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

Increased activity of the Na(+)-H+ exchanger (NHE-1 isoform) has been observed in cells and tissues from hypertensive humans and animals, including the spontaneously hypertensive rat (SHR). No mutation in NHE-1 DNA sequence or alteration in NHE-1 mRNA and protein expression has been demonstrated in hypertension, indicating that alterations in proteins that regulate NHE-1 activity are responsible for increased activity. The recent finding that NHE-1 phosphorylation in SHR vascular smooth muscle cells (VSMCs) was greater than in Wistar-Kyoto rat (WKY) VSMCs suggested that NHE-1 kinases may represent an abnormal regulatory pathway present in hypertension. To define NHE-1 kinases altered in the hypertensive phenotype. We measured NHE-1 kinase activity by an in-gel-kinase assay using a recombinant glutathione S-transferase NHE-1 fusion protein as a substrate. At least 7 NHE-1 kinases (42 to 90 kD) were present in VSMCs. We studied a 90-kD kinase because it was the major NHE-1 kinase and exhibited differences between SHR and WKY. Comparison of 90-kD kinase activity revealed that SHR VSMCs had increased activity in growth-arrested cells and in cells stimulated by angiotensin II (100 nmol/L for 5 minutes). Activation of the 90-kD kinase by angiotensin II was Ca2+ dependent, PKC independent, and partially dependent on the mitogen-activated protein kinase pathway. These findings indicate that increased activity of a 90-kD NHE-1 kinase is a characteristic of SHR VSMCs in culture and suggest that alterations in the 90-kD NHE-1 kinase and/or proteins that regulate its activity may be a pathogenic component in hypertension in the SHR.
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PMID:A 90-kD Na(+)-H+ exchanger kinase has increased activity in spontaneously hypertensive rat vascular smooth muscle cells. 918 Jun 27

We identified and cloned a homolog of mammalian mitogen-activated protein kinase-activated protein kinase (MAPKAPK)-2 and -3 from sea urchin, Hemicentrotus pulcherrimus. The obtained cDNA clone was composed of 350 amino acid residues which contain MAPK phosphorylation sites and the bipartite nuclear localization signal sites in its C-terminal domain. The clone showed 65.4 and 66.7% amino acid residue identity to human MAPKAPK-2 and -3, respectively. Phylogenetic analysis revealed that the homolog can be classified into a distinct group of MAPKAPK and, therefore, the identified homolog was designated as MAPKAPK-4. Biochemical characterization was performed using recombinant glutathione S-transferase (GST)-MAPKAPK-4 fusion protein. The protein kinase activity of GST-MAPKAPK-4 was activated by MAPK and this enabled the kinase to phosphorylate both glycogen synthase N-terminal peptide and the regulatory light chain of myosin II in vitro. Northern blot analysis showed that MAPKAPK-4 was expressed throughout the development of sea urchin embryos. These observations suggest that MAPKAPK-4 may play an important role in the regulation of myosin II activity during the development of sea urchin.
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PMID:Identification of MAPKAPK homolog (MAPKAPK-4) as a myosin II regulatory light-chain kinase in sea urchin egg extracts. 921 Jun 46

An activated form of the human cytokine-suppressive anti-inflammatory drug-binding protein 2 (CSBP2) kinase was expressed in Spodoptera frugiperda (SF9) cells from a baculovirus vector. To maximize expression and to facilitate purification of the recombinant protein, CSBP2 kinase was expressed as a carboxy-terminal fusion protein to glutathione S-transferase (GST). Under optimal conditions, 2-3 mg of GST-CSBP2 could be obtained per liter of infected cell culture. The fusion protein was easily purified from the soluble fraction of the total cell lysate under nondenaturing conditions by using a glutathione-Sepharose 4B affinity resin. As expected, the purified GST-CSBP2 fusion protein was approximately 68 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and reacted with antibodies directed toward either the GST or the CSBP amino terminus. To obtain activated CSBP2, SF9 cells were coinfected with two recombinant baculovirus vectors: one that directed the synthesis of the GST-CSBP2 fusion protein and a second vector that directed the synthesis of a constitutively active form of the CSBP activating kinase, MKK3. Coexpression of GST-CSBP2 kinase with the MKK3 activator increased GST-CSBP2 activity 8- to 10-fold based on the ability of GST-CSBP2 to phosphorylate the substrate, myelin basic protein (MBP), and the ATF2 transcription factor, in vitro. Moreover, activated GST-CSBP2 was capable of activating a bacterially derived mitogen-activated protein kinase-activating protein kinase 2 in vitro. The activity of insect-derived GST-CSBP2 was also inhibited by the CSBP inhibitor, SB202190. We anticipate that the preparation and purification techniques described in this study will facilitate further biochemical characterization of this kinase.
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PMID:Expression, purification, and characterization of an activated cytokine-suppressive anti-inflammatory drug-binding protein 2 (CSBP2) kinase from baculovirus-infected insect cells. 922 23

