Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

MAP kinase-activated protein kinase-2 (MAPKAP kinase-2) phosphorylates the serine residues in murine heat shock protein 25 (hsp25) and human heat shock protein 27 (hsp27) which are phosphorylated in vivo in response to growth factors and heat shock, namely Ser15 and Ser86 (hsp25) and Ser15, Ser78 and Ser82 (hsp27). Ser86 of hsp25 and the equivalent residue in hsp27 (Ser82) are phosphorylated preferentially in vitro. The small heat shock protein is present in rabbit skeletal muscle and hsp25 kinase activity in skeletal muscle extracts co-purifies with MAPKAP kinase-2 activity throughout the purification of the latter enzyme. These results suggest that MAPKAP kinase-2 is the enzyme responsible for the phosphorylation of these small heat shock proteins in mammalian cells.
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PMID:Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. 133 86

Phosphorylation of heat shock protein 27 (HSP27) can modulate actin filament dynamics in response to growth factors. During heat shock, HSP27 is phosphorylated at the same sites and by the same protein kinase as during mitogenic stimulation. This suggests that the same function of the protein may be activated during growth factor stimulation and the stress response. To determine the role of HSP27 phosphorylation in the heat shock response, several stable Chinese hamster cell lines that constitutively express various levels of the wild-type HSP27 (HU27 cells) or a nonphosphorylatable form of human HSP27 (HU27pm3 cells) were developed. In contrast to HU27 cells, which showed increased survival after heat shock, HU27pm3 cells showed only slightly enhanced survival. Evidence is presented that stabilization of microfilaments is a major target of the protective function of HSP27. In the HU27pm3 cells, the microfilaments were thermosensitized compared with those in the control cells, whereas wild-type HSP27 caused an increased stability of these structures in HU27 cells. HU27 but not HU27pm3 cells were highly resistant to cytochalasin D treatment compared with control cells. Moreover, in cells treated with cytochalasin D, wild-type HSP27 but not the phosphorylated form of HSP27 accelerated the reappearance of actin filaments. The mutations in human HSP27 had no effect on heat shock-induced change in solubility and cellular localization of the protein, indicating that phosphorylation was not involved in these processes. However, induction of HSP27 phosphorylation by stressing agents or mitogens caused a reduction in the multimeric size of the wild-type protein, an effect which was not observed with the mutant protein. We propose that early during stress, phosphorylation-induced conformational changes in the HSP27 oligomers regulate the activity of the protein at the level of microfilament dynamics, resulting in both enhanced stability and accelerated recovery of the filaments. The level of protection provided by HSP27 during heat shock may thus represent the contribution of better maintenance of actin filament integrity to overall cell survival.
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PMID:Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27. 779 59

We have previously reported the presence of a 28-kDa protein in human mammary adenocarcinoma MCF-7 cells, whose phosphorylation by phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and permeant diacylglycerol 1,2-dioctanoyl-sn-glycerol was correlated to growth arrest induced by the protein kinase C (PKC) activators. We now investigate the possible identity of this protein with the estrogen-regulated "24-kDa" protein shown as related to the mammalian heat shock protein 27 (Fuqua, S. A. W., Blum-Salingaros, M., and McGuire, W. L. (1989) Cancer Res 49, 4126-4129). 32P-Labeled 28-kDa protein from TPA-treated MCF-7 cells was immunoprecipitated with a 24-kDa-specific monoclonal antibody. Immunoblots from cell extracts fractionated by two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis demonstrated that TPA induced the conversion of a 28-kDa isoform "a" (pI 6.7) to a more acidic isoform "b" (pI 6.2). Two-dimensional gel analysis of [3H]leucine-labeled MCF-7 cell extracts demonstrated that conversely to TPA, which induced only phosphorylation of 28-kDa protein, heat shock induced both synthesis (increase of isoform a) and phosphorylation (conversion of isoforms a to b) of the protein. 32P labeling of MCF-7 cells allowed demonstration of the presence of an extra phosphoisoform "c" (pI 5.9) upon TPA as well as heat shock treatment. When cells were pretreated with the bisindolylmaleimide GF109203X, a selective inhibitor of PKC, the heat shock-induced phosphorylation was unchanged, while the TPA effect was almost abolished, suggesting that the heat shock-activated protein kinase was very likely different from PKC. However, peptide mapping of the 28-kDa phosphoprotein suggested identical sites of phosphorylation upon TPA and heat shock stimulation. Partial amino acid sequencing of the 28-kDa protein revealed identity with both the 24-kDa protein and the mammalian HSP27. The fact that estrogens and PKC, respectively, regulate expression and phosphorylation of this 24/28-kDa protein strongly argues for its key role in MCF-7 cell proliferation and differentiation.
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PMID:The 28-kDa protein whose phosphorylation is induced by protein kinase C activators in MCF-7 cells belongs to the family of low molecular mass heat shock proteins and is the estrogen-regulated 24-kDa protein. 832 90

