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)

The Rel-associated protein pp40 is functionally related to I kappa B, an inhibitor of the transcription factor NF-kappa B. Purified pp40 inhibits the DNA binding activity of the NF-kappa B protein complex (p50:p65 heterodimers), p50:c-Rel heteromers, and c-Rel homodimers. The sequence of the complementary DNA encoding pp40 revealed similarity to the gene encoding MAD-3, a protein with mammalian I kappa B-like activity. Protein sequencing of I kappa B purified from rabbit lung confirmed that MAD-3 encodes a protein similar to I kappa B. The sequence similarity between MAD-3 and pp40 includes a casein kinase II and consensus tyrosine phosphorylation site, as well as five repeats of a sequence found in the human erythrocyte protein ankyrin. These results suggest that rel-related transcription factors, which are capable of cytosolic to nuclear translocation, may be held in the cytosol by interaction with related cytoplasmic anchor molecules.
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PMID:Rel-associated pp40: an inhibitor of the rel family of transcription factors. 189 14

Modification of cellular proteins via phosphorylation is known to be a major regulatory mechanism whereby external stimuli control intracellular events. We demonstrated that bacterial LPS induced a distinct set of phosphorylated protein (pp) in murine peritoneal macrophages, and that the LPS-induced pp were specifically located in cytosol and/or membrane fractions. One of the most heavily phosphorylated substrate proteins with a molecular mass of 65 kDa (pp65) was purified to homogeneity via SDS-PAGE analysis and autoradiography by sequential chromatography on Sephacryl S-200, HPLC anion exchange, and hydroxyapatite HPLC. Our pp65 is apparently the first purified LPS-induced pp, and is thought to be a novel protein. Serine residues on pp65 were found to be exclusively phosphorylated, indicating a contribution by LPS-inducible serine kinase. Interestingly, LPS-induced phosphorylation of pp65 was not observed in macrophages from a LPS-nonresponsive C3H/HeJ strain of mice, although their macrophages had about the same amounts of unphosphorylated p65 as normal macrophages when detected under Western blot analysis by using polyclonal anti-pp65 antibodies. This suggests that the functional defect of C3H/HeJ macrophages exists somewhere in the process before the pp65 phosphorylation. Moreover, the degree of the pp65 phosphorylation in macrophages stimulated with LPS or lipd A correlated well to that of cellular responses such as IL-1 production in the same macrophages. Considering these observations, the pp65 seems to play a crucial role in macrophage activation, and the studies on the structure and function of the pp65 should lead to progress in our understanding of the mechanisms of macrophage activation by LPS.
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PMID:Purification and characterization of the 65-kDa protein phosphorylated in murine macrophages by stimulation with bacterial lipopolysaccharide. 202 84

Antisera raised against two mitosis-specific protein kinases from human cells recognized a single 65-kDa polypeptide (p65) that is present in similar amounts in interphase and mitotic cell extracts. Immunoblot analysis of reduced and unreduced extracts revealed that p65 exists as a 65-kDa monomer during interphase but forms a 130-kDa disulfide-linked homodimer during mitosis. Several different antibodies recognizing the p34cdc2 protein kinase and cyclin B components of M phase-promoting factor (MPF) coprecipitated p65 from mitotic but not from interphase extracts. In addition, an anti-p65 immunoaffinity column substantially depleted mitotic extracts of histon H1 kinase activity assayed under conditions diagnostic for MPF. These results suggest that active human MPF may be a complex of p34cdc2, cyclin B, and dimeric p65. A sulfhydryl cycle, proposed in the earlier literature on the biochemistry of mitosis, might underlie the dimerization of p65 and formation of active MPF.
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PMID:Identification of mitosis-specific p65 dimer as a component of human M phase-promoting factor. 217 7

