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)

Tristetraprolin (TTP) is a potential transcription factor that contains three PPPPG repeats and two putative CCCH zinc fingers. TTP is encoded by the early response gene Zfp-36, which is highly expressed in response to growth factors and in several hematopoietic cell lines. In the present studies, we investigated the possibility that TTP is phosphorylated in intact cells. In NIH/3T3 cells that were made to overexpress TTP constitutively, we found that the protein was phosphorylated on serine residues, and that this phosphorylation was rapidly (within 10 min) stimulated by several mitogens. In cell-free assays, recombinant mouse TTP was a substrate for the mitogen-activated protein (MAP) kinase. By a combination of protease digestion experiments and site-directed mutagenesis strategies, we found that serine 220 was phosphorylated by p42 MAP kinase in vitro. Expression of mutant TTP in fibroblasts confirmed that serine 220 was one of the major, mitogen-stimulated phosphorylation sites on the protein in intact cells. These results suggest that TTP may be phosphorylated by MAP kinases in vivo and that this phosphorylation may regulate its function.
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PMID:Phosphorylation of tristetraprolin, a potential zinc finger transcription factor, by mitogen stimulation in intact cells and by mitogen-activated protein kinase in vitro. 776 35

Ste5 is a Zn2+ finger-like protein thought to function before three kinases, Ste11 (a MEKK), Ste7 (a MEK), and Fus3 (a MAPK), in a conserved MAP kinase cascade required for mating in S. cerevisiae. Here, we present evidence that Ste5 forms a multikinase complex that joins these kinases for efficient Fus3 activation. By two-hybrid analysis, Ste11, Ste7, and Fus3 associate with different domains of Ste5, while Kss1, another MAPK, associates with the same domain as Fus3, thus implying that Ste5 simultaneously binds a MEKK, MEK, and MAPK. Ste5 copurifies with Ste11, Fus3, and a hypophosphorylated form of Ste7, and all four proteins cosediment in a glycerol gradient as if in a large complex. Ste5 also increases the amount of Ste11 complexed to Ste7 and Fus3 and is required for Ste11 to function. These results substantiate a novel signal transduction component that physically links multiple kinases within a single cascade.
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PMID:Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae. 806 90

PTP2C, a widely distributed protein tyrosine phosphatase (PTP) containing two SH2 domains, was expressed as a recombinant enzyme in Escherichia coli and purified to near homogeneity. The purified enzyme and a truncated form lacking the SH2 domains (delta SH2-PTP2C) have been characterized with four commonly used substrates. Both forms showed pH optima of around neutrality for protein substrates but below 5.5 for a peptide substrate and para-nitrophenylphosphate. The dependence of the enzymes on ionic strength varied with the nature of the substrates involved. Like its analog PTP1C, PTP2C displayed a specific activity of less than 0.1% of that observed with other known PTPs toward protein substrates. Deletion of the SH2 domains increased its activity by 12-45-fold, depending on the substrates used. Limited trypsinolysis which cleaved about 4 kDa from the carboxyl terminus resulted in a 2-5-fold activation of the full-length enzyme but was essentially without effect on the truncated enzyme. Both forms showed similar responses to effectors including activators (e.g. anionic phospholipids) or inhibitors (e.g. vanadate, molybdate, or Zn2+). PTP2C and delta SH2-PTP2C were phosphorylated in vitro by mitogen-activated protein kinase, protein kinase C, and various protein tyrosine kinases; in the latter case, they underwent autodephosphorylation. No significant effect of the phosphorylation reactions on enzyme activity could be observed in vitro.
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PMID:Purification and characterization of PTP2C, a widely distributed protein tyrosine phosphatase containing two SH2 domains. 813 10

Nur77 represents a unique class within the steroid receptor superfamily since its synthesis is tightly regulated by extracellular signals and it is capable of potent transactivation activity in the absence of an exogenously added ligand. In this study, we sought to dissect the functional domains regulating the activities of Nur77 by deletion mapping. We demonstrate that whereas the transactivation activity of Nur77 resides in the amino-terminal domain, the carboxy-terminal domain regulates this activity. A short deletion from the carboxy terminus eliminates transactivation activity while a further deletion restores the activity. Deletion of the domain immediately carboxyl to the zinc fingers motif eliminates both DNA binding activity and nuclear localization, thus abolishing transactivation. Nur77 is posttranslationally modified predominantly by phosphorylation, which occurs primarily at the N-terminal domain. The growth-related kinase pp90rsk, but neither the pp44mapk nor the pp70s6k, can phosphorylate recombinant Nur77 in vitro. Furthermore, we have identified a site within the region required for sequence-specific DNA binding, Ser-354, that is phosphorylated by pp90rsk in vitro; this site is also phosphorylated in vivo. The possibility that phosphorylation might affect DNA binding is discussed.
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PMID:Functional domains and phosphorylation of the orphan receptor Nur77. 823 15