Monoclonal antibody MPM-2 recognizes a large family of mitotic phosphoproteins in a phosphorylation-dependent manner. The antigenic phosphoepitope, designated the MPM-2 epitope, putatively consists of hydrophobic residue-Thr/Ser-Pro-hydrophobic residue-uncharged/basic residue. In this study, we addressed whether this sequence motif contains all the information necessary for recognition and phosphorylation by the kinase that phosphorylates most MPM-2 antigens. A fusion protein between glutathione S-transferase and a 19-residue peptide that contained two representative MPM-2 epitope sequences overlapping with two potential MAP kinase phosphorylation sites was constructed. Both the MPM-2 epitope sequences in the fusion protein (GST-MPM2) were phosphorylated by Xenopus egg extract, making the fusion protein MPM-2 reactive. However, while MAP kinase phosphorylated both the MPM-2 epitope sequences, neither ME kinase-H, a good candidate for a major MPM-2 epitope kinase, nor mitotic cdc2 kinase, which is known to phosphorylate certain MPM-2 antigens in vitro, phosphorylated GST-MPM2 to any significant extent. Furthermore, depletion of MAP kinase activity removed most, if not all, of the GST-MPM2 phosphorylating activity from crude Xenopus egg extracts. These results suggest that additional or different structural information than that provided by the deduced MPM-2 epitope sequence is required for recognition and phosphorylation by ME kinase-H or other major MPM-2 epitope kinases. They also offer a valid explanation for selective phosphorylation of certain MPM-2 antigens by MAP kinase as well as selective recognition of certain phosphorylated MAP kinase substrates by MPM-2.
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PMID:MPM-2 epitope sequence is not sufficient for recognition and phosphorylation by ME kinase-H. 930 47

Ste5 is a scaffold for the mitogen-activated protein kinase (MAPK) cascade components in a yeast pheromone response pathway. Ste5 also associates with Ste4, the beta subunit of a heterotrimeric guanine nucleotide-binding protein, potentially linking receptor activation to stimulation of the MAPK cascade. A RING-H2 motif at the Ste5 amino terminus is apparently essential for function because Ste5(C177S) and Ste5(C177A C180A) mutants did not rescue the mating defect of a ste5Delta cell. In vitro Ste5(C177A C180A) bound each component of the MAPK cascade, but not Ste4. Unlike wild-type Ste5, the mutant did not appear to oligomerize; however, when fused to a heterologous dimerization domain (glutathione S-transferase), the chimeric protein restored mating in an ste5Delta cell and an ste4Delta ste5Delta double mutant. Thus, the RING-H2 domain mediates Ste4-Ste5 interaction, which is a prerequisite for Ste5-Ste5 self-association and signaling.
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PMID:Ste5 RING-H2 domain: role in Ste4-promoted oligomerization for yeast pheromone signaling. 931 11

