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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)

Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases implicated in the control of cell proliferation and differentiation. We have found that activated p42mapk is a target for the phosphoepitope antibody MPM-2, a monoclonal antibody that recognizes a cell cycle-regulated phosphoepitope. We have determined that the MPM-2 antibody recognizes the regulatory region of p42mapk. Binding of the MPM-2 antibody to active p42mapk in vitro results in a decrease in p42mapk enzymatic activity. The MPM-2 phosphoepitope can be generated in vitro on bacterially expressed p42mapk by phosphorylation with either isoform of MAP kinase kinase (MKK), MKK1, or MKK2. Analysis of p42mapk proteins mutated in their regulatory sites shows that phosphorylated Thr-183 is essential for the binding of the MPM-2 antibody. MPM-2 binding to Thr-183 is affected by the amino acid present in the other regulatory site, Tyr-185. Substitution of Tyr-185 with phenylalanine results in strong binding of the MPM-2 antibody, whereas substitution with glutamic acid substantially diminishes MPM-2 antibody binding. The MPM-2 phosphoepitope antibody recognizes an amino acid domain incorporating the regulatory phosphothreonine on activated p42mapk in eggs during meiosis and in mammalian cultured cells during the G0 to G1 transition.
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PMID:The MPM-2 antibody inhibits mitogen-activated protein kinase activity by binding to an epitope containing phosphothreonine-183. 753 73

ERF (ETS2 Repressor Factor) is a novel member of the ets family of genes, which was isolated by virtue of its interaction with the ets binding site (EBS) within the ETS2 promoter. The 2.7 kb ubiquitously expressed ERF mRNA encodes a 548 amino acid phosphoprotein that exhibits strong transcriptional repressor activity on promoters that contain an EBS. The localization of the DNA-binding domain of the protein at the N-terminus and th repression domain at the C-terminus is reminiscent of the organization of ELK1-like members of the ets family; however, there is no significant homology between ERF and ELK1 or any other ets member outside the DNA-binding domain. The repressor activity of ERF can antagonize the activity of other ets genes that are known transcriptional activators. Furthermore, ERF can suppress the ets-dependent transforming activity of the gag-myb-ets fusion oncogene of ME26 virus. Although ERF protein levels remain constant throughout the cell cycle, the phosphorylation level of the protein is altered as a function of the cell cycle and after mitogenic stimulation. The ERF protein is also hyperphosphorylated in cells transformed by the activated Ha-ras and v-src genes and the transcription repressor activity of ERF is decreased after co-transfection with activated Ha-ras or the kinase domain of the c-Raf-1 gene, indicating that ERF activity is probably regulated by the ras/MAPK pathway. Consistent with the in vivo phosphorylation and inactivation by ras, ERF is efficiently phosphorylated in vitro by Erk2 and cdc2/cyclin B kinases, at sites similar to those detected in vivo. Furthermore, a single mutation at position 526 results in the loss of a specific phosphopeptide both in in vivo and in vitro (by Erk2) labeling. Substitution of Thr526 for glutamic acid also decreases the repression ability of ERF. Our data suggest a model in which modulation of ERF activity is involved in the transcriptional regulation of genes activated during entry into G1 phase. Obstruction of the ERF repressor function by the transactivating members of the ets family of genes (i.e.gag-myb-ets) may be essential for the control of genes involved in cell proliferation and may also underlie their tumorigenic effects.
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PMID:ERF: an ETS domain protein with strong transcriptional repressor activity, can suppress ets-associated tumorigenesis and is regulated by phosphorylation during cell cycle and mitogenic stimulation. 758 8

A recently described downstream target of mitogen-activated protein kinases (MAPKs) is the MAPK-activated protein (MAPKAP) kinase 2 which has been shown to be responsible for small heat shock protein phosphorylation. We have analyzed the mechanism of MAPKAP kinase 2 activation by MAPK phosphorylation using a recombinant MAPKAP kinase 2-fusion protein, p44MAPK and p38/40MAPK in vitro and using an epitope-tagged MAPKAP kinase 2 in heat-shocked NIH 3T3 cells. It is demonstrated that, in addition to the known phosphorylation of the threonine residue carboxyl-terminal to the catalytic domain, Thr-317, activation of MAPKAP kinase 2 in vitro and in vivo is dependent on phosphorylation of a second threonine residue, Thr-205, which is located within the catalytic domain and which is highly conserved in several protein kinases. Constitutive activation of MAPKAP kinase 2 is obtained by replacement of both of these threonine residues by glutamic acid. A constitutively active form of MAPKAP kinase 2 is also obtained by deletion of a carboxyl-terminal region containing Thr-317 and the A-helix motif or by replacing the conserved residues of the A-helix. These data suggest a dual mechanism of MAPKAP kinase 2 activation by phosphorylation of Thr-205 inside the catalytic domain and by phosphorylation of Thr-317 outside the catalytic domain involving an autoinhibitory A-helix motif.
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PMID:Constitutive activation of mitogen-activated protein kinase-activated protein kinase 2 by mutation of phosphorylation sites and an A-helix motif. 759 79