We have examined the negative regulation of the 44-kDa mitogen-activated protein kinase (MAP kinase), also known as extracellular signal-regulated protein kinase 1 (ERK1), in NIH3T3 cells transfected with an expression plasmid encoding the human insulin receptor (NHIR cells). In these cells ERK1 activation is induced by two distinct stimuli, insulin and tumor-promoting agent (TPA). While insulin was found to be more potent than TPA for ERK1 activation, both stimuli produced the same transient activation pattern with a rapid peak (reached within 5 min) followed by a fast decrease within 20 min. By performing reconstitution experiments with immunoprecipitated ERK1 and lysates from NHIR cells, we showed that extracts from untreated cells exhibit an ERK1 inhibitory activity. Interestingly, this inhibitor was found to be regulated by insulin and TPA with a profile that is the mirror image of ERK1 activity. This repressing activity was sensitive to tyrosine phosphatase inhibitors, such as sodium orthovanadate and zinc acetate, but it was not affected by serine/threonine phosphatase inhibitors, such as sodium fluoride and okadaic acid. Moreover, it was possible to observe in extracts of NHIR cells an activity dephosphorylating ERK1. The time course of this phosphatase activity was comparable to that of the ERK1 inhibition, suggesting that the repressing activity could reflect a dephosphorylating action. Interestingly, phosphatase 2A treatment of extracts from 5-min TPA-treated cells (where the ERK1 inhibitor was weak) was able to induce an increase in the ERK1 repressing activity. This suggests that serine/threonine dephosphorylation of ERK1 inhibitor leads to an increase in its activity. In summary, we have shown that NHIR cells contain a regulatable ERK1 inhibitor, which is likely to be due to tyrosine phosphatase(s). We would like to suggest that such activities are key components in the fine-tuning of the MAP kinase cascade.
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PMID:Insulin and tumor-promoting agent regulate an inhibitor of the 44-kDa mitogen-activated protein kinase/extracellular signal-regulated protein kinase 1 in fibroblasts. 828 32

In PC12 cells, extracellular signal-regulated kinase-1 (ERK1 or pp44/mitogen-activated protein kinase) is stimulated in response to epidermal growth factor (EGF) and nerve growth factor (NGF). This stimulation is rapid and short-lived after EGF activation. In contrast, NGF promotes a swift, but persistent, ERK1 stimulation. We took advantage of this difference in activation pattern to study the negative regulation of ERK1. Using antibodies to the C-terminus of ERK1, we performed in vitro reconstitution experiments with immunoprecipitated ERK1 from stimulated cells and extracts from PC12 cells incubated with EGF or NGF for various periods of times. Using this approach, we showed that extracts from unstimulated cells reduce ERK1 activity. Upon exposure of cells to NGF or EGF, we found that the inhibitory activity had a pattern opposite that of ERK1 phosphorylation and activity. Indeed, the highest ERK1 activation was associated with the lowest ERK1-repressing activity and vice versa. This ERK1 inhibitory activity was found to be sensitive mainly to sodium orthovanadate and to a lesser extent to zinc acetate. Interestingly, okadaic acid decreased ERK1-repressing activity from unstimulated cells when tested with ERK1 from 5-min NGF-treated cells, but not with ERK1 from 5-min EGF-treated cells. Hence, ERK1 appears to be regulated differently after stimulation of cells with EGF compared to NGF. We show that cell extracts promote ERK1 dephosphorylation. Indeed, we were able to detect a phosphatase activity toward in vivo phosphorylated ERK1 that was regulated differently after NGF and EGF treatments of the cells, and that has a profile of regulation similar to that of the ERK1 inhibitory activity. This regulatable phosphatase activity was also observed using in vitro phosphorylated ERK1. Taken together, our data provide evidence that ERK1 is negatively controlled by a phosphatase(s) that can undergo differential modulation depending on the stimuli used.
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PMID:Regulation of extracellular signal-regulated protein kinase-1 (ERK-1; pp44/mitogen-activated protein kinase) by epidermal growth factor and nerve growth factor in PC12 cells: implication of ERK1 inhibitory activities. 838 83

The growth factor-like effect of zinc in vitro and in vivo, which has long been recognized was investigated with respect to its mechanisms of action. Addition of zinc chloride to bombesin-sensitive Swiss 3T3 mouse fibroblasts induced a fourfold stimulation in the cytosolic myelin basic protein kinase activity. The response was dose- and time-dependent, with an ED50 of around 100 microM and a peak at 5 min. The kinase activity coeluted with p42 MAP kinase using chromatography on Mono-Q ion exchange. Intracellular loading of cells with the heavy metal chelator BTC-5N did not attenuate the response to zinc. The action of zinc was not suppressed by long-term pretreatment with 4-beta-phorbol dibutyrate (48 h). Addition of 0.3 mM vanadate alone did not increase the kinase activity, but prolonged the action of zinc when added simultaneously. Addition of zinc (0.3 mM) or epidermal growth factor for 1 min resulted in a marked increase in tyrosine phosphorylation of proteins with apparent molecular weights of approximately 100, 105-120, 215, and 240 kDa in whole cell extracts. Immunoprecipitation against the p85 subunit of phosphatidylinositol 3-kinase resulted in the appearance of two phosphotyrosine-containing proteins, 100 and 115 kDa, in extracts from cells treated with zinc or epidermal growth factor, indicating that the tyrosine phosphorylation was recognized by the corresponding SH2-domains. The present study demonstrates that extracellular zinc has the potential to partially mimic the action of growth factors on intracellular MAP kinase activation and protein tyrosine phosphorylation.
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PMID:Extracellular zinc ions induces mitogen-activated protein kinase activity and protein tyrosine phosphorylation in bombesin-sensitive Swiss 3T3 fibroblasts. 864 99

