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)

The receptors for polypeptide growth factors and proteins coded by oncogenes of the src family are endowed with protein kinase activity and share the uncommon property of autophosphorylating at tyrosine residues. It is unclear whether the tyrosine kinase activity is also directed towards other targets of physiological significance. In this work, phosphotyrosine antibodies were used to detect, by Western blots and immunoprecipitation, proteins phosphorylated at tyrosine in fibroblasts either stimulated by growth factors (PDGF and EGF) or transformed by oncogene-coded tyrosine kinases. In stimulated cells the antibodies detected the autophosphorylated receptors, but only trace amounts of other proteins phosphorylated at tyrosine. In fibroblasts transformed by retroviral oncogenes (v-src, v-abl, v-fps or v-fes) proteins other than the corresponding oncogene-coded kinase, were found. A p70 was found to be heavily phosphorylated in fibroblasts transformed by v-src, v-fes and v-fps. A p130 and a p36 were found in cells transformed by v-src and v-abl. A unique p70 was phosphorylated in v-abl-transformed fibroblasts. These proteins were also phosphorylated in vitro in an immunocomplex kinase reaction. This reaction was blocked by the specific kinase inhibitors. These data strongly suggest that tyrosine kinases phosphorylate protein targets other than themselves. These targets are barely detectable in normal cells stimulated by growth factors, where the kinase activity is triggered rapidly and transiently. By contrast, a number of intracellular proteins phosphorylated at tyrosine accumulate in cells transformed by v-onc-coded kinases, endowed with constitutive and non-regulated enzymatic activity.
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PMID:Immunological detection of proteins phosphorylated at tyrosine in cells stimulated by growth factors or transformed by retroviral-oncogene-coded tyrosine kinases. 242 7

We have reported previously that the addition of dexamethasone to cultured quiescent suckling rat hepatocytes in the presence of insulin, a culture condition which does not cause growth activation, induces a selective increase in the synthesis of the 49-kD/55-kD cytokeratin (CK49/CK55) pair over a 24-h period. This increased synthesis coincides with the formation of dense filament networks reminiscent of those observed in situ at the cell periphery (Marceau, N., H. Baribault, and I. Leroux-Nicollet. 1985. Can. J. Biochem. Cell Biol. 63:448-457). We show here for the first time that when EGF is added 48 h after insulin and dexamethasone, there is an early preferential phosphorylation of the CK55 of the CK49/CK55 pair, an induced filament rearrangement from the cell periphery to the cytoplasm, and a subsequent entry into S phase and mitosis after a lag period of 8 h. Indirect immunofluorescence microscopy with monoclonal antibodies to CK49 and CK55 indicate that, while before EGF treatment the cytokeratin filaments were mainly distributed near the cell periphery, the addition of EGF resulted in their reorganization to a predominantly cytoplasmic localization within less than 3 h. Antitubulin and anti-actin antibodies showed no detectable alteration in the distribution of microtubules and microfilaments. Pulse-chase measurements with [35S]methionine showed no apparent change in the turnover of either CK49 or CK55 during the period that precedes the initiation of DNA synthesis. 32P-labeling in vivo followed by SDS-PAGE demonstrated that CK55 was phosphorylated at a much higher level than CK49 in nonstimulated hepatocytes, and that the addition of EGF resulted in a selective stimulation of 32P-CK55 labeling within less than 30 min. Comparative analyses by two-dimensional PAGE of [35S]methionine and 32P-labeled cytokeratins at various times after EGF stimulation demonstrated a rapid increase in a first phosphorylated form of CK55 and the appearance of a second phosphorylated form at 30 min poststimulation. The changes in the relative proportion of nonphosphorylated and phosphorylated forms were confirmed by immunoblotting with the anti-CK55 monoclonal antibody. Determinations of the 32P-labeled phosphoamino acids of CK55 extracted from the gels demonstrated that the radioactivity was mostly in serine residues. Labeling of Triton-permeabilized hepatocytes with gamma 32P-ATP after treatment with EGF for 30 min to 3 h at 37 degrees C, also demonstrated a phosphorylation of CK55 and CK49 as well, implying that the EGF-responsive serine protein kinase is detergent insoluble and probably part of the surface membrane skeleton.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Epidermal growth factor-induced selective phosphorylation of cultured rat hepatocyte 55-kD cytokeratin before filament reorganization and DNA synthesis. 247 79