The coordinated cellular responses to physiological stress are known to be effected in part by the activation of heat-shock factor 1, a transcriptional activator protein capable of binding to, and inducing transcription from genes containing heat shock elements. Other stress responsive signal transduction pathways also exist including the stress activated protein kinase cascade that regulates the activity of the transcription factor AP1. We have examined the expression of the low molecular stress proteins, heat shock protein 27 and alpha B-crystallin in astrocytes in response to physiological stress of different types and asked what component of this induction is effected at the transcriptional level and whether activation of heat shock factor 1 and AP1 might account for these events. We have found that stress regulated induction of alpha B-crystallin has a strong transcriptional component and that it may be effected by at least two different transcriptional mechanisms. In one set of phenomena, represented here by cadmium exposure, alpha B-crystallin and heat shock protein 27 are coordinately regulated and this occurs in the presence of activated heat shock factor 1. In the second series of phenomena, represented here by hypertonic stress, alpha B-crystallin is induced in the absence of heat shock factor activation and in the absence of any corresponding change in heat shock protein 27 expression. Although hypertonic stress does activate an AP1-like binding activity, the AP1 consensus binding site in the alpha B-crystallin promoter does not appear to be a target for this hypertonic stress inducible activity. These data suggest that the hypertonic stress response is effected through a heat shock factor independent mechanism and that hypertonic stress regulated induction of alpha B-crystallin does not directly depend on the SAPK pathway and AP1 activity.
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PMID:Transcription regulation of alpha B-crystallin in astrocytes: analysis of HSF and AP1 activation by different types of physiological stress. 874 50

The presence of a novel 38 kDa protein that is tyrosine phosphorylated in human neutrophils, a terminally differentiated cell, upon stimulation of these cells with low concentrations of lipopolysaccharide (LPS) in combination with serum has been demonstrated. This 38 kDa protein was identified as the mammalian homologue of HOG1 in yeast, the p38 mitogen-activated protein (MAP) kinase. This conclusion is based on the experimental findings that anti-phosphotyrosine (anti-PY) antibody immunoprecipitates a 38 kDa protein that is recognized by anti-p38 MAP kinase antibody, and conversely, anti-p38 MAP kinase antibody immunoprecipitates a 38 kDa protein that can be recognized by anti-PY antibody. Moreover, this tyrosine phosphorylated protein is found associated entirely with the cytosol. It was also found that this p38 MAP kinase is activated following stimulation of these cells with low concentrations of LPS in combination with serum. This conclusion is based on three experimental findings. First, soluble fractions isolated from LPS-stimulated cells phosphorylate heat shock protein 27 (hsp27) in an in vitro assay, and this effect is not inhibited by protein kinase C and protein kinase A inhibitor peptides. This effect is similar to the effect produced by the commercially available phosphorylated and activated MAPKAP kinase-2 (MAP kinase activated protein kinase-2). Secondly, a 27 kDa protein that aligns with a protein recognized by anti-hsp27 antibody is phosphorylated upon LPS stimulation of intact human neutrophils prelabelled with radioactive phosphate. Lastly, immune complex protein kinase assays, using [gamma-32P]ATP and activating transcription factor 2 (ATF2) as substrates, showed increased p38 MAP kinase activity from LPS-stimulated human neutrophils. The phosphorylation and activation of this p38 MAP kinase can be affected by both G-protein-coupled receptors such as platelet-activating factor (PAF) and non-G-protein-coupled receptors such as the cytokine-coupled receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor alpha (TNF-alpha). The effect of low concentrations of PAF is greatly increased in cells pretreated with LPS. The tyrosine phosphorylation of the p38 MAP kinase is not restricted to stimuli that mediate their actions through membrane-associated receptors, but it can be affected by agents that bypass membrane-associated receptors such as the protein translation blocker anisomycin. While anisomycin is known to increase the tyrosine phosphorylation of the 54 kDa SAPK (stress-activated protein kinase), this is the first report that shows that anisomycin also tyrosine phosphorylates the p38 MAP kinase. Cytokine receptors that increase the tyrosine phosphorylation and activation of the erk1 and erk2 MAP kinases have less effect on this p38 MAP kinase than those that do not affect the erk1 and erk2 MAP kinases. The possible role of the p38 MAP kinase in the phosphorylation of cytosolic phospholipase A2 is discussed.
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PMID:Tyrosine phosphorylation and activation of a new mitogen-activated protein (MAP)-kinase cascade in human neutrophils stimulated with various agonists. 876 79

