UNIPROT:P51812 - FACTA Search

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Query: UNIPROT:P51812 (mitogen-activated protein)
10,636 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Deletion of the yeast Ser/Thr protein phosphatase PPZ1 results in increased tolerance to sodium and lithium. PPZ1 is also important for cell integrity, as ppz1Delta cells undergo lysis under caffeine stress and PPZ1 overexpression overrides the lytic defect of mutants in the protein kinase C/mitogen-activated protein (MAP) kinase pathway. The PPZ1 protein can be dissected in two halves. The COOH-terminal half is related to type 1 phosphatases, whereas the NH2-terminal half is unrelated to phosphatases and contains a consensus site for N-myristoylation. Several mutated versions of PPZ1 have been constructed and tested for complementation of ppz1Delta mutants. We show that PPZ1 can be myristoylated in vivo and that change of Gly-2 to Ala results in lack of myristoylation and loss of complementation of salt tolerance. Removal of the entire NH2-terminal half results in complete loss of function, although it does not abolish the phosphatase activity of the protein expressed in Escherichia coli. The deletion of a large region of the NH2-terminal half (residues 17-193) does not affect the ability to complement the salt tolerance phenotype but abolish complementation of caffeine sensitivity, whereas the opposite behavior is observed upon removal of residues from 241 to 318. Mutation of Arg-451 to Leu results in both complete loss of function and of phosphatase activity. These results indicates that the NH2-terminal half of the protein contains structural determinants that are specific for certain functions and that the phosphatase activity is required but not sufficient for full PPZ1 function.
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PMID:The NH2-terminal extension of protein phosphatase PPZ1 has an essential functional role. 882 89

The Mpk1 (Slt2) mitogen-activated protein (MAP) kinase has been implicated in several biological processes in Saccharomyces cerevisiae. The Rlm1 protein, a member of the MADS box family of transcription factors, functions downstream of Mpk1 in the pathway. To characterize the role of Rlm1 in mediating the transcriptional activation by the Mpk1 pathway, we constructed a LexA-Rlm1 deltaN chimera in which sequences, including the MADS box domain of the Rlm1 protein, were replaced by the LexA DNA binding domain and tested the ability of this chimera to activate a LexA operator-controlled reporter gene. In this assay, the Rlm1 protein was found to activate transcription in a manner regulated by the Mpk1 pathway. The Mpk1 protein kinase phosphorylated Rlm1 deltaN in vitro and the LexA-Rlm1 deltaN chimera protein was phosphorylated in vivo in a Mpk1-dependent manner. These results suggest that Mpk1 regulates the transcriptional activity of Rlm1 by directly phosphorylating it. We identified a Mpk1-like protein kinase, Mlp1, as an Rlm1-associated protein by using the yeast two-hybrid system. Overexpression of MLP1 suppresses the caffeine-sensitive phenotype of the bck1 delta mutation. The additivity of the mlp1 delta defect with the Mpk1 delta defect with regard to the caffeine sensitivity, combined with the results of genetic epistasis experiments, suggested that the activity of Rlm1 is regulated independently by Mpk1 MAP kinase and the Mlp1 MAP kinase-like kinase.
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PMID:Characterization of a serum response factor-like protein in Saccharomyces cerevisiae, Rlm1, which has transcriptional activity regulated by the Mpk1 (Slt2) mitogen-activated protein kinase pathway. 911 31

We have investigated in rat pheochromacytoma PC12 cells the activation of the mitogen-activated protein kinases ERK1 and ERK2 by the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). This treatment slowly decreases ATP levels to 30% of control, whereas the internal calcium level rises very rapidly to 250% of control, derived from internal stores. Tyrosine phosphorylation of ERK1 and ERK2 increases gradually, starting after 5 min of treatment, to reach a maximum at 30 min; the kinase activity reaches 250% when measured after 1 hr of treatment. The drop in ATP levels is slower still. Comparison of the time courses of the rapid rise in cytosolic calcium with the slower increase in ERK1 and ERK2 activation suggests one or more intermediate stages in this pathway. Chelation of cytosolic calcium with dimethyl bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the FCCP-stimulated rise in internal calcium, as well as the tyrosine phosphorylation and the activation of the ERKs. Surprisingly, caffeine, which releases calcium from different internal stores, did not increase the tyrosine phosphorylation and did not activate the ERKs. The FCCP effect on calcium storage may be related to mitochondrial dysfunction in Alzheimer disease, which might result in ineffective buffering of cytosolic calcium that leads to mitogen-activated protein kinase activation and subsequent protein phosphorylations.
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PMID:Compromised mitochondrial function leads to increased cytosolic calcium and to activation of MAP kinases. 927 88

