Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacterially expressed, dual specificity phosphatase VHR protein induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes, albeit with slower kinetics than that observed in progesterone- or insulin-induced maturation. A mutant VHR protein missing an essential cysteine residue for its in vitro phosphatase activity completely lacked activity in injected oocytes. VHR injection done in conjunction with progesterone or insulin treatment resulted in highly synergized GVBD responses showing much faster kinetics than that produced by VHR or either hormone alone. The delayed kinetics of VHR-induced GVBD and the synergistic responses obtained in the presence of hormones suggested that this protein may be promoting G2/M transition by weakly mimicking the action of cdc25, the dual specificity phosphatase that physiologically activates the maturation promotion factor. Various experimental observations are consistent with such a role for the injected VHR in oocytes: 1) as opposed to hormone-treated oocytes, histone H1 kinase activation is not preceded by MAPK activation in the process of GVBD in VHR-injected oocytes; 2) incubation of purified VHR with highly concentrated cell-free extracts of untreated oocytes resulted in activation of histone H1 kinase activity in the lysates; 3) coinjection of VHR with activated Ras proteins resulted in synergized responses, faster than those produced by either protein alone; 4) coinjection of VHR with the purified amino-terminal SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (which blocks insulin-induced GVBD) does not affect VHR-induced maturation. The biological actions of VHR in oocytes clearly distinguish it from other dual specificity phosphatases, which have shown inhibitory effects when tested in oocytes. We speculate that VHR may represent a dual specificity phosphatase responsible for activation of cdk-cyclin complex(es) at a still undetermined stage of the cell cycle.
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PMID:Human dual specificity phosphatase VHR activates maturation promotion factor and triggers meiotic maturation in Xenopus oocytes. 777 84

An expression and purification method was developed to obtain the recombinant human dual-specific protein tyrosine phosphatase (PTPase) VHR in quantities suitable for both kinetic studies and crystallization. Physical characterization of the homogeneous recombinant protein verified the mass to be 20,500 +/- 100 by matrix-assisted laser desorption mass spectrometry, confirmed the anticipated NH2-terminal amino acid sequence and demonstrated that the protein exists as a monomer. Conditions were developed to obtain crystals which were suitable for x-ray structure determination. Using synthetic diphosphorylated peptides corresponding to MAP177-189 (mitogen-activated protein) kinase (DHTG-FLpTEpYVATR), an assay was devised which permitted the determination of the rate constants for dephosphorylation of the diphosphorylated peptide on threonine and tyrosine residues. The diphosphorylated peptides are preferred over the singly phosphorylated on tyrosine by 3-8-fold. The apparent second-order rate constant kcat/Km for dephosphorylation of phosphotyrosine on DHTGFLpTEpYVATR was 32,000 M-1 S-1 while dephosphorylation of phosphothreonine was 14 M-1 S-1 (pH 6). The reaction of DHTGFLpTEpYVATR with VHR is ordered, with rapid dephosphorylation on tyrosine occurring first followed by slow dephosphorylation on threonine. Similar results were obtained with F(NLe)(N-Le)pTPpYVVTR, a peptide corresponding to a MAP kinase-like protein (JNK1(180-189)) which is involved in the stress response signaling pathway.
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PMID:The purification and characterization of a human dual-specific protein tyrosine phosphatase. 787 21

