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Results for “Protein phosphorylation”

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PubMed1989

Protein phosphorylation during 1-methyladenine-induced maturation of Asterias oocytes.

Maturation was induced in Asterias oocytes with 1-methyladenine (1-MA) at a final concentration of 2 microM. At 5, 10, and 30 min of treatment, oocytes were homogenized and the cytosolic fraction was prepared. The cytosol was incubated with [gamma-32P]ATP and [gamma-32P]GTP. The phosphorylated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the radioactivity in the gels was determined by autoradiography. The cytosol prepared from 1-MA-treated oocytes incubated with [gamma-32P]ATP showed a marked increase in the radiolabeling of proteins with estimated molecular weights of 70,000 and 62,000 Da. With [gamma-32P]GTP a 56,000-Da protein showed increased radiolabeling. The present finding suggests that an early biochemical event of 1-MA-induced oocyte maturation in Asterias is the stimulation of phosphorylation of specific proteins.

Adenine↗

PubMed1993

Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus.

In vitro phosphorylation reactions using extracts of Streptomyces griseus cells and gamma-[32P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1984

Effect of phorbol 12-myristate 13-acetate and its analogue 4 alpha-phorbol 12,13-didecanoate on protein phosphorylation and lysosomal enzyme release in rabbit neutrophils.

The co-carcinogenic compound phorbol 12-myristate 13-acetate but not its inactive analogue 4 alpha-phorbol 12,13-didecanoate causes the phosphorylation of several rabbit neutrophil polypeptides whose molecular weights and isoelectric points (pI) are as follows: Mr = 40,000, pI = 6.4; Mr = 50,000, pI = 4.9; Mr = 55,000, pI = 6.3; Mr = 64,000, pI = 6.0; Mr = 70,000, pI = 5.6; Mr = 90,000, pI = 6.0. Most of these phosphorylated proteins are located exclusively in the cytosol; the 64,000 molecular weight protein is found both in the cytosol and the cytoskeleton, and the 40,000 molecular weight protein is found in the nuclear pellet. The 50,000 molecular weight protein is also phosphorylated in whole cells by the chemotactic peptide fMet-Leu-Phe and in cell-free systems by protein kinase C. Using limited proteolysis, one phosphopeptide fragment was phosphorylated by the three stimuli. In addition, phorbol 12-myristate 13-acetate but not 4 alpha-phorbol 12,13-didecanoate causes cell aggregation and the exocytotic release of the specific granules of rabbit neutrophils. In contrast, both compounds increase the amount of actin associated with the cytoskeleton. The divalent cation ionophore A23187 at low concentration and the compound phorbol 12-myristate 13-acetate act synergistically in causing neutrophil degranulation. Lysosomal enzyme release and the phosphorylation of the 50,000 molecular weight polypeptide produced by phorbl 12-myristate 13-acetate are inhibited by trifluoperazine, and these two responses seem to be causally related. These results are discussed in terms of the role of 1,2-diacylglycerol and activation of protein kinase C in specific granule release from rabbit neutrophils.

Animals↗

PubMed1999

Sequence and structure-based prediction of eukaryotic protein phosphorylation sites.

Protein phosphorylation at serine, threonine or tyrosine residues affects a multitude of cellular signaling processes. How is specificity in substrate recognition and phosphorylation by protein kinases achieved? Here, we present an artificial neural network method that predicts phosphorylation sites in independent sequences with a sensitivity in the range from 69 % to 96 %. As an example, we predict novel phosphorylation sites in the p300/CBP protein that may regulate interaction with transcription factors and histone acetyltransferase activity. In addition, serine and threonine residues in p300/CBP that can be modified by O-linked glycosylation with N-acetylglucosamine are identified. Glycosylation may prevent phosphorylation at these sites, a mechanism named yin-yang regulation. The prediction server is available on the Internet at http://www.cbs.dtu.dk/services/NetPhos/or via e-mail to NetPhos@cbs. dtu.dk.

Amino Acid Motifs↗

PubMed1991

Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine.

A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine. The gene was designated SPK1 for serine-protein kinase. Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.

Amino Acid Sequence↗

PubMed1994

Specific protein phosphorylation induced in Xanthomonas campestris pv. oryzae by bacteriophage Xp12.