The proteins Msb3p (Ynl293p) and Msb4p (Yol112p) of the yeast Saccharomyces cerevisiae are very similar in sequence and share a highly conserved domain called TBC. To characterize the cellular functions of these proteins, we constructed single and double yeast mutants by disrupting the MSB3 gene, the MSB4 gene, or both. Co-deletion of the MSB3 and MSB4 coding regions caused growth inhibition in the presence of 10 mM caffeine and 4% dimethyl sulphoxide (DMSO), increased the sensitivity of the yeast strain to latrunculin-A, produced a random budding pattern in diploid cells, and affected the organization of the actin cytoskeleton. Caffeine sensitivity is often associated with defects in mitogen-activated protein (MAP) kinase pathways, highly conserved mechanisms mediating transduction of external signals. The biological effect of DMSO in S. cerevisiae is unclear. The msb3 msb4 mutant's increased sensitivity to latrunculin-A suggests that the double mutation causes destabilization of the actin cytoskeleton. Microscopic observations confirmed this: in haploid and diploid msb3 msb4 mutant cells, polymerized actin was delocalized from the budding sites. Complementation studies suggested that MSB3 and MSB4 encode overlapping activities in the yeast cells. We thus propose that both Msb3p and Msb4p act in budding site selection, probably via their involvement in the organization of the actin cytoskeleton.
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PMID:Co-deletion of the MSB3 and MSB4 coding regions affects bipolar budding and perturbs the organization of the actin cytoskeleton. 1092 23

The gene pzl-1 from the filamentous fungus Neurospora crassa encodes a putative Ser/Thr protein phosphatase that is reminiscent of the Ppz1/Ppz2 and Pzh1 phosphatases from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The entire PZL-1 protein, as well as its carboxyl-terminal domain, have been expressed in Escherichia coli as active protein phosphatases. To characterize its cellular role, PZL-1 was also expressed in Sz. pombe and in S. cerevisiae. Expression of PZL-1 in S. cerevisiae from the PPZ1 promoter was able to rescue the altered sensitivity to caffeine and lithium ions of a ppz1 strain. Furthermore, high copy number expression of PZL-1 alleviated the lytic phenotype of a S. cerevisiae slt2/mpk1 mitogen-activated protein (MAP) kinase mutant, similarly to that described for PPZ1, and mimicked the effects of high levels of Ppz1 on cell growth. Expression of PZL-1 in fission yeast from a weak version of the nmt1 promoter fully rescued the growth defect of a pzh1Delta strain in high potassium, but only partially complemented the sodium-hypertolerant phenotype. Strong overexpression of the N. crassa phosphatase in Sz. pombe affected cell growth and morphology. Therefore, PZL-1 appears to fulfil every known function carried out by its S. cerevisiae counterpart, despite the marked divergence in sequence within their NH(2)-terminal moieties.
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PMID:Functional analysis of the Neurospora crassa PZL-1 protein phosphatase by expression in budding and fission yeast. 1116 54

When MCF-7 cells were incubated with 10 or 20 microM CdCl(2), p53 protein level increased after 18 h. Among serines in p53 protein immunoprecipitated from cells treated with CdCl(2), only Ser 15 was phosphorylated. No clear phosphorylation was found on Ser 6, 9, 20, 37, and 392. Accumulation of p53 protein phosphorylated at Ser 15 was also found after 18 h exposure. While phosphorylation of extracellular signal-regulated protein kinase, c-Jun NH2-terminal kinase and p38 was found in cells treated with CdCl(2), treatment with U0126, LL-Z1640-2, or SB203580 did not suppress Ser 15 phosphorylation. On the other hand, treatment with wortmannin or caffeine suppressed CdCl(2)-induced Ser 15 phosphorylation and accumulation of p53 protein. The present results showed that cadmium induces phosphorylation of p53 at Ser 15 in MCF-7 cells depending on phosphatidylinositol 3-kinase related kinases, but not on mitogen-activated protein kinases.
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PMID:Cadmium induces phosphorylation of p53 at serine 15 in MCF-7 cells. 1130 31