Vascular endothelial cells are constantly in contact with oxyradicals and must be especially well equipped to resist their toxic effects and generate appropriate physiological responses. Despite the importance of oxyradicals in the physiopathology of the vascular endothelium, the mechanisms regulating the oxidative response of endothelial cells are poorly understood. In the present study, we observed that H2O2 in concentrations that induced severe fragmentation of F-actin in fibroblasts rather induced a reorganization of F-actin in primary cultures of human umbilical vein endothelial cells (HUVECs) that was characterized by the accumulation of stress fibers, the recruitment of vinculin to focal adhesions, and the loss of membrane ruffles, H2O2 also induced in these cells a strong (10- to 14-fold) activation of the p38 mitogen-activated protein (MAP) kinase, which resulted in activation of MAP kinase-activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). The MAP kinases extracellular-regulated kinase, and c-Jun N-terminal kinase/stress-activated protein kinase were only slightly increased by these treatments. Inhibiting p38 activity with the highly specific inhibitor SB203580 blocked the H2O2-induced endothelial microfilament responses. Moreover, fibroblasts acquired an endothelium-like SB203580-sensitive actin response when HSP27 concentration was increased by gene transfection to the same high level as found in HUVECs. The results indicate that activation of p38 MAP kinase in cells such as endothelial cells, which naturally express high level of HSP27, plays a central role in modulating microfilament responses to oxidative stress. Consequently, the p38 MAP kinase pathway may participate in the several oxyradical-activated functions of the endothelium that are associated with reorganization of microfilament network.
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PMID:Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. 904 59

The role of p38 mitogen-activated protein kinase (MAPK) in responses of human fibroblasts and vascular endothelial cells to IL-1 was investigated by use of a pyridinyl imidazole compound (SB 203580), which specifically inhibits the enzyme. SB 203580 inhibited (50% inhibitory concentration approximately 0.5 microM) IL-1-induced phosphorylation of heat shock protein 27 (an indicator of p38 MAPK activity) in fibroblasts without affecting the other known IL-1-activated protein kinase pathways (p42/p44 MAPK, p54 MAPK/c-Jun N-terminal kinase and beta-casein kinase). SB 203580 significantly inhibited IL-1-stimulated IL-6, (30 to 50% at 1 microM) but not IL-8 production from human fibroblasts (gingival and dermal) and umbilical vein endothelial cells. IL-1 induction of steady state level of IL-6 mRNA was not significantly inhibited, which is consistent with p38 MAPK regulating IL-6 production at the translational level. SB 203580 strongly inhibited IL-1-stimulated PG production by fibroblasts and human umbilical vein endothelial cells. This was associated with the inhibition of the induction of PGH synthase-2 protein and mRNA. SB 203580 also inhibited the stimulation of collagenase-1 and stromelysin-1 production by IL-1 without affecting synthesis of the tissue inhibitor of metalloproteinases (TIMP)-1. SB 203580 prevented the increase in collagenase-1 and stromelysin-1 mRNA stimulated by IL-1. In a model of cartilage breakdown, short-term IL-1-stimulated proteoglycan resorption and inhibition of proteoglycan synthesis were unaffected by SB 203580, while longer term collagen breakdown was prevented. It is concluded that 1) p38 MAPK plays an important role in the regulation of some, but not all, responses to IL-1, and 2) it is involved in the regulation of mRNA levels of some IL-1-responsive genes.
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PMID:Actions of IL-1 are selectively controlled by p38 mitogen-activated protein kinase: regulation of prostaglandin H synthase-2, metalloproteinases, and IL-6 at different levels. 912 Feb 70