Tristetraprolin (TTP) is the prototype of a group of potential transcription factors that contain two or more unusual CCCH zinc fingers. TTP is encoded by the immediate-early response gene Zfp-36, which is rapidly induced in fibroblasts in response to insulin and other growth factors. Indirect evidence suggests that TTP might function as an inhibitory transcription factor. The present studies evaluated the effect of mitogens on the subcellular localization of TTP using Western blotting of cellular nuclear and cytosolic fractions. In NIH/3T3 mouse fibroblasts that constitutively express TTP, 70% of the protein was located in the nucleus of quiescent, serum-deprived cells. Immunoreactive TTP began to increase in the cytosolic compartment within 1 min of serum stimulation of the cells; this increase in cytosolic protein was essentially complete within 5 min of serum stimulation (81% of total) and was accompanied by a commensurate decrease in nuclear TTP. This translocation was complete well before the increase in TTP synthesis that occurred after serum stimulation. Similar experiments in cells expressing a mutant TTP, in which the major mitogen-activated protein kinase site (serine 220) had been mutated to alanine, revealed normal nuclear to cytosolic translocation after serum stimulation, indicating that phosphorylation of this site is not necessary for this translocation to occur. These results suggest that TTP is rapidly modified in response to mitogens so that it is rapidly released from the nucleus to the cytosol, or that proteins retaining TTP in the nucleus are modified to release it into the cytosol. Thus, TTP's proposed function as a transcription factor, possibly an inhibitory one, may be regulated in cells in part by a novel mechanism, i.e. that of rapid, mitogen-stimulated translocation out of the cellular nucleus.
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PMID:Mitogens stimulate the rapid nuclear to cytosolic translocation of tristetraprolin, a potential zinc-finger transcription factor. 882 54

Dystrophin is a protein product of the gene responsible for Duchenne and Becker muscular dystrophy. The protein is localized to the inner surface of sarcolemma and is associated with a group of membrane (glyco)proteins. Dystrophin links cytoskeletal actins via the dystrophin-associated protein complex to extracellular matrix protein, laminin. This structural organization implicates the role of dystrophin in stabilizing the sarcolemma of muscle fibers. Precisely how dystrophin functions is far from clear. The presence of an array of isoforms of the C-terminal region of dystrophin suggests that dystrophin may have functions other than structural. In agreement, many potential phosphorylation sites are found in the C-terminal region of dystrophin, and the C-terminal region of dystrophin is phosphorylated both in vitro and in vivo by many protein kinases, including MAP kinase, p34cdc2 kinase, CaM kinase, and casein kinase, and is dephosphorylated by calcineurin. The C-terminal domain of dystrophin is also a substrate for hierarchical phosphorylation by casein kinase-2 and GSK-3. These observations, in accordance with the finding that the cysteine-rich region binds to Ca2+, Zn2+, and calmodulin, suggest an active involvement of dystrophin in transducing signals across muscle sarcolemma. Phosphorylation-dephosphorylation of the C-terminal region of dystrophin may play a role in regulating dystrophin-protein interactions and (or) transducing signal from the extracellular matrix via the dystrophin molecule to the cytoskeleton.
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PMID:Phosphorylation of the carboxyl-terminal region of dystrophin. 896 Mar 49

A critical role of the 289-residue (289R) E1A protein of human adenovirus type 5 during productive infection is to transactivate expression of all early viral transcription. Sequences within and proximal to conserved region 3 (CR3) promote expression of these viral genes through interactions with a variety of transcription factors requiring the zinc binding motif in CR3 and in some cases a region at the carboxy-terminal end of CR3, including residues 183 to 188. It is known that 3',5' cyclic AMP (cAMP) reduces the level of phosphorylation of the 289R E1A protein through the activation of protein phosphatase 2A by the E4orf4 protein. This study was designed to identify the E1A phosphorylation sites affected by E4orf4 expression and to determine their importance in regulation of E1A activity. We report here that two previously unidentified sites at Ser-185 and Ser-188 are the targets for decreased phosphorylation in response to cAMP. At least one of these sites, presumably Ser-185, is phosphorylated in vitro by purified mitogen-activated protein kinase (MAPK), and both are hyperphosphorylated in cells which express a constitutively active form of MAPK kinase. Analysis of E1A-mediated transactivation activity indicated that elevated phosphorylation at these sites increased expression of the E4 promoter but not that of E3. We have recently shown that one or more E4 products induce cell death due to p53-independent apoptosis, and thus it seems likely that one role of the E4orf4 protein is to limit production of toxic E4 products by limiting expression of the E4 promoter.
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PMID:Phosphorylation within the transactivation domain of adenovirus E1A protein by mitogen-activated protein kinase regulates expression of early region 4. 909 26


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