Recently, some groups including ours found that some ganglioside species could specifically modulate the certain protein kinase activities on the cell surface membrane. We show here two representative cases. One is the ganglioside-dependent modulation of autophosphorylation of EGF or PDGF receptors, by which altered the cell growth. Another is the ganglioside dependent ecto-type protein kinase activities on the cell surface of a human neuroblastoma cell line, GOTO. The latter activities may be closely correlated with the ganglioside GQ1b dependent neuritogenesis.
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PMID:[Ganglioside modulation of protein kinase activities: it's implication in cell growth and differentiation]. 251 1

The insulin receptor, a glycoprotein consisting of two extracellular alpha- and two transmembrane beta-subunits, is thought to mediate hormone action by means of its tyrosine-specific protein kinase activity. To explore the mechanism of insulin receptor phosphorylation we have used NIH3T3 cells transfected with two receptor constructs: one encoding a chimeric receptor composed of the extracellular domain of the human EGF receptor and the cytosolic domain of the human insulin receptor beta-subunit, and a second construct encoding a kinase-defiecient human insulin receptor. Stimulation of these cells with EGF induced tyrosine autophosphorylation of the EGF-insulin receptor chimera (150 kd) and tyrosine phosphorylation of the beta-subunit of the kinase-deficient insulin receptor (95 kd). The phosphopeptides of the autophosphorylated cytoplasmic domain of the EGF-insulin receptor chimera were comparable to those of the transphosphorylated beta-subunit of the kinase-deficient insulin receptor and of the wild-type human insulin receptor. When immunoaffinity purified EGF-insulin receptor hybrids and kinase-deficient insulin receptors were used in a cell lysate phosphorylation assay, it was found that addition of EGF produced 32P-labeling of both receptor species. We conclude that EGF acting directly through the EGF-insulin receptor chimera causes transphosphorylation of the kinase-deficient insulin receptor. These data support the notion that autophosphorylation of the insulin receptor may proceed by an intermolecular mechanism.
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PMID:Intermolecular transphosphorylation between insulin receptors and EGF-insulin receptor chimerae. 258

The effect of the human rIL-1 alpha and rTNF-alpha on the binding of 125I-labeled epidermal growth factor ([125I]EGF) to its receptor (EGF-R) has been studied in human gingival fibroblasts (HuGi). Incubation of these cells with recombinant cytokines at 37 degrees C caused a rapid, dose-dependent decrease in their ability to subsequently bind subsaturating levels of [125I]EGF at 4 degrees C. Inhibition was evident at 5 min after addition of cytokines, reached a maximal level (60-70% reduction) after 15 to 30 min, and declined thereafter. Normal EGF binding was attained by 2 h. Half-maximal inhibition of EGF binding occurred at 10 pM IL-1 and 50 pM TNF. The two cytokines were not additive in their effect. Competition experiments at 4 degrees C showed that the cytokines did not interact directly with EGF-R; Scatchard analysis of binding of [125I]EGF to HuGi after treatment with IL-1 and TNF revealed an increase in EGF-R Kd from 0.75 nM to 2.9 nM with no change in receptor number. The effect of IL-1 and TNF on EGF-R was compared with that of the tumor-promotor PMA which is known to "transmodulate" EGF-R affinity by activating protein kinase C which then phosphorylates EGF-R. PMA caused a greater inhibition of EGF binding to HuGi (80 to 85% inhibition; ED50 = 500 pM), and recovery of binding was much slower. Importantly, in HuGi made deficient in protein kinase C by prolonged incubation with PMA, addition of fresh PMA no longer affected EGF binding, while the response to IL-1 and TNF was intact. Cytokine- but not PMA-mediated EGF-R transmodulation was partially reversed by treatment of the cells with millimolar concentrations of the kinase inhibitor amiloride. HuGi were incubated with H3 32PO4, stimulated with PMA or cytokines, and EGF-R were immunoprecipitated; IL-1 and TNF, like PMA, caused a 2- to 5-fold increase in receptor phosphorylation. We conclude that occupation of IL-1 and TNF-R activates a protein kinase, distinct from kinase C, for which EGF-R is a substrate.
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PMID:IL-1 and TNF transmodulate epidermal growth factor receptors by a protein kinase C-independent mechanism. 278 20