The impact of disruption of the PI3K (phosphatidylinositol 3-kinase) pathway on the response of human leukemia cells to pharmacological cyclin-dependent kinase (CDK) inhibitors has been examined. Exposure of U937 monocytic leukemia cells to minimally toxic concentrations of flavopiridol (FP), roscovitine, or CGP74514A for 3 h in conjunction with the PI3K inhibitor LY294002 (abbreviated LY in the article) resulted in a marked decrease in Akt phosphorylation. Coexposure of cells to LY and CDK inhibitors also resulted in an early (i.e., within 3 h) and striking increase in mitochondrial damage [e.g., cytochrome c, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis (IAP)-binding protein with low isoelectric point (Smac/DIABLO), and apoptosis-initiating factor (AIF) release], caspase activation, and apoptosis. Similar interactions were observed in a variety of other leukemia cell types (e.g., HL-60, Jurkat, Raji, and NB4). Apoptosis, induced by FP/LY, was substantially blocked by ectopic expression of Bcl-2, but to a considerably lesser extent by dominant-negative caspase-8. FP-induced apoptosis was not enhanced by agents that inhibited protein kinase (PK) A (H89), PKC (GFX), mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK1/2; U0126), p38 MAP kinase (MAPK; SB202190), m-target of rapamycin (TOR; rapamycin), or ataxia-telangiectasia mutation (ATM; caffeine), whereas the PI3K inhibitor wortmannin exerted effects similar to those of LY. The dramatic potentiation of CDK inhibitor-induced apoptosis by LY was accompanied by diminished Bad phosphorylation, induction of Bcl-2 cleavage, and down-regulation of X-linked IAP (XIAP) and Mcl-1. Cells exposed to CDK inhibitors + LY also exhibited reduced phosphorylation of glycogen synthase kinase (GSK)-3, forkhead transcription factor (FKHR), p70(S6K), and ERK, but increased activation of p34(cdc2) and p38 MAPK. LY/CDK inhibitor-treated cells also displayed diminished pRb dephosphorylation on CDK2- and CDK4-specific sites, retinoblastoma protein cleavage, and down-regulation of cyclin D(1). Inducible expression of constitutively active (myristolated) Akt significantly, albeit partially, attenuated apoptosis in Jurkat leukemia cells treated with either FP alone or the combination of FP and LY. Finally, cotreatment with LY and FP resulted in a dramatic increase in apoptosis in primary leukemic blasts obtained from a patient with acute myeloblastic leukemia. Together, these findings suggest that the PI3K/Akt pathway plays a major role in regulating the apoptotic response of human leukemia cells to pharmacological CDK inhibitors and raise the possibility that combined interruption of CDK- and PI3K-related pathways may represent a novel therapeutic strategy in hematological malignancies.
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PMID:The lethal effects of pharmacological cyclin-dependent kinase inhibitors in human leukemia cells proceed through a phosphatidylinositol 3-kinase/Akt-dependent process. 1270 69

Caffeine is a natural purine analogue that elicits pleiotropic effects leading ultimately to cell's death by a largely uncharacterized mechanism. Previous works have shown that this drug induces a rapid phosphorylation of the Mpk1p, the final mitogen-activated protein (MAP) kinase of the Pkc1p-mediated cell integrity pathway. In this work, we showed that this phosphorylation did not necessitate the main cell wall sensors Wsc1p and Mid2p, but was abolished upon deletion of ROM2 encoding a GDP/GTP exchange factor of Rho1p. We also showed that the caffeine-induced phosphorylation of Mpk1p was accompanied by a negligible activation of its main downstream target, the Rlm1p transcription factor. This result was consolidated by the finding that the loss of RLM1 had no consequence on the increased resistance of caffeine-treated cells to zymolyase, indicating that the cell wall modification caused by this drug is largely independent of transcriptional activation of Rlm1p-regulated genes. Additionally, the transcriptional programme elicited by caffeine resembled that of rapamycin, a potent inhibitor of the TOR1/2 kinases. Consistent with this analysis, we found that the caffeine-induced phosphorylation of Mpk1p was lost in a tor1Delta mutant. Moreover, a tor1Delta mutant was, like mutants defective in components of the Pkc1p-Mpk1p cascade, highly sensitive to caffeine. However, the hypersensitivity of a tor1 null mutant to this drug was rescued neither by sorbitol nor by adenine, which was found to outcompete caffeine effects specially on mutants in the PKC pathway. Altogether, these data indicated that Tor1 kinase is a target of caffeine, whose inhibition incidentally activates the Pkc1p-Mpk1p cascade, and that the caffeine-dependent phenotypes are largely dependent on inhibition of Tor1p-regulated cellular functions. Finally, we found that caffeine provoked, in a Rom2p-dependent manner, a transient drop in intracellular levels of cAMP, that was followed by change in expression of genes implicated in Ras/cAMP pathway. This result may pose Rom2p as a mediator in the interplay between Tor1p and the Ras/cAMP pathway.
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PMID:Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. 1692 51