We examined the effect of insulin on nuclear factor kappa B (NF-kappa B) activity in Chinese ovary (CHO) cells overexpressing wild-type (CHO-R cells) or -defective insulin receptors mutated at Tyr1162 and Tyr1163 autophosphorylation sites (CHO-Y2 cells). In CHO-R cells, insulin caused a specific, time-, and concentration-dependent activation of NF-kappa B. The insulin-induced DNA-binding complex was identified as the p50/p65 heterodimer. Insulin activation of NF-kappa B: 1) was related to insulin receptor number and tyrosine kinase activity since it was markedly reduced in parental CHO cells which proved to respond to insulin growth factor-1 and phorbol 12-myristate 13-acetate (PMA) activation, and was dramatically decreased in CHO-Y2 cells; 2) persisted in the presence of cycloheximide and was blocked by pyrrolidine dithiocarbamate, aspirin and sodium salicylate, three compounds interfering with I kappa B degradation and/or NF-kappa B.I kappa B complex dissociation; 3) was independent of both PMA-sensitive and atypical (zeta) protein kinases C; and 4) was dependent on Raf-1 kinase activity since insulin-stimulated NF-kappa B DNA binding activity was inhibited by 8-bromo-cAMP, a Raf-1 kinase inhibitor. Moreover, insulin activation of NF-kappa B-driven luciferase reporter gene expression was blocked in CHO-R cells expressing a Raf-1 dominant negative mutant. This is the first evidence that insulin activates NF-kappa B in mammalian cells through a post-translational mechanism requiring both insulin receptor tyrosine kinase and Raf-1 kinase activities.
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PMID:Insulin activates nuclear factor kappa B in mammalian cells through a Raf-1-mediated pathway. 759 58

T-cell activation requires two different signals. The T-cell receptor's recognition of a specific antigen on antigen-presenting cells provides one, and the second signal comes from costimulatory molecules such as CD28. In contrast, T cells that are stimulated with antigen in the absence of the CD28 costimulatory signal can become anergic (nonresponsive). The CD28 response element (CD28RE) has been identified as the DNA element mediating interleukin 2 (IL-2) gene activation by CD28 costimulation. Our previous work demonstrates that the Rel/NF-kappa B family proteins c-Rel, RelA (p65), and NFKB1 (p50) are involved in the complex that binds to the CD28RE. We also showed that c-Rel, but not NFKB1 (p50), can bind to the CD28RE and activate CD28RE-driven transcription in cotransfection assays. However, the role of RelA (p65) in CD28 signaling has not yet been addressed. We provide evidence that RelA (p65) itself bound directly to the CD28RE of the IL-2 promoter and other lymphokine promoters. In addition, RelA (p65) was a potent transcriptional activator of the CD28RE in vivo. We show that a RelA (p65)-c-Rel heterodimer bound to the CD28RE and synergistically activated the CD28RE enhancer activity. We also demonstrate that activated Raf-1 kinase synergized with RelA (p65) in activating the CD28RE enhancer activity. Interestingly, a soluble anti-CD28 monoclonal antibody alone, in the absence of other stimuli, also synergized with RelA (p65) in activating the CD28RE. Furthermore, we show that RelA (p65) activated expression of the wild-type IL-2 promoter but not the CD28RE-mutated IL-2 promoter. A combination of RelA (p65) and NFKB1 (p50) also activated the IL-2 promoter through the CD28RE site. These results demonstrate the functional regulation of the CD28RE, within the IL-2 promoter, by Rel/NF-kappa B transcription factors.
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PMID:RelA is a potent transcriptional activator of the CD28 response element within the interleukin 2 promoter. 762 20