Protamine sulfate (PS), a specific blocker of PDGF action, inhibited the proliferative response of tsKSV-NRK cells to a reactivated, temperature-sensitive viral Ki-RAS protein, but it did not affect the proliferative response of tsASV-NRK cells to a reactivated pp60v-src protein kinase. The inhibition by PS of the proliferation response of tsKSV-NRK cells to reactivated Ki-RAS protein was overcome by serum growth factors, notably EGF, and concentrated serum-free conditioned medium from cultured NRK cells infected with wild-type KSV, but not by a combination of PDGF and insulin. These observations suggest that the viral Ki-RAS protein, but not pp60v-src, stimulates proliferation exclusively by inducing the host cells to produce PDGF or PDGF-like mitogenic factors.
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PMID:Evidence that the viral Ki-ras protein, but not the pp60v-src protein of ASV, stimulates proliferation through the PDGF receptor. 282 9

During the course of studies on protein kinases in psoriatic epidermis, a novel histone-activated protein kinase activity was identified. This activity (referred to as PK-II because it was the second peak of protein kinase activity eluted from a DEAE column) was partially purified from the supernatant of an epidermal homogenate by DEAE-cellulose column chromatography. Although histone was not a substrate for phosphorylation, in the presence of histone, endogenous proteins of Mr 105 and 95 kDa were phosphorylated. Activity was not affected by Ca2+/phospholipid, cAMP, cGMP, cAMP-dependent kinase inhibitor, spermine, spermidine, calmodulin, EGF, or phorbol ester. Phosphorylation was specific for serine and threonine residues. A major peak of PK-II activity eluted from sepharose 6B with an apparent Mr of 100 kDa, suggesting that histone may stimulate autophosphorylation. The properties of PK-II resemble those recently described for a class of polypeptide-dependent protein kinases isolated from placenta, Ehrlich ascites tumor cells, and bakers' yeast. PK-II was significantly higher in psoriatic involved epidermis (32.6 +/- 11.6 pmol/min/mg protein) compared to psoriatic uninvolved epidermis (5.7 +/- 0.6 pmol/min/mg; p = 0.03) and normal epidermis (9.5 +/- 2.2 pmol/min/mg; p = 0.05). The function of histone stimulated protein kinase in epidermal function and its role in the pathophysiology of psoriasis remain to be explored.
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PMID:A novel histone-stimulated protein kinase in normal and psoriatic epidermis. 291 42