The aim of this study was to investigate different protein kinase inhibitors (secondary metabolite-derived substances, synthetic compounds, and substrate-based peptides) for their potency to inhibit the mammalian small heat shock protein (HSP25) kinase (E.C. 2.7.1.37) isolated from Ehrlich ascites tumor cells. Among the secondary metabolite-derived inhibitors (staurosporine, K-252a, K-252b, KT5926, KT5720, erbstatin analog, and quercetin) and synthetic compounds (H-9, H-89, HA 1004, KN-62, ML-7, tyrphostin A25, and tyrphostin B42), KT5926, staurosporine, and K-252a inhibited HSP25 kinase most efficiently. Kinetic analysis revealed that inhibition by staurosporine (Ki = 32.4 nM) and K-252a (Ki = 13.7 nM) was competitive with ATP. Inhibition by KT5926 was competitive with the substrate peptide KKKALNRQLSVAA (Ki = 27.2 nM) and noncompetitive with respect to ATP (Ki = 38.8 nM). In comparison with other protein kinases, HSP25 kinase was relatively resistant to most of the inhibitors. KT5926 was the only tested inhibitor with certain preference for HSP25 kinase when compared with protein kinases A, C, and G. Among the tested substrate-based peptides, we identified one peptide (KKKALNRQLGVAA), which preferentially inhibited HSP25 kinase in comparison with protein kinases A and C and mitogen-activated protein kinase. This peptide inhibited HSP25 kinase competitively with the substrate peptide (Ki = 8.1 microM) and noncompetitively with ATP (Ki = 134 microM). A peptide (SRVLKEDKERWEDVK) derived from the putative autoinhibitory domain of the closely related human mitogen-activated protein kinase-activated protein kinase-2 did not inhibit HSP25 kinase activity, suggesting the existence of several species of HSP25 kinases. Furthermore, the data identified structural requirements for inhibitors of HSP25-kinase.
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PMID:Effect of protein kinase inhibitors on activity of mammalian small heat-shock protein (HSP25) kinase. 921 84

Vascular endothelial growth factor (VEGF) is a potent chemotactic agent for endothelial cells. Yet the signalling pathways that modulate the motogenic effects of VEGF in vascular endothelial cells are still ill defined. In the present study, we found in primary cultures of human umbilical vein endothelial cells (HUVEC) that VEGF increased cell migration and induced a marked reorganization of the microfilament network that was characterized by the formation of stress fibers and the recruitment of vinculin to focal adhesions. VEGF also stimulated the mitogen activated protein (MAP) kinases ERK (extracellular signal-regulated kinase) and p38 (stress activated protein kinase-2), but not SAPK1/JNK (stress activated protein kinase-1/c-Jun NH2-terminal kinase). Activation of p38 resulted in activation of MAP kinase activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). Inhibiting the VEGF-induced activation of ERK with PD098059 did not influence actin organization or cell migration but totally inhibited the VEGF-induced incorporation of thymidine into DNA. Inhibition of p38 activity by the specific inhibitor SB203580 led to an inhibition of HSP27 phosphorylation, actin reorganization and cell migration. The results indicate that the p38 pathway conveys the VEGF signal to microfilaments inducing rearrangements of the actin cytoskeleton that regulate cell migration. By modulating cell migration, p38 may thus be an important regulator of angiogenesis.
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PMID:p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. 939 75

Stress-activated protein kinase 2a, also called p38, is inhibited by SB 203580 and this drug has been used widely to implicate this enzyme in the regulation of many physiological processes. Here, we introduce a novel method of general application, which can be used to establish whether the effects of SB 203580 are mediated via inhibition of stress-activated protein kinase 2a/p38 or whether they result from 'non-specific' effects. Four events thought to occur upon activation of stress-activated protein kinase 2a/p38 have been established unequivocally. These are the activation of mitogen-activated protein kinase-activated protein kinase-2 and mitogen- and stress-activated protein kinase-1 and the phosphorylation of their presumed substrates, heat shock protein 27 and the transcription factor cyclic AMP response element binding protein, respectively. In contrast, the SB 203580-induced activation of c-Raf is independent of stress-activated protein kinase 2a/p38 inhibition.
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PMID:Use of a drug-resistant mutant of stress-activated protein kinase 2a/p38 to validate the in vivo specificity of SB 203580. 1037 Nov 63


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