The transcription factor Elk-1 is a component of ternary complex factor and regulates gene expression in response to a wide variety of extracellular stimuli. Phosphorylation of the C-terminal domain of Elk-1, especially at serine 383, is important for its transactivation activity. Recently mitogen-activated protein kinases, such as extracellular signal-regulated kinase, stress-activated protein kinase, and p38 mitogen-activated protein kinase have been demonstrated to be Elk-1 kinases. However, negative regulators of Elk-1, such as protein phosphatases, still remain to be identified. Here we report that COS cell lysates were able to dephosphorylate an extracellular signal-regulated kinase-phosphorylated glutathione S-transferase-Elkc fusion protein, including serine 383. The phosphatase activity was inhibited by cyclosporin A (a calcineurin inhibitor) but not by okadaic acid (a PP1 and PP2A inhibitor). Purified calcineurin also could efficiently dephosphorylate glutathione S-transferase-Elkc in vitro. Pretreatment of COS cells with cyclosporin A significantly enhanced epidermal growth factor-induced serine 383 Elk-1 phosphorylation whereas ionomycin inhibited the Elk-1 phosphorylation. These data provide both in vitro and in vivo evidence that calcineurin is the major Elk-1 phosphatase and plays a critical role in Elk-1 regulation. The identification of calcineurin as the major Elk-1 phosphatase may provide a mechanism for Elk-1 regulation by Ca2+ signals as well as a possible biochemical basis for the neurotoxicity and nephrotoxicity of the immunosuppressant drug cyclosporin A.
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PMID:The calcium/calmodulin-dependent protein phosphatase calcineurin is the major Elk-1 phosphatase. 936 95

Recently, TAP42 was isolated as a high copy suppressor of sit4-, a yeast phosphatase related to protein phosphatase 2A (PP2A). TAP42 is related to the murine alpha4 protein, which was discovered independently by its association with Ig-alpha in the B cell receptor complex. Herein we show that a glutathione S-transferase (GST)-alpha4 fusion protein bound the catalytic subunit (C) of human PP2A from monomeric or multimeric preparations of PP2A in a "pull-down" assay. In an overlay assay, the GST-alpha4 protein bound to the phosphorylated and unphosphorylated forms of C that were separated in two-dimensional gels and immobilized on filters. The results show direct and exclusive binding of alpha4 to C. This is unusual because all known regulatory B subunits, or tumor virus antigens, bind stably only to the AC dimer of PP2A. The alpha4-C form of PP2A had an increased activity ratio compared with the AC form of PP2A when myelin basic protein phosphorylated by mitogen-activated protein kinase and phosphorylase a were used as substrates. Recombinant alpha4 cleaved from GST was phosphorylated by p56(lck) tyrosine kinase and protein kinase C. A FLAG-tagged alpha4 expressed in COS7 cells was recovered as a protein containing phosphoserine and coimmunoprecipitated with the C but not the A subunit of PP2A. Treatment of cells with rapamycin prevented the association of PP2A with FLAG-alpha4. The results reveal a novel heterodimer alpha4-C form of PP2A that may be involved in rapamycin-sensitive signaling pathways in mammalian cells.
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PMID:B cell receptor-associated protein alpha4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A. 938 Jun 85

Growth hormone (GH) signaling requires activation of the GH receptor (GHR)-associated tyrosine kinase, JAK2. JAK2 activation by GH is believed to facilitate initiation of various pathways including the Ras, mitogen-activated protein kinase, STAT, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase systems. In the present study, we explore the biochemical and functional involvement of the Src homology 2 (SH2)-containing protein-tyrosine phosphatase, SHP-2, in GH signaling. GH stimulation of murine NIH 3T3-F442A fibroblasts, cells that homologously express GHRs, resulted in tyrosine phosphorylation of SHP-2. As assessed specifically by anti-SHP-2 coimmunoprecipitation and by affinity precipitation with a glutathione S-transferase fusion protein incorporating the SH2 domains of SHP-2, GH induced formation of a complex of tyrosine phosphoproteins including SHP-2, GHR, JAK2, and a glycoprotein with properties consistent with being a SIRP-alpha-like molecule. A reciprocal binding assay using IM-9 cells as a source of SHP-1 and SHP-2 revealed specific association of SHP-2 (but not SHP-1) with a glutathione S-transferase fusion incorporating GHR cytoplasmic domain residues 485-620, but only if the fusion was first rendered tyrosine-phosphorylated. GH-dependent tyrosine phosphorylation of SHP-2 was also observed in murine 32D cells (which lack IRS-1 and -2) stably transfected with the GHR. Further, GH-dependent anti-SHP-2 coimmunoprecipitation of the Grb2 adapter protein was detected in both 3T3-F442A and 32D-rGHR cells, indicating that biochemical involvement of SHP-2 in GH signaling may not require IRS-1 or -2. Finally, GH-induced transactivation of a c-Fos enhancer-driven luciferase reporter in GHR- and JAK2-transfected COS-7 cells was significantly reduced when a catalytically inactive SHP-2 mutant (but not wild-type SHP-2) was coexpressed; in contrast, expression of a catalytically inactive SHP-1 mutant allowed modestly enhanced GH-induced transactivation of the reporter in comparison with that found with expression of wild-type SHP-1. Collectively, these biochemical and functional data imply a positive role for SHP-2 in GH signaling.
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PMID:Involvement of the Src homology 2-containing tyrosine phosphatase SHP-2 in growth hormone signaling. 944 80