Treatment of PC12 cells with nerve growth factor (NGF) results in neural differentiation of the cells, inducing neurite outgrowth. Ras protein has been shown to play an essential role in this process. To examine whether or not the MAP kinase (MAPK) cascade mediates the NGF- and Ras-induced neural differentiation process, we injected PC12 cells with constitutive active forms of each components of the MAPK cascade. When a moderately active mutant of Xenopus MAPK kinase (S222E-MAPKK) in which Ser 222 was changed into glutamic acid was injected, the neurite outgrowth of PC12 cells occurred to some extent. Injection of an N-terminal truncated STE11 protein (delta N-STE11), a constitutively active form of STE11 which is a yeast MAPKK kinase, induced neurite outgrowth in PC12 cells. Furthermore, injection of thiophosphorylated MAPK, but not purified active MAPK, into PC12 cells resulted in neurite outgrowth. Thiophosphorylated MAPK was resistant to protein phosphatase 2A treatment, while purified active MAPK was inactivated by this treatment. All these results have suggested that sustained activation of MAPK is sufficient for PC12 cell differentiation. In accord with this, the delta N-STE11- or S222E- MAPKK-induced neurite outgrowth was inhibited by coinjection of CL-100 protein, a dual-specificity phosphatase that is capable of inactivating MAPK.
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PMID:Induction of neurite outgrowth by MAP kinase in PC12 cells. 762 41

Stathmin, a probable relay protein possibly integrating multiple intracellular regulatory signals [reviewed in Sobel (1991) Trends Biochem. Sci. 16, 301-305], was expressed in Escherichia coli at levels as high as 20% of total bacterial protein. Characterization of the purified recombinant protein revealed that it had biochemical properties very similar to those of the native protein. It is a good substrate for both cyclic AMP-dependent protein kinase (PKA) and p34cdc2, on the same four sites as the native eukaryotic protein. As shown by m.s., the difference in isoelectric points from the native protein is probably due to the absence of acetylation of the protein produced in bacteria. C.d. studies indicate that stathmin probably contains about 45% of its sequence in an alpha-helical conformation, as also predicted for the sequence between residues 47 and 124 by computer analysis. Replacement of Ser-63 by alanine by in vitro mutagenesis resulted in a ten times less efficient phosphorylation of stathmin by PKA which occurred solely on Ser-16, confirming that Ser-63 is the major target of this kinase. Replacement of Ser-25, the major site phosphorylated by mitogen-activated protein kinase in vitro and in vivo, by the charged amino acid glutamic acid reproduced, in conjunction with the phosphorylation of Ser-16 by PKA, the mobility shift on SDS/polyacrylamide gels induced by the phosphorylation of Ser-25. This result strongly suggests that glutamic acid in position 25 is able to mimic the putative interactions of phosphoserine-25 with phosphoserine-16, as well as the resulting conformational changes that are probably also related to the functional regulation of stathmin.
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PMID:Molecular characterization of human stathmin expressed in Escherichia coli: site-directed mutagenesis of two phosphorylatable serines (Ser-25 and Ser-63). 800 36

The signal transduction kinase MEK (mitogen-activated protein (MAP) or extracellular signal-regulated (Erk) kinase)-1 is activated via phosphorylation by MEKK (MEK kinase) and raf kinases. We show here that these two kinases phosphorylate rat MEK-1 exclusively on two serine codons, Ser218 and Ser222. Phosphorylation of MEK-1 on serines 218 and 222 is both necessary and sufficient for MEK-1 to be activated and able to phosphorylate MAP kinase. A mutant form of MEK-1 that replaces these two codons with alanine cannot be activated, and one that substitutes glutamic acid residues in place of these 2 serines is active independent of activation by phosphorylation. These sites of activation occur in a region of MEK-1 that is similar to sites of activating phosphorylation in several other serine/threonine kinases, suggesting that this region may represent a conserved "activating domain" of many kinases. MEKK and raf display differences in site preference between these two codons, with MEKK showing preference for the amino acid at codon 218 and raf phosphorylating each residue approximately equally. This site preference might result in differences in the temporal or subsequent substrate patterns of MEK activation that result from these two activation pathways.
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PMID:Identification of 2 serine residues of MEK-1 that are differentially phosphorylated during activation by raf and MEK kinase. 803 65