RNA-dependent protein kinase is a M(r) 68,000 protein in human cells (p68 kinase) or a M(r) 65,000 protein in murine cells (p65 kinase). p65/p68 is a serine/threonine kinase induced by interferon treatment and generally activated by double-stranded RNAs. Once activated, the known function of this kinase is inhibition of protein synthesis through phosphorylation of the eukaryotic initiation factor 2. Here we have investigated the potential for tumorigenicity in mice of murine NIH 3T3 clones expressing human p68 kinase, either the wild-type or a mutant inactive kinase with a single amino acid substitution in the invariant lysine-296 in the catalytic domain II. Expression of the mutant p68 kinase was correlated with a malignant transformation phenotype, giving rise to the production of large tumors of at least 1 cm in diameter within 7-12 days in all inoculated mice. In contrast, no tumor growth was observed for several weeks in mice inoculated with NIH 3T3 cell clones expressing either the wild-type recombinant p68 kinase or only the endogenous p65 kinase, the murine analogue of the p68 kinase. These results suggest that functional p65/p68 kinase (recently called PKR), by a still undefined mechanism, may also act as a tumor suppressor. Consequently, one of the pathways by which interferon inhibits tumor growth might be through its capacity to induce the enhanced expression of this kinase.
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PMID:Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase. 767 39

Intracellular protein phosphorylation is thought to be the initial step in cell activation. Bacterial lipopolysaccharide (LPS) induces a special set of the protein phosphorylation in the murine peritoneal macrophages, including p65 (molecular mass of 65 kDa) which is a substrate of serine kinase and the most dominant phosphorylated cytosolic protein. This article deals with the relation between the LPS-induced protein phosphorylation in the murine peritoneal macrophages and their productions of IL-1 beta and TNF-alpha. LPS-induced p65 phosphorylation seems to be dependent on protein kinase C (PKC) and calmodulin (CaM), because it diminishes in the presence of inhibitors to PKC or CaM. Tyrosine kinase inhibitors do not affect the p65 phosphorylation. The PKC inhibitors also affect the mRNA expressions and the productions of active molecules of IL-1 beta and TNF-alpha. Though the CaM inhibitor inhibits the mRNA expression and the active molecule production of IL-1 beta, it does not affect those of TNF-alpha. These results suggest that LPS-induced p65 phosphorylation is closely related to PKC and CaM, and that IL-1 beta production depends on PKC and CaM, while the TNF-alpha production is not dependent on CaM. These findings indicate the existence of multiple pathways and different regulatory mechanisms for transduction of LPS signal in the macrophages. Furthermore, LPS-induced phosphorylation is not observed in endotoxin tolerant macrophages after re-stimulation with LPS, suggesting that the LPS-stimulus signal is blocked at a site in the signal transduction-pathway before the point of phosphorylation of proteins in the tolerant macrophages.
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PMID:Intracellular protein phosphorylation in murine peritoneal macrophages in response to bacterial lipopolysaccharide (LPS): effects of kinase-inhibitors and LPS-induced tolerance. 768 35

Protein hyper- or hypophosphorylation induced by okadaic acid (OA) treatment was examined using quiescent cultures of the BALB/MK-2 mouse epidermal keratinocytes. Treatment with OA enhanced the phosphorylation of five proteins with molecular weights of 65,000, 55,000, 50,000, 28,000 and 15,000 (p65, p55, p50, p28, and p15, respectively) and decreased that of two proteins with molecular weights of 22,000 and 20,000 (p22 and p20, respectively). The two major phosphorylated proteins, p65 and p55, were identified as type II and type I keratins, respectively, by immunoblotting and immunoprecipitation with keratin specific antibodies. Serine was the only phosphoamino acid residue in hydrolysates of the 32P-labeled keratins purified from OA-treated cells. Two-dimensional tryptic peptide maps of the phosphorylated keratins showed that the hyperphosphorylation was largely due to phosphorylation at several additional sites in both keratins. The hyperphosphorylation of keratins induced by OA treatment resulted in a drastic change in their solubility. This change closely correlated with reorganization of the keratin filament network, which finally collapsed into large perinuclear aggregates. Concomitantly the cells changed from a typical epithelial shape to a round shape. Of several protein kinase inhibitors tested, only staurosporine interfered with this OA-induced morphological change and reorganization of the keratin network.
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PMID:Hyperphosphorylation of keratins by treatment with okadaic acid of BALB/MK-2 mouse keratinocytes. 769 89