Vaccinia phosphatase VH-1 and its mammalian counterparts, including protein-tyrosine phosphatases (PTPase) CL100 and VHR, constitute a novel subfamily of protein-tyrosine phosphatases that exhibits dual substrate specificity for phosphotyrosine- and phosphoserine/threonine-containing substrates. The expression of human VH-1-like PTPase CL100 is rapidly inducible by mitogen stimulation and oxidative stress, suggesting that this gene is transcriptionally regulated. In order to study the mechanism underlying this transcriptional regulation, we isolated the first human gene of this subfamily, the CL100 gene, and characterized its promoter. The gene consists of four exons intervened by three short introns 400-500 base pairs in length. Analysis of the protein sequence encoded by each exon revealed that there is a second region of similarity between CL100 protein and cdc25 in addition to the PTPase catalytic domain. Promoter analysis of the CL100 gene indicates that an 800-base pair region flanking the transcriptional initiation site is sufficient to confer a transcriptional response to serum and 12-O-tetradecanoylphorbol-13-acetate stimulation. The CL100 gene is expressed in numerous tissues, including nonmitotic cells in the brain. Within the brain, CL100 mRNA is localized in discrete neuronal populations, suggesting that this PTPase is likely to play a key role in neurotransmission as well as in mitotic signaling. Finally, although extracellular signal-regulated kinase has recently been shown to act as substrate for CL100 in vitro, we find no clear correspondence between the distribution of extracellular signal-regulated kinase and CL100 mRNA in the brain. The potential significance of a second cdc25 homology domain of CL100 is discussed.
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PMID:Isolation and characterization of a human dual specificity protein-tyrosine phosphatase gene. 810 4

A cDNA encoding a novel human extracellularly-regulated kinase (ERK) phosphatase, designated B59, was isolated from a B5/589 human mammary epithelial cell cDNA library. The 1104 nucleotide open reading frame encodes 368 amino acids including the highly conserved catalytic site sequence of protein phosphotyrosine phosphatases (PTPs), VXVHCXXGXXR, at amino acid position 276-287. The predicted 70 amino acid stretch surrounding the HC motif shares significant sequence identity with other human dual specificity PTPs (dsPTPs), including the known ERK PTPs CL100, PAC1, B23, as well as the dsPTPs VH-1 and VHR. B59 protein synthesized in vitro in a rabbit reticulocyte lysate dephosphorylated rat ERK1 and ERK2 proteins whose phosphorylation had been stimulated by v-mos kinase added to the lysate. Ectopic expression of B59 in NIH3T3 fibroblasts inhibited the induction of an oncogene-responsive promoter by the dominant-activating raf mutant, raf-BXB. Moreover, cotransfection of NIH3T3 cells with B59 inhibited morphological transformation by H-ras and v-raf oncogenes. These results suggest that B59 suppresses the transforming activity of H-ras or v-raf oncogenes through ERK dephosphorylation and inactivation.
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PMID:A novel human ERK phosphatase regulates H-ras and v-raf signal transduction. 917 61

The mammalian dual-specificity protein-tyrosine phosphatase VHR (for VH1-related) has been identified as a novel regulator of extracellular regulated kinases (ERKs). To identify potential cellular substrates of VHR, covalently immobilized mutant VHR protein was employed as an affinity trap. A tyrosine-phosphorylated protein(s) of approximately 42 kDa was specifically adsorbed by the affinity column and identified as ERK1 and ERK2. Subsequent kinetic analyses and transfection studies demonstrated that VHR specifically dephosphorylates and inactivates ERK1 and ERK2 in vitro and in vivo. Only the native structure of phosphorylated ERK was recognized by VHR and was inactivated with a second-order rate constant of 40,000 M-1 s-1. VHR was found to dephosphorylate endogenous ERK, but not p38 and JNK. Immunodepletion of endogenous VHR eliminated the dephosphorylation of cellular ERK. Transfection studies in COS-1 cells demonstrated that in vivo phosphorylation of epidermal growth factor-stimulated ERK depended on VHR protein levels. Overexpression above endogenous levels of VHR led to accelerated ERK inactivation, but did not alter the normal activation of ERK. Unique among reported mitogen activated protein kinase phosphatases, VHR is constitutively expressed, localized to the nucleus, and tyrosine-specific. This study is the first to report the identification of authentic substrates of dual-specificity phosphatases utilizing affinity absorbents and is the first to identify a nuclear, constitutively expressed, and tyrosine-specific ERK phosphatase. The data strongly suggest that VHR is responsible for the rapid inactivation of ERK following stimulation and for its repression in quiescent cells.
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PMID:Extracellular regulated kinases (ERK) 1 and ERK2 are authentic substrates for the dual-specificity protein-tyrosine phosphatase VHR. A novel role in down-regulating the ERK pathway. 1022 87