Despite a lot of gap junction research, the complex connection between gap junction and cell proliferation remains an exciting area of investigation. Thus, we examined the effect of connexin 43 (Cx43) on the migration and proliferation of embryonic stem (ES) cells and its related signaling pathways following stimulation with the adenosine analogue 5'-N-ethylcarboxamide (NECA). NECA increased phosphorylation of Cx43 which was blocked by caffeine, a non-selective adenosine receptor antagonist. In experiment to measure the gap junctional intercellular communication, NECA blocked transfer of Lucifer yellow to neighboring cells in a scrape loading/dye transfer (SL/DT) assay. In addition, NECA-induced phosphorylation of phosphoinositide 3-kinase (PI3K)/Akt, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and nuclear factor-kappa B (NF-kappaB) signal pathways. Inhibition of these signaling pathways reduced NECA-induced phosphorylation of Cx43. Moreover, NECA-treated cells demonstrated phosphorylation of Src, which was blocked by caffeine. In this experiment, a disruption of Cx43 using Cx43-specific small interfering RNA (siRNA) also enhanced Src phosphorylation. In a further study, phosphorylations of integrin beta1, focal adhesion kinase (FAK), and paxillin by NECA were restrained by caffeine as well as the Src blocker, PP2. Finally, we identified that NECA-stimulated cell migration and expressions of cell-cycle regulatory proteins [cyclin D1, cyclin-dependent kinase (CDK) 4, cyclin E, and CDK2]; these increases were inhibited by caffeine, or PP2. We conclude that NECA-stimulated Cx43 phosphorylation mediated by PI3K/Akt, PKC, MAPKs, and NF-kappaB, which subsequently stimulated cell migration and proliferation through Src, integrin beta1, FAK, and paxillin signal pathways.
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PMID:Involvement of Cx43 phosphorylation in 5'-N-ethylcarboxamide-induced migration and proliferation of mouse embryonic stem cells. 2023 18

Caffeine treatment of ovine oocytes increases the activity of maturation-promoting factor (MPF) and mitogen-activated protein kinases (MAPKs) and, in somatic cell nuclear transfer (SCNT) embryos, increases the frequency of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). At the blastocyst stage, caffeine-treated SCNT embryos have increased cell numbers. One explanation for this is that NEBD and PCC release chromatin-bound somatic factors, allowing greater access of oocyte factors involved in DNA synthesis and nuclear reprogramming to donor chromatin. This could advance DNA replication and cleavage in the first cell cycle, resulting in increased cell numbers. Alternatively, increased MAPK activity may affect localisation of heat shock proteins (HSPs) and reduce apoptosis. To investigate these possibilities, we investigated chromatin accessibility, the timing of DNA synthesis and first cleavage, the localisation of HSP27 during early development and the frequency of apoptotic nuclei at the blastocyst stage. Compared with control SCNT (non-caffeine treatment), caffeine treatment (10 mM caffeine for 6 h prior to activation) increased the accessibility of DNase I to donor chromatin (P < 0.05 at 1.5 h post activation (h.p.a.)), advanced DNA synthesis (43.5% v. 67.6%, respectively; P < 0.01 at 6 h.p.a.) and first cleavage (27.3% v. 40.5% at 20 h.p.a., respectively) and increased nuclear localisation of HSP27. Although development to the blastocyst stage was not affected, caffeine increased total cell numbers (98.5 v. 76.6; P < 0.05) and reduced the frequency of apoptotic nuclei (11.27% v. 20.3%; P < 0.05) compared with control SCNT group.
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PMID:Treatment of ovine oocytes with caffeine increases the accessibility of DNase I to the donor chromatin and reduces apoptosis in somatic cell nuclear transfer embryos. 2059 34

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