Stimulation of the protein kinase A (PKA) signalling pathway exerts an inhibitory effect on the proliferation of numerous cells, including T lymphocytes. In CD4+ T helper cells, stimulation of PKA leads to suppression of interleukin 2 (IL-2) induction, while induction of the genes coding for the lymphokines IL-4 and IL-5 is enhanced. We show that the differential effect of PKA activity on induction of the IL-2 and IL-4 genes is mediated through their promoters. One major target of the suppressive effect of PKA is the kappa B site in the IL-2 promoter. A kappa B site is missing in the IL-4 promoter. Mutations preventing factor binding to the IL-2 kappa B site result in a loss of PKA-mediated suppression of IL-2 promoter activity. Furthermore, activation of the PKA signalling pathway impairs the inducible activity of multiple kappa B sites of the IL-2 promoter, but not of other factor binding sites. The reduction in activity of kappa B sites in activated and PKA-stimulated T cells is accompanied by changes in the concentration and DNA binding of Rel/NF-kappa B factors. Stimulation of the PKA pathway in Jurkat T cells with the PKA activator forskolin leads to an increase in synthesis of c-Rel and p105/p50, while synthesis of p65/RelA remains unchanged. However, nuclear translocation and DNA binding of p65 is distinctly impaired, probably due to a retarded degradation of I kappa B-alpha. In a similar way, stimulation of the PKA signalling pathway inhibits nuclear translocation of p65 and generation of nuclear kappa B complexes in peripheral T lymphocytes from murine lymph nodes. These results indicate that PKA-mediated suppression of NF-kappa B activity plays an important role in the control of activation of peripheral T lymphocytes.
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PMID:RelA/p65 is a molecular target for the immunosuppressive action of protein kinase A. 774 6

Nuclear factor kappa B (NF-kappa B), consisting of p50 and p65, is bound to a cytoplasmic retention protein, I kappa B, in a resting state, and the stimulation of cells with a variety of inflammatory stimuli induces the dissociation of NF-kappa B from I kappa B and the nuclear translocation of NF-kappa B, thereby activating several genes involved in inflammatory responses, such as interleukin (IL)-6, IL-8, and tumor necrosis factor alpha. In order to elucidate the precise mechanism of NF-kappa B activation, we have established lipopolysaccharide (LPS)-dependent NF-kappa B activation in a cell-free system using plasma membrane-enriched, cytosol, and nuclear fractions extracted from a human monocytic cell line, THP-1, by disruption with sonication followed by a differential centrifugation. The combination of plasma membrane-enriched fraction and cytosol was sufficient to activate NF-kappa B in a LPS/CD14-dependent manner only in the presence of ATP as judged by the binding of NF-kappa B to the IL-8 gene kappa B site on an electrophoretic mobility shift assay. LPS-dependent NF-kappa B activation was inhibited by protein kinase inhibitors, such as staurosporine, herbimycin A, tyrphostin, and genistein, but not mitogen-activated protein kinase substrate, cGMP-dependent protein kinase, cAMP-dependent protein kinase, protein kinase C, and calmodulin-dependent protein kinase II inhibitory peptides, suggesting that staurosporine-sensitive kinase(s) as well as tyrosine kinase(s) are involved in LPS-mediated NF-kappa B activation. In addition, LPS induced the phosphorylation of I kappa B-alpha, starting at 5 min after the stimulation in a cell-free system. Moreover, the phosphorylation was inhibited by herbimycin A and tyrphostin, but not staurosporine, suggesting that these protein kinase inhibitors act at distinct steps of signal transmission. Establishment of ligand-dependent activation of NF-kappa B in a cell-free system will facilitate identification of protein kinase(s) and its substrate(s) involved in LPS-mediated NF-kappa B activation.
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PMID:Establishment of lipopolysaccharide-dependent nuclear factor kappa B activation in a cell-free system. 787 68


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