Five putative dual specificity protein phosphatases (DSPs), designated LMW-DSP1, -DSP4, -DSP6, -DSP10, and -DSP11, were cloned with a combination of RT-PCR and cDNA library screening strategies. Sequencing analysis revealed that all lacked the cdc25 homology domain that is conserved in most known DSPs/MAP kinase phosphatases (MKPs). LMW-DSP1 exhibited the highest similarity to plant DSPs. LMW-DSP4 exhibited the highest similarity to human YVH1 and rat GKAP, but its C-terminal region was much shorter than that of the human and rat clones. LMW-DSP6 was found to be identical to recently cloned TMDP, and LMW-DSP11 seemed to be a mouse ortholog of human VHR. LMW-DSP10 was found to have a DSP catalytic-like domain, but the critical cysteine residue for catalytic activity was missing. Recombinant LMW-DSP1, -DSP6, and -DSP11 exhibited obvious and strong activity against an artificial low molecular substrate, para-nitrophenyl phosphate (pNPP). Recombinant LMW-DSP4 exhibited slight but significant activity, whereas no activity was detected for LMW-DSP10. The phosphatase activity of the recombinant LMW-DSPs was inhibited by orthovanadate but not sodium fluoride. However, none of the DSPs could dephosphorylate MAP kinases such as ERK1, p38, and SAPK/JNK in transiently transfected COS7 cells under the conditions used. Northern blot analysis revealed that LMW-DSP1, -DSP6, -DSP10, and -DSP11 were specifically expressed in testis, while LMW-DSP4 was broadly expressed. The testis-specific expression and apparent absence of dephosphorylation action on MAP kinases suggest that LMW-DSP1, -DSP6, -DSP10, and -DSP11 play specific roles in testis. Taken together, it is conceivable that a distinct class of low molecular mass DSPs is present and plays a role in dephosphorylating unknown molecules other than MAP kinases.
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PMID:A growing family of dual specificity phosphatases with low molecular masses. 1143 89

Human VHR (vaccinia H1 related phosphatase) is a member of the dual-specificity phosphatases (DSPs) that often act on bisphosphorylated protein substrates. Unlike most DSPs, VHR displays a strong preference for dephosphorylating phosphotyrosine residues over phosphothreonine residues. Here we describe the 2.75 A crystal structure of the C124S inactive VHR mutant in complex with a bisphosphorylated peptide corresponding to the MAP kinase activation lip. This structure and subsequent biochemical studies revealed the basis for the strong preference for hydrolyzing phosphotyrosine within bisphosphorylated substrates containing -pTXpY-. In the structure, the two phospho residues are oriented into distinct pockets; the phosphotyrosine is bound in the exposed yet deep active site cleft while the phosphothreonine is loosely tethered into a nearby basic pocket containing Arg(158). As this structure is the first substrate-enzyme complex reported for the DSP family of enzymes, these results provide the first glimpse into how DSPs bind their protein substrates.
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PMID:Structural basis for the recognition of a bisphosphorylated MAP kinase peptide by human VHR protein Phosphatase. 1186 39