Treatment of [32P]phosphate prelabeled intact human A431 epidermoid carcinoma cells with epidermal growth factor (EGF, 100 ng/ml) or 12-O-tetradecanoylphorbol-13-acetate (TPA, 10(-7)M) resulted in a selective enhancement in the phosphorylation of the following soluble acidic proteins: a phosphoprotein with a molecular weight of 17,000 (pp17; similar notation used throughout) pI approximately 5.5); pp27 (pI approximately 5.5); pp34 (pI approximately 6.2); and pp80 (pI approximately 4.5) as detected by two-dimensional gel electrophoresis. EGF or TPA induced a 4- to 6-fold increase in the phosphorylation of pp17 and a 2- to 4-fold increase in the phosphorylation of pp27, pp34, and pp80 within 15 min after treatment of subconfluent A431 cells. Alkali treatment of the gels removed most of the incorporated [32P] phosphate from the phosphoproteins, including pp27, pp34, and pp80; however, the phosphoester bond in pp17 was stable to alkaline hydrolysis since there was no removal of [32P]phosphate from this protein. Treatment of A431 cells with dibutyryl cyclic adenosine 3':5'-monophosphate (1 mM) also increased the phosphorylation of pp17, pp27, and pp34 but not of pp80. Activation of endogenous calcium- and phospholipid-dependent protein kinase C in the cytosol of A431 cells in a cell-free system resulted in the enhanced phosphorylation of pp27, pp34, and pp80 but not of pp17 while exogenous addition of the catalytic subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase to cytosol preparations resulted in the phosphorylation of pp17, pp27, and pp34, but not of pp80. These results demonstrate that at least four soluble acidic proteins are phosphorylated in A431 cells in response to either EGF or TPA in vivo suggesting that these two agents may exert part of their biological effects on A431 cells through a biochemical pathway involving the phosphorylation of several common proteins; moreover, the studies suggest that these four acidic proteins may be substrates in vitro for protein kinase C and/or a cyclic adenosine 3':5'-monophosphate-dependent protein kinase.
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PMID:Effect of epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate on the phosphorylation of soluble acidic proteins in A431 epidermoid carcinoma cells. 301 83

Plasma membranes have been isolated from the human epidermoid carcinoma cell line A431 by a rapid fractionation of lysate on Percoll density gradient at pH 9.6. Endoplasmic reticulum, lysosomes and mitochondria sedimented at the bottom of gradient whereas plasma membranes focused at low density, as shown with specific markers. Plasma membranes displayed a 4.5- and 4.4-fold enrichment in [3H]concanavalin A and 5'-nucleotidase, respectively. This proteic fraction was further characterized by its lipid composition and phospholipid analysis. The cholesterol/phospholipid molar ratio was 0.45 in plasma membranes against 0.19 in lysate. Sphingomyelin increased from 7.5% of total phospholipids in lysate to 16.2% in plasma membranes, as well as phosphatidylserine which displayed a 1.5-fold enrichment in the plasma membrane fraction. This was at the expense of phosphatidylcholine (45.2% in lysate, against 35% in plasma membranes). Electron microscopy of the isolated material showed vesicles essentially free from endoplasmic reticulum and organelles. These plasma membranes retained the ability to bind 125I-labelled epidermal growth factor (125I-EGF) with a Kd = 4.7 nM and Bmax = 63 pmol/mg protein. EGF binding resulted in a stimulation of the phosphorylation protein reaction in the presence of [gamma-32P]ATP and sodium dodecyl sulfate polyacrylamide gels of phosphorylated proteins indicated that the radioactivity of the major band of molecular weight 170,000 was clearly enhanced by EGF binding. These results indicate that the EGF receptor and its intrinsic protein kinase activity were preserved during our plasma membrane isolation procedure.
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PMID:Characterization of plasma membranes from A431 cells, isolated by self-generating Percoll gradient: a rapid isolation procedure to obtain plasma membranes with functional epidermal growth factor receptors. 325 34

Insulin-like growth factor 1 and insulin reduced the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells by 15-20% at 37 degrees C, but not at 4 degrees C. Scatchard analysis indicated that IGF-1 and insulin affected the higher-affinity component of EGF binding, an effect previously associated with the activation of protein kinase C. However, the inhibition of 125I-EGF binding by IGF-1 and insulin was increased, not reduced, when the cells were treated with high concentrations of phorbol esters to down-modulate protein kinase C. We suggest that IGF-1 and insulin activate a protein kinase with similar or overlapping specificity to that of protein kinase C.
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PMID:Insulin-like growth factor 1 and insulin reduce epidermal growth factor binding to Swiss 3T3 cells by an indirect mechanism that is apparently independent of protein kinase C. 328 69


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