We have investigated the endogenous phosphorylation patterns of phosphorylated proteins of Xanthomonas campestris pv. oryzae induced by its bacteriophages. For bacteriophage Xp12-infected cells, at least three phosphoproteins with apparent molecular weights of 28, 28.5 and 45 kDa were detected by in vitro labeling with [gamma-32P]-ATP. These Xp12-specific phosphoproteins only occurred with Xp12 infection, and were not shown in uninfected or Xp10-infected cells. The protein kinase(s) responsible could use either ATP or GTP as the nucleotide substrate with nearly the same efficiency. Magnesium was proved to be an essential factor for the phosphorylation. EGTA treatment excluding the possibility that the presumed protein kinase was calcium-dependent. Under our reaction conditions, the optimal phosphorylation occurred at pH 7 to 8, for 30 to 40 min at 25 to 37 degrees C. The Xp12-specific protein phosphorylation hint the existence of a physiological regulation mechanism involved in the life cycle of bacteriophage Xp12. Furthermore, the presumed protein kinase was shown to be encoded by the genome of Xp12 rather than indirectly induced by Xp12 infection.

Adenosine Triphosphate↗

PubMed2005

Phospholipid metabolism and protein kinase C mediated protein phosphorylation in dietary protein deficiency in rat lung.

Nutritional deprivation of proteins decreases the protein kinase C (PKC) activity in rat lung. The activity of (PKC) is influenced by lipid metabolism. Changes in PKC activity may influence phosphorylation of its substrate proteins in the tissues. Therefore, alterations in phospholipid metabolism and PKC mediated protein phosphorylation in dietary protein deficiency in rat lung were envisaged. The study was conducted on rats fed on three different types of diet viz., casein (20% protein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threoning). Feeding of protein deficient diet caused reduction in incorporation of [3H] myo-inositol in the total phosphoinositides in lungs and an increase in total inositol phosphate pool. There was a significant reduction in the contents and turnover rate of phosphatidyl inositol and phosphatidyl inositol monophosphate. Supplementation of diet with L-lysine and DL-threonine had a reversing effect on total pool of phosphoinositides and, the metabolism of phosphatidyl inositol bisphosphate and phosphatidyl inositol. In phosphatidyl choline metabolism, the dietary protein deficiency led to a decrease in incorporation of [14C-methyl] choline-chloride in total phospholipids. In contrast, its incorporation increased in phosphatidyl choline pool. The contents of phosphatidyl choline and residue, incorporation of [14C-methyl] choline-chloride in them and their turnover rate also increased. Supplementation of diet had a reversal effect on most of these parameters. Phosphorylation of proteins of 84, 47, 35 and 16 kDa was identified to be mediated by PKC. In dietary protein deficiency, phosphorylation of all these proteins, except that of 47 kDa, increased. Supplementation of diet reversed the pattern except that of 84 kDa. The findings suggest that changes in phospholipid metabolism in dietary protein deficiency may effect the activity of PKC thereby influencing the phosphorylation of its substrate proteins and hence associated functions that may lead to pathophysiology of lung.

Animals↗

PubMed1990

A filter-based protein kinase assay selective for alkali-stable protein phosphorylation and suitable for acid-labile protein phosphorylation.

Alkali-stable phosphorylation of proteins, particularly phosphotyrosine and phosphohistidine, is an important phenomenon in cells. In the case of phosphohistidine and some other phosphoamino acids, the phosphorylation is acid-labile and in these cases studies have been severely limited by the absence of a rapid assay suitable for acid-labile phosphorylation. The assay presented here involves a conventional kinase assay reaction followed by mild alkaline hydrolysis and adsorption of the product to washed Nytran paper at high pH. After further washing, at pH 9, the radioactivity on the papers is determined by liquid scintillation counting. Hence, acid-labile phosphorylation is preserved. The assay is selective for alkali-stable phosphorylation but not fully specific, mainly due to the need to balance the severity of the partial alkaline hydrolysis with the stability of the protein-peptide bonds. The assay has been used for the purification and characterization of a protein histidine kinase from Saccharomyces cerevisiae.

Histidine↗

PubMed2006

Modulation of protein phosphorylation by Mr 25,000 protein partially overlapping phosvitin and lipovitellin 2 in Xenopus laevis vitellogenin B1 protein.

A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. In an attempt to elucidate the physiological role of pp25, its effect on protein phosphorylation was studied. In vitro phosphorylation of some endogenous proteins from the cytoplasm and membrane fraction of Xenopus oocytes by casein kinase II and protein kinase C (PKC) was inhibited by increasing the concentration of pp25. By Western blot analysis using an antibody against phospho-(Ser/Thr) PKC substrate, phosphorylation of some endogenous proteins, especially in the cytoplasm, of Xenopus embryos was seen to increase when pp25 disappeared during developmental stages 35-45. These results suggest that pp25 may have a role as an inhibitory modulator of some protein phosphorylation in Xenopus oocytes and embryos.

Animals↗

PubMedDate not supplied

Changes in the patterns of protein phosphorylation associated with granulocytic and monocytic-induced differentiation of HL-60 cells.

Using two-dimensional gel electrophoresis we have analyzed the pattern of phosphorylated proteins in HL-60 leukemia cells and changes associated with their differentiation into granulocyte and monocyte-like cells. In undifferentiated cells 18 spots with MW ranging from 110 to 17, kDa were individualized with high resolution and reproducibility. Myelocytic differentiation induced by dimethyl sulfoxide (DMSO) and retinoic acid (RA) resulted in a decrease of the overall phosphorylation, the disappearance of two proteins of 42 and 17 kDa, and the appearance of one new acidic protein of 46-48 kDa. These changes seem to be specifically related to this differentiation pathway since they are not found in two HL-60 subclones resistant to DMSO or RA-induced differentiation. Monocytic differentiation induced by 1,25-dihydroxyvitamin D3 [1,25 (OH)2D3] and the combination of RA + 1-B-D-arabinofuranosyl cytosine (Ara-C) was associated with the appearance of 2 proteins of 68 kDa and 2 proteins of 80 kDa located in the acidic region of the gel. The protein of 17 kDa, when disappeared completely in granulocytic-like cells was present in monocytic cells, this suggesting that its phosphorylation state may be involved in the control of the differentiation pathway of HL-60 cells. Data concerning the effect of phorbol myristate acetate (PMA) and histamine on the level of phosphorylation of various proteins in HL-60 cells have also been obtained and discussed. Our results show that the myelocytic and monocytic phenotypes are characterized by a specific pattern of phosphoproteins involving both phosphorylation and dephosphorylation reactions.

Calcitriol↗

PubMed1991

Protein phosphorylation and metalloproteinase synthesis by lapine articular chondrocytes.

Interleukin 1 (IL-1) increases the synthesis of neutral metalloproteinases (NMP) and prostaglandin E2 (PGE2) by primary monolayer cultures of lapine articular chondrocytes. Two-dimensional gel electrophoresis of 32P-labelled cells confirmed that IL-1 strongly altered the pattern of phosphorylated proteins in chondrocytes. This presumably reflects modulation of the activities of protein phosphatases, kinases or both. Despite considerable experimental effort, it proved impossible to implicate activation of protein kinase C (PKC) in the responses of these cells to IL-1. Additional evidence appears also to eliminate protein kinases activated by cyclic AMP and Ca2+/calmodulin. Moreover, recent data further suggest that the 80 kDa IL-1 receptor is not a protein kinase. These findings increase the likelihood that a novel kinase is involved in IL-1 signalling in chondrocytes, as proposed previously for fibroblasts.

Animals↗

PubMedDate not supplied

Cyclosporin treatment alters protein phosphorylation in kidney membranes.

Phosphorylation, protein carboxyl methylation, and ADP-ribosylation were assayed in renal basolateral membranes and brush border membranes isolated from rats treated by subcutaneous administration of 5 or 10 mg/(kg.day) of cyclosporin A (CsA) for 10 days to investigate potential alterations in signal transduction in kidney cortex. Protein carboxyl methylation of class II measured in membranes and in cytosolic fraction was not affected by CsA treatment. ADP-ribosylation performed in the presence of pertussis or cholera toxin was also similar in control and treated rats. However, changes in phosphorylation of endogenous substrates were observed in membranes and cytosol isolated from rats treated with 10 mg/(kg.day) of CsA. Phosphorylation was increased for two brush border membrane proteins (56 and 77 kilodaltons (kDa)) by 47 and 24% and for two basolateral membrane proteins (51 and 80 kDa) by 28 and 29%, respectively. In the cytosolic fraction, phosphorylation of two proteins (31 and 65 kDa) was increased by 37% and that of 25- and 43-kDa proteins was reduced by 29%. Protein kinase A, protein kinase C, and tyrosine protein kinase activities were also determined in membranes. Increases in protein kinase C and tyrosine protein kinase activities were observed in basolateral membranes, but not in brush border membranes after cyclosporin A administration. Endogenous substrates for tyrosine kinase were also detected with an antiphosphotyrosine (PY20) monoclonal antibody. Densitometric analysis indicated that the phosphorylation of three proteins of high molecular masses (61, 132, and 183 kDa) was stimulated by CsA in basolateral membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗

PubMed1982

Hormone-induced protein phosphorylation. I. Relationship between secretagogue action and endogenous protein phosphorylation in intact cells from the exocrine pancreas and parotid.

We undertook studies to determine whether secretagogue action on the exocrine pancreas and parotid is accompanied by phosphorylation of proteins in intact cells. For this purpose, rat pancreatic, and parotid lobules were preincubated with 32Pi for 45 min at 37 degrees C, washed, and then incubated at 37 degrees C in the presence or absence of secretagogues that effect discharge through different second messengers. Among a variety of polypeptides exhibiting enhanced phosphorylation in pancreatic lobules upon a 30-s incubation in the presence of the secretagogues carbamylcholine, cholecystokinin octapeptide, or secretin, one species with an Mr of 29,000 was especially notable for three reasons: (a) its enhanced level of phosphorylation was dependent on the dose of secretagogue used and was still apparent after incubation for 30 min at 37 degrees C; (b) an analogous phosphorylated polypeptide was observed in isoproterenol-stimulated parotid lobules; and (c) in both tissues its selective dephosphorylation was observed upon termination of stimulation by administration of atropine to carbamylcholine-stimulated pancreatic lobules and propranolol to isoproterenol-stimulated parotid lobules. These results suggest that the phosphorylation of one protein with an Mr of 29,000 is closely correlated both temporally and in a dose-dependent fashion with secretagogue action in both the exocrine pancreas and parotid.

Animals↗

PubMed1987

Protein phosphorylation in rat mast cell granules. Cyclic AMP dependent phosphorylation of a 44K protein associated with broken granules.

When rat mast cells are prelabeled with 32PO4 and exposed to non-immunologic or immunologic stimuli under conditions that lead to mediator release from granules, they show rapid increases in labeling of a number of high molecular weight proteins. To determine if granule membrane proteins are subject to protein phosphorylation and perhaps participate in this response, granules with intact or broken membranes were isolated from sonicated, purified rat serosal mast cells on a Percoll gradient. When the granules with broken membranes were incubated with [gamma-32P]ATP and Mg2+ in the absence of exogenous protein kinases, one major radioactive band was recovered in the 44K area after electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. The phosphorylation reaction with ATP required Mg2+, was enhanced by 0.05 to 0.5 microM cyclic AMP, and was inhibited by Ca2+ (0.5 mM and higher). The initial reaction was rapid, and the maximal response was seen at 30 degrees. The 44K band was absent in granules with intact membranes incubated with [gamma-32P]ATP but present when intact granules were lysed with distilled water before adding the [gamma-32P]ATP. These observations indicate that granules have an endogenous phosphorylating system and that the phosphorylation response is on the inner surface of the granule membranes. The possibility was not excluded that a portion of the phosphorylating activity was derived from the cytosol and became firmly associated with broken granules when the intact cells were sonicated. Analysis for possible phosphorylated amino acids in the 44K band after acid hydrolysis showed both phosphoserine and phosphothreonine, indicating that the radioactivity was in a phosphorylated protein or glycoprotein. The 44K phosphorylated protein was made up of several components ranging in pI from approximately 7.6 to 6.6. While the identity of the phosphorylated 44K polypeptide is uncertain, one important possibility is that it is part of an autophosphorylated cAMP dependent protein kinase. The cyclic AMP dependent phosphorylating activity present in granules provides a mechanism by which the granules might respond rapidly to cyclic AMP during mediator release.

Animals↗

PubMed1989

Prostaglandin F2 alpha administered in vivo induces Ca2+-dependent protein phosphorylation in rat luteal tissue.

The present study was performed in order to further elucidate the mechanism of action of PGF2 alpha in luteolysis in the rat ovary. Seven days after priming with superovulatory doses of pregnant mare serum gonadotropin and human chorionic gonadotropin to induce luteal tissue formation, the rats were injected with a luteolytic dose of the prostaglandin F2 alpha analogue cloprostenol. The ovaries were then homogenized, a 30,000 x g supernatant and pellet were prepared, whereafter aliquots of the preparations were incubated in the presence of [gamma-32P]ATP with or without Ca2+. The phosphorylated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and localized by autoradiography. The presence of Ca2+ caused an increased phosphorylation of a 45 kDa protein band in the particulate, but not in the cytosol, fraction. Furthermore, PGF2 alpha rapidly increased the 32P incorporation into the same protein band of 45 kDa. Thus, the PGF2 alpha-stimulated 32P incorporation was Ca2+-dependent and seen only in the particulate fraction. These results suggest that PGF2 alpha in its role as a luteolytic agent stimulates a Ca2+-dependent phosphorylation of a specific protein in luteal membranes of the rat ovary.

Animals↗

PubMed1993

Inhibition of chemotactic peptide-induced development of cell polarity and locomotion by the protein kinase C inhibitor CGP 41 251 in human neutrophils correlates with inhibition of protein phosphorylation.

Several protein kinase inhibitors of the staurosporine type displaying different enzyme specificity were used to study the role of protein kinase C (PKC) in motile neutrophil functions. Effects on protein phosphorylation, F-actin localization, morphology, and locomotion were determined. Only staurosporine, but not another inhibitor more specific for PKC (CGP 41 251) or inhibitors of cyclic nucleotide-dependent kinases (KT 5720, KT 5822), induced formation of F-actin-enriched surface protrusions at nanomolar concentrations in initially spherical neutrophils. The inhibitor with the highest specificity for PKC, CGP 41 251, had no effect on morphology and F-actin localization of resting cells up to 1 microM. However, this inhibitor suppressed front-tail polarity and locomotion in chemotactic peptide-stimulated cells. The cells became nonpolar with surface projections instead. Inhibition of front-tail polarity induced by CGP 41 251 strongly correlated with inhibition of in situ phosphorylation of a 67-kDa protein (IC50 = 0.2 microM), whereas induction of polarity by chemotactic peptide was associated with increased phosphorylation of this protein. Kinase inhibitors inactive on PKC (KT 5720, KT 5822) did not affect development of front-tail polarity. We conclude that a kinase involved in the phosphorylation of a 67-kDa protein, very likely a PKC isoform, may be important for the development of cell polarity and locomotion. In contrast, chemotactic peptide-induced formation of actin-containing protrusions may not be mediated by this enzyme.

Actins↗

PubMedDate not supplied

[Tyrosine-phosphorylated proteins in human breast carcinoma].

Proteins phosphorylated on tyrosine are detectable by antibodies against phosphotyrosine (P-Tyr antibodies) in cells transformed by oncogene-encoded tyrosine kinases. We used P-Tyr antibodies to investigate the existence of abnormal levels of phosphoproteins in human breast cancer. Three human breast cancer cell lines (SK-BR-3, MCF-7 and CG-5) and 37 human breast cancer specimens were examined by Western blot analysis and "in vitro" kinase assay. In the SK-BR-3 cell line three major phosphoproteins of the approximate Mr of 185,000 (p185), 135,000 (p135) and 110,000 (p110) were detected. The former was identified as the HER-2 gene product by specific antibodies against HER-2 encoded protein. In the other cell lines, a product of the approximate Mr of 170,000 (p170), together with a p135 and a p110, were phosphorylated on tyrosine. P185 and p170 were shown to have an associated tyrosine kinase activity. Two proteins, comigrating with p135 and p110, were found to be highly phosphorylated on tyrosine in 50% of the breast cancer samples, but not in samples harvested from 12 human tumors of the gastro-intestinal tract. These data show that 50% of human breast cancer samples display an abnormal level of tyrosine phosphorylated proteins.

Breast Neoplasms↗

PubMed2003

The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites.

Protein phosphorylation analysis is an enormous challenge. This review summarises the currently used techniques, which are based on radiolabelling and mass spectrometry as well as electrophoretic and chromatographic separation. Many methods exist, but there is still no single procedure applicable to all phosphoproteins. MS is able to deliver information about the location of phosphorylation sites, but phosphospecific properties with respect to ionisation present obstacles. Therefore, multidimensional approaches involving several analytical methods are often necessary to conquer phosphorylation site identification.

Chromatography, High Pressure Liquid↗