The JNK group (for c-Jun N-terminal kinase) of mitogen-activated protein kinases (MAP kinases) is activated in cells in response to environmental stress and cytokines. Activation of JNK is the result of dual phosphorylation by specific upstream kinases which phosphorylate the TxY motif. Much less is known concerning the down-regulation by protein phosphatases. Here, we demonstrate that the tyrosine-specific and constitutively-expressed phosphatase VHR (for VH1-Related) down-regulates the JNK signaling pathway at the level of JNK dephosphorylation. VHR was shown to efficiently dephosphorylate JNK and to form a tight complex with activated JNK when the catalytically-inactive C124S VHR mutant was employed as an in vivo substrate trap. Utilizing an in vitro assay, the transcription factor c-Jun specifically inhibited the ability of VHR to dephosphorylate JNK, likely by sterically blocking access to the phosphorylation sites when JNK and c-Jun form a complex. c-Jun has no effect on the ability of VHR to inactivate the ERK MAP kinases or to hydrolyze artificial substrates. The c-Jun inhibition results are discussed in terms of the resistant-nature of JNK dephosphorylation in cellular extracts and in terms of a general model in which VHR may be a general MAP kinase phosphatase whose specificity and activity are dictated by the presence of MAP kinase-associated proteins that inhibit dephosphorylation.
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PMID:Dual-specificity protein tyrosine phosphatase VHR down-regulates c-Jun N-terminal kinase (JNK). 1197 Nov 92

The extracellular signal-regulated protein kinase 2 (ERK2) is the founding member of a family of mitogen-activated protein kinases (MAPKs) that are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. The MAPKs are unique among the Ser/Thr protein kinases in that they require both Thr and Tyr phosphorylation for full activation. The dual phosphorylation of Thr-183 and Tyr-185 in ERK2 is catalyzed by MAPK/ERK kinase 1 (MEK1). However, the identity and relative activity of protein phosphatases that inactivate ERK2 are less well established. In this study, we performed a kinetic analysis of ERK2 dephosphorylation by protein phosphatases using a continuous spectrophotometric enzyme-coupled assay that measures the inorganic phosphate produced in the reaction. Eleven different protein phosphatases, many previously suggested to be involved in ERK2 regulation, were compared, including tyrosine-specific phosphatases (PTP1B, CD45, and HePTP), dual specificity MAPK phosphatases (VHR, MKP3, and MKP5), and Ser/Thr protein phosphatases (PP1, PP2A, PP2B, PP2C alpha, and lambda PP). The results provide biochemical evidence that protein phosphatases display exquisite specificity in their substrate recognition and implicate HePTP, MKP3, and PP2A as ERK2 phosphatases. The fact that ERK2 inactivation could be carried out by multiple specific phosphatases shows that signals can be integrated into the pathway at the phosphatase level to determine the cellular response to external stimuli. Important insights into the roles of various protein phosphatases in ERK2 kinase signaling are obtained, and further analysis of the mechanism by which different protein phosphatases recognize and inactivate MAPKs will increase our understanding of how this kinase family is regulated.
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PMID:The specificity of extracellular signal-regulated kinase 2 dephosphorylation by protein phosphatases. 1208 7

We have identified a novel dual-specificity phosphatase (DSP), called LDP-2 (low-molecular-mass DSP-2), composed of 220 amino acid residues showing high sequence homology to VHR and LDP-1/TMDP, which belong to a family of DSPs with low molecular masses. The LDP-2 gene is ubiquitously expressed, and LDP-2 is localized in the cytoplasm. The main structural feature of LDP-2 is that the serine-156 residue located in the common active site sequence motif, HCXXGXXRS, for DSP is naturally substituted with an alanine residue. The recombinant LDP-2 protein showed extremely low phosphatase activity towards p-nitrophenyl phosphate (pNPP). Back-mutation of Ala-156 in LDP-2 to a serine (A156S mutation) conferred significant phosphatase activity towards pNPP. However, both LDP-2 and LDP-2 (A156S) exhibited substantial phosphatase activities towards both phospho-seryl/threonyl and -tyrosyl residues of myelin basic protein, with similar specific activities. Ala-156 of LDP-2 might be crucially involved in the recognition of a physiological substrate. We analyzed the effect of VHR and LDP-2 on mitogen-activated protein kinases (MAPKs) in vivo. We first found that VHR inhibits the activation of p38 as well as ERK and JNK, with similar efficiency. Under the conditions used, LDP-2 specifically suppressed JNK activation.
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PMID:A novel low-molecular-mass dual-specificity phosphatase, LDP-2, with a naturally occurring substitution that affects substrate specificity. 1220 17


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