By interference of the yeast pheromone mitogen-activated protein kinase (MAPK) pathway with an alfalfa cDNA expression library, we have isolated the MP2C gene encoding a functional protein phosphatase type 2C. Epistasis analysis in yeast indicated that the molecular target of the MP2C phosphatase is Ste11, a MAPK kinase kinase that is a central regulator of the pheromone and osmosensing pathways. In plants, MP2C functions as a negative regulator of the stress-activated MAPK (SAMK) pathway that is activated by cold, drought, touch, and wounding. Although activation of the SAMK pathway occurs by a posttranslational mechanism, de novo transcription and translation of protein factor(s) are necessary for its inactivation. MP2C is likely to be this or one of these factors, because wound-induced activation of SAMK is followed by MP2C gene expression and recombinant glutathione S-transferase-MP2C is able to inactivate extracts containing wound-induced SAMK. Wound-induced MP2C expression is a transient event and correlates with the refractory period, i.e., the time when restimulation of the SAMK pathway is not possible by a second stimulation. These data suggest that MP2C is part of a negative feedback mechanism that is responsible for resetting the SAMK cascade in plants.
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PMID:MP2C, a plant protein phosphatase 2C, functions as a negative regulator of mitogen-activated protein kinase pathways in yeast and plants. 946 21

SHPS-1 is a receptor-like protein that undergoes tyrosine phosphorylation and binds SHP-2, an SH2 domain-containing protein tyrosine phosphatase, in response to insulin and other mitogens. The overexpression of wild-type SHPS-1, but not of a mutant SHPS-1 in which all four tyrosine residues in its cytoplasmic region were mutated to phenylalanine, markedly enhanced insulin-induced activation of mitogen-activated protein kinase in Chinese hamster ovary cells that overexpress the human insulin receptor. Mutation of each tyrosine residue individually revealed that the major sites of tyrosine phosphorylation of SHPS-1 in response to insulin are Tyr449 and Tyr473. In addition, mutation of either Tyr449 or Tyr473 abolished the insulin-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Surface plasmon resonance analysis showed that glutathione S-transferase fusion proteins containing the NH2-terminal or COOH-terminal SH2 domains of SHP-2 bound preferentially to phosphotyrosyl peptides corresponding to the sequences surrounding Tyr449 or Tyr473, respectively, of SHPS-1. Furthermore, phosphotyrosyl peptides containing Tyr449 or Tyr473 were effective substrates for the phosphatase activity of recombinant SHP-2 in vitro. Together, these results suggest that insulin may induce phosphorylation of SHPS-1 at Tyr449 and Tyr473, to which SHP-2 then binds through its NH2-terminal and COOH-terminal SH2 domains, respectively. SHPS-1 may play a crucial role both in the recruitment of SHP-2 from the cytosol to a site near the plasma membrane and in increasing its catalytic activity, thereby positively regulating the RAS-mitogen-activated protein kinase signaling cascade in response to insulin.
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PMID:Roles of the complex formation of SHPS-1 with SHP-2 in insulin-stimulated mitogen-activated protein kinase activation. 953 15


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