ii) Ca2+-sensitive [3H]ryanodine binding. These studies were performed in the presence and absence of AIP (autocamtide-2-related inhibitory peptide), a highly specific inhibitor of CaMKII. At 1 µM AIP Ca2+ spark duration, frequency and width were decreased significantly. Similarly, 1 µM AIP decreased [3H]ryanodine binding. At 5 µM AIP, a more profound inhibition of Ca2+ sparks and a decrease in [3H]ryanodine binding was observed. Separate measurements showed that AIP (1–5 µM) did not affect sarcoplasmic reticulum Ca2+-ATPase-mediated Ca2+ uptake. These results suggest the existence of an endogenous CaMKIIδ that associates directly with RyR2 and specifically modulates RyR2 activity.
N-Formyl peptide receptor subtypes in human neutrophils activate l-plastin phosphorylation through different signal transduction intermediates
Authors: PACLET Marie-Hélène , DAVIS Clare , KOTSONIS Peter , GODOVAC-ZIMMERMANN Jasminka , SEGAL Anthony W. , DEKKER Lodewijk V.
Published: January 2004

We investigated the coupling of the fMLP (N-formyl-l-methionyl-l-leucyl-l-phenylalanine; ‘chemotactic peptide’) receptor with phosphorylation of the actin-binding protein l-plastin in neutrophils. Using two-dimensional IEF (isoelectric focusing)/PAGE and MALDI–TOF (matrix-assisted laser desorption ionization–time-of-flight)-MS, l-plastin was identified as a major phosphoprotein in fMLP-stimulated neutrophils whose phosphorylation was dependent on phosphoinositide 3-kinase, PLD (phospholipase D) and PKC (protein kinase C) activity. Two fMLP receptor subtypes were identified in neutrophils, characterized by a distinct sensitivity to fMLP and antagonistic peptides. Both receptor subtypes induced the phosphorylation of l-plastin. l-plastin phosphorylation induced by low-affinity fMLP receptors involves an action of phosphoinositide 3-kinase, PLD and PKC isotypes. In contrast, none of these intermediates are utilized by high-affinity fMLP receptors in the phosphorylation of l-plastin. However, the PKC inhibitor Ro-31-8220 inhibits l-plastin phosphorylation induced by the high-affinity fMLP receptor. Thus, an as yet unknown Ro-31-8220-sensitive kinase regulates l-plastin phosphorylation in response to the high-affinity fMLP receptor. The results suggest a model in which receptor subtypes induce a similar endpoint event through different signal-transduction intermediates. This may be relevant in the context of cell migration in which one receptor subpopulation may become desensitized in a concentration gradient of chemoattractant.
Activator protein-2gamma (AP-2gamma) expression is specifically induced by oestrogens through binding of the oestrogen receptor to a canonical element within the 5′-untranslated region
Authors: ORSO Francesca , COTTONE Erika , HASLETON Mark D. , IBBITT J. Claire , SISMONDI Piero , HURST Helen C. , DE BORTOLI Michele
Published: January 2004

The activator protein 2 (AP-2) transcription factors are essential proteins for oestrogenic repression of the ERBB2 proto-oncogene in breast cancer cells. In the present study, we have examined the possible oestrogenic regulation of AP-2 genes themselves in breast-tumour-derived lines. As early as 1 h after oestrogen treatment, AP-2γ mRNA was markedly increased, whereas AP-2α was down-regulated, but with slower kinetics, and AP-2β was not affected at all. Addition of anti-oestrogens ablated these effects. Modulation of the protein levels corresponded to changes in the transcript levels, thus suggesting that in oestrogen-treated cells, an inversion of the balance between AP-2α and AP-2γ isoforms occurs. The 5´-untranslated region (5´-UTR) of the human AP-2γ gene contains one consensus and one degenerate oestrogen-responsive element (ERE). Reporter constructs carrying the AP-2γ promoter and the 5´-UTR were up-regulated by oestrogens in transient transfection assays. Deletion of the most conserved (but not of the degenerate) ERE from reporter constructs abrogated the oestrogenic response, although both ERE-containing segments were footprinted in DNaseI protection assays. In vitro binding assays demonstrated the ability of oestrogen receptor α (ERα) to bind to this site, and chromatin immunoprecipitation analysis of the endogenous gene showed that ERα occupies this region in response to oestrogens. We conclude that AP-2γ is a primary oestrogen-responsive gene and suggest that AP-2 proteins may mediate some oestrogenic responses.
Interferon regulatory factor-2 drives megakaryocytic differentiation
Authors: STELLACCI Emilia , TESTA Ugo , PETRUCCI Eleonora , BENEDETTI Eleonora , ORSATTI Roberto , FECCIA Tiziana , STAFSNES Marit , COCCIA Eliana M. , MARZIALI Giovanna , BATTISTINI Angela
Published: January 2004

IRFs [IFN (interferon) regulatory factors] constitute a family of transcription factors involved in IFN signalling and in the development and differentiation of the immune system. IRF-2 has generally been described as an antagonist of IRF-1-mediated transcription of IFN and IFN-inducible genes; however, it has been recently identified as a transcriptional activator of some genes, such as those encoding histone H4, VCAM-1 (vascular cell adhesion molecule-1) and Fas ligand. Biologically, IRF-2 plays an important role in cell growth regulation and has been shown to be a potential oncogene. Studies in knock-out mice have also implicated IRF-2 in the differentiation and functionality of haematopoietic cells. Here we show that IRF-2 expression in a myeloid progenitor cell line leads to reprogramming of these cells towards the megakaryocytic lineage and enables them to respond to thrombopoietin, as assessed by cell morphology and expression of specific differentiation markers. Up-regulation of transcription factors involved in the development of the megakaryocytic lineage, such as GATA-1, GATA-2, FOG-1 (friend of GATA-1) and NF-E2 (nuclear factor-erythroid-2), and transcriptional stimulation of the thrombopoietin receptor were also demonstrated. Our results provide evidence for a key role for IRF-2 in the induction of a programme of megakaryocytic differentiation, and reveal a remarkable functional diversity of this transcription factor in the regulation of cellular responses.