Many growth factors whose receptors are protein tyrosine kinases stimulate the MAP kinase pathway by activating first the GTP-binding protein Ras and then the protein kinase p74raf-1. p74raf-1 phosphorylates and activates MAP kinase kinase (MAPKK). To understand the mechanism of activation of MAPKK, we have identified Ser217 and Ser221 of MAPKK1 as the sites phosphorylated by p74raf-1. This represents the first characterization of sites phosphorylated by this proto-oncogene product. Ser217 and Ser221 lie in a region of the catalytic domain where the activating phosphorylation sites of several other protein kinases are located. Among MAPKK family members, this region is the most conserved, suggesting that all members of the family are activated by the phosphorylation of these sites. A 'kinase-dead' MAPKK1 mutant was phosphorylated at the same residues as the wild-type enzyme, establishing that both sites are phosphorylated directly by p74raf-1, and not by autophosphorylation. Only the diphosphorylated form of MAPKK1 (phosphorylated at both Ser217 and Ser221) was detected, even when the stoichiometry of phosphorylation by p74raf-1 was low, indicating that phosphorylation of one of these sites is rate limiting, phosphorylation of the second then occurring extremely rapidly. Ser217 and Ser221 were both phosphorylated in vivo within minutes when PC12 cells were stimulated with nerve growth factor. Analysis of MAPKK1 mutants in which either Ser217 or Ser221 were changed to glutamic acid, and the finding that inactivation of maximally activated MAPKK1 required the dephosphorylation of both serines, shows that phosphorylation of either residue is sufficient for maximal activation.
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PMID:Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. 815

Epidermal growth factor (EGF) receptor tyrosine kinase activity is down-regulated by a number of growth-modulating agents that activate protein kinase C and/or mitogen-activated protein (MAP) kinases. Although the mechanism is unclear, it has been hypothesized that phosphorylation of specific threonine residues leads to inhibition of the EGF receptor tyrosine kinase. Two sites phosphorylated on the EGF receptor in response to phorbol esters are possible mediators of this effect: threonine 654, the target of protein kinase C, and threonine 669, the target of MAP kinase and the major site of phosphorylation on the EGF receptor. In order to investigate the role of these residues in receptor regulation, we substituted glutamic acid to mimic the negative charge introduced by phosphorylation at these sites. The wild-type and mutant receptor cDNAs were then transfected into CHO cells that lack endogenous EGF receptor. The EGF binding properties of the mutant receptors were similar to those of the wild-type EGF receptors. EGF stimulated tyrosine kinase activity and DNA synthesis in cells expressing both mutant receptors, indicating that the mutant EGF receptors are biologically active. Treatment of cells with phorbol esters inhibited the high affinity EGF binding and tyrosine kinase activities of both mutant and wild-type EGF receptors. These results indicate that acidic residues at either the Thr-654 or Thr-669 site modulate but do not block EGF receptor signalling. Furthermore, this data demonstrates that the mutant EGF receptors are still a target for inhibition by phorbol esters. Thus, events other than phosphorylation of Thr-654 or Thr-669 appear to be required for receptor down-regulation by protein kinase C or MAP kinase.
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PMID:Role of threonine residues in regulation of the epidermal growth factor receptor by protein kinase C and mitogen-activated protein kinase. 839 47

The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.
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PMID:Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase. 855 85

Human heat shock transcription factor 1 (HSF1) is responsible for stress-induced transcription of heat shock protein genes. The activity of the HSF1 transcriptional activation domains is modulated by a separate regulatory domain, which confers repression at control temperature and heat inducibility. We show here that two specific proline-directed serine motifs are important for function of the regulatory domain: Mutation of these serines to alanine derepresses HSF1 activity at control temperature, and mutation to glutamic acid, mimicking a phosphorylated serine, results in normal repression at control temperature and normal heat shock inducibility. Tryptic mapping shows that these serines are the major phosphorylation sites of HSF1 at control temperature in vivo. Stimulation of the Raf/ERK pathway in vivo results in an increased level of phosphorylation of these major sites and the regulatory domain is an excellent substrate in vitro for the mitogen-activated MAPK/ERK. We conclude that phosphorylation of the regulatory domain of HSF1 decreases the activity of HSF1 at control temperature, and propose a mechanism for modification of HSF1 activity by growth control signals.
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PMID:Repression of human heat shock factor 1 activity at control temperature by phosphorylation. 894 18


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