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Results for “Protein phosphorylation”

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At least 199 records · Page 11Linked to original sources

PubMed1990

Protein phosphorylation and kinase activities in tumour cells after hyperthermia.

Phosphorylation of various proteins and the activities of specific kinases were studied in tumour cells after hyperthermia. P388 lymphoid tumour cells were treated at 40-45 degrees C for 1 h in vitro. Immediately after heat treatment, particulate and cytosol cell fractions were isolated, phosphorylated proteins separated and various kinase activities were measured. Hyperthermic treatment of the cells caused a significant decrease in protein kinase C activity while the activity of calcium-ion and phospholipid-independent protein kinases increased. Phosphorylation of cytosol proteins of 120, 80, 33, 25 and 14 kDa increased significantly after hyperthermia, and protein kinase C selectively phosphorylated the last three of these proteins. The phosphorylation of three heat shock proteins (44, 70 and 85 kDa) was not changed after hyperthermic treatment. Four tyrosine kinase activities were separated. The protein tyrosine kinase activity decreased to one-tenth of the control value after 45 degrees C for 1 h hyperthermia. The changes in kinase activities and protein phosphorylation induced by hyperthermia proved to be temperature- and time-dependent.

Animals↗

PubMed2005

Bidirectional regulation of cytoplasmic polyadenylation element-binding protein phosphorylation by Ca2+/calmodulin-dependent protein kinase II and protein phosphatase 1 during hippocampal long-term potentiation.

Induction of hippocampal long-term potentiation (LTP) requires activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), whereas maintenance of LTP additionally requires protein synthesis. We recently reported that CaMKII stimulates protein synthesis in depolarized hippocampal neurons through phosphorylation of the mRNA translation factor cytoplasmic polyadenylation element-binding protein (CPEB), and this phosphorylation is rapidly reversed by protein phosphatase 1 (PP1). Protein synthesis-dependent late-phase LTP (L-LTP) in the hippocampus requires calcium influx through the NMDA-type glutamate receptor (NMDA-R) to activate CaMKII as well as concomitant inhibition of PP1 mediated by protein kinase A. Therefore, we investigated the regulation of CPEB phosphorylation during L-LTP. Pharmacological stimulation of the NMDA-R in hippocampal slices to produce chemical long-term depression induced a brief dephosphorylation of CPEB. Modest LTP induction (once at 100 Hz), which induces a protein synthesis-independent early-phase LTP (E-LTP), resulted in a transient phosphorylation of CPEB. However, stronger stimulation (four times at 100 Hz), known to induce protein synthesis-dependent L-LTP, elicited a prolonged phosphorylation of CPEB. Furthermore, CPEB phosphorylation correlated with phosphorylation of PP1 inhibitor dopamine- and cAMP-regulated phosphoprotein, a known substrate for protein kinase A. These results evoke the hypothesis that bidirectional regulation of CPEB phosphorylation by CaMKII and protein phosphatases may serve as a mechanism to convert E-LTP into protein synthesis-dependent L-LTP by stimulating protein synthesis and thereby stabilizing synaptic enhancement.

Animals↗

PubMed1983

Stimulation and inhibition by magnesium ions of intrinsic protein phosphorylating systems in synaptosomal membrane fragments from rat brain.

Synaptosomal membrane fragments from rat brain were incubated with [gamma-32P]ATP in the presence of cyclic AMP or Ca2+ plus calmodulin and a range of Mg2+ concentrations. Incorporation of 32P into membrane polypeptides was examined by electrophoresis and radioautography. Cyclic AMP-stimulated reactions were stimulated by low concentrations and inhibited to varying degrees by high concentrations of Mg2+ in the range 1-50 mM. In general the Ca2+ plus calmodulin-stimulated reactions were maximally active in the range 30-50 mM Mg2+, but the Ca2+ plus calmodulin dependent phosphorylation of Protein I was progressively inhibited by concentrations of Mg2+ above 5 mM. These results emphasize the importance of establishing optimum Mg2+ concentrations in the study of specific membrane protein phosphorylating systems.

Animals↗

PubMed1994

Growth hormone (GH) induces tyrosine-phosphorylated proteins in mouse L cells that express recombinant GH receptors.

Porcine and bovine GH receptor (GHR) cDNAs were stably expressed in mouse L cells, which normally do not possess detectable levels of mouse GHR. Expression of the GHR cDNAs resulted in specific binding of 125I-labeled GH by these cell lines. To study GHR-related signaling events in these cells, protein tyrosine phosphorylation was examined. In GH-treated cells, a tyrosine-phosphorylated protein with a molecular mass of approximately 95 kDa (pp95) was increased dramatically (approximately 100-fold) relative to non-GH-treated cells. The amount of pp95 within the cells after GH treatment was positively correlated with the number of GHRs on the cells. Tyrosine phosphorylation of pp95 could not be induced by prolactin, insulin, insulin-like growth factor I, interleukin 2, epidermal growth factor, platelet-derived growth factor, or fibroblast growth factor. Phosphorylation of pp95 was found to be a rapid event that could be observed 60 sec after GH treatment. Also, pp95 appears to exist as a complex of two proteins, i.e., pp95 and pp96. The GH-induced response by these cells may be of use in screening GH analogs for biological activity.

Animals↗

PubMed2001

Protein phosphorylation degree: determination by capillary liquid chromatography and inductively coupled plasma mass spectrometry.

Capillary liquid chromatography (muLC) interfaced to inductively coupled plasma mass spectrometry (ICPMS) is introduced as a new micromethod to determine the phosphorylation degree in phosphoproteins and phosphopeptides containing cysteine and/or methionine residues. The stoichiometric phosphorus to sulfur (31P to 32S) ratio is experimentally determined by muLC-ICPMS and converted into the degree of phosphorylation using protein/ peptide sequence information. The method is applied to the phosphoproteins beta-casein, beta-casein, and recombinant protein kinase A catalytic subunit and to synthetic phosphopeptides. The accurate data obtained by muLC-ICPMS allow quantitative assessment of the compound-specific discrimination of the electrospray ionization process between nonphosphorylated and phosphorylated proteins and peptides.

Amino Acid Sequence↗

PubMed2003

[Analysis of protein phosphorylation by combination of IMAC, phosphatase with biological mass spectrometry].

Protein phosphorylation is the most important reversible post-translational modification in cells. Analysis of phosphorylated proteins and identification of their phosphorylation sites is helpful for understanding their biological functions. MALDI-TOF-MS and ESI-Q-TOF-MS play important roles in protein phosphorylation analysis. In this work, immobilized metal affinity chromatography (IMAC) was used to selectively enrich phosphopeptides from protein digest mixtures, and treatment of phosphopeptides with alkaline phosphatase was used to confirm the phosphorylation. Finally, the phosphorylation sites were determined by tandem mass spectrometry analysis and database searching.

Chromatography, Affinity↗

PubMed1978

Cytoplasmic control of histone messenger RNA translation is not mediated through protein phosphorylation.

It was investigated whether the cytoplasmic control of histone synthesis, i.e. the selective and rapid degradation of histone mRNA following interruption of DNA replication, is linked to the phosphorylation of specific proteins of the translational apparatus or other cytoplasmic proteins. No specific protein phosphorylation was observed when synchronized HeLa cells in the S-phase were blocked with hydroxyurea or cytosine arabinoside and pulse-labeled simultaneously with [32P]orthophosphate. Protein phosphorylation was also studied in cell-free protein-synthesizing systems (S16 extracts) from blocked and unblocked S-phase cells under a variety of conditions. No protein could be detected that becomes specifically phosphorylated with [gamma-32P]ATP in a cell-free system derived from S-phase cells in which DNA replication and histone mRNA translation was interrupted with hydroxyurea.

Cytarabine↗

PubMedDate not supplied

Oral aluminum alters in vitro protein phosphorylation and kinase activities in rat brain.

Chronic, oral administration of aluminum to rats increases the in vivo concentration of cyclic AMP and the phosphorylation of microtubule-associated protein-2 (MAP-2) and the 200 kD neurofilament subunit (15,16). In the present study, the effect of this treatment on endogenous protein phosphorylation in soluble and particulate fractions prepared from cerebral cortices was examined. Chronic aluminum treatment significantly elevated the basal and cyclic AMP-dependent phosphorylation of 11-12 endogenous proteins in the soluble fraction prepared from cerebral cortices. Endogenous protein phosphorylation in the soluble fraction occurring in the presence of Ca++ alone or Ca++, phorbol 12-myristate 13-acetate and phosphatidylserine was not significantly altered by aluminum treatment. In the particulate fraction the phosphorylation of several proteins was significantly decreased by aluminum administration; however, the phosphorylation of the majority of protein substrates remained unaltered. Aluminum treatment did not alter the activities of cyclic AMP-dependent protein kinase or protein tyrosine kinase in the soluble and particulate fractions. The activity of Ca++/phospholipid-dependent protein kinase (protein kinase C) was increased in the particulate fraction of aluminum-fed rats. These results clearly demonstrate that specific effects on protein phosphorylation and protein kinase activities result from in vivo aluminum administration.

Administration, Oral↗

PubMed1997

Arachidonic acid inhibits myelin basic protein phosphorylation in cultured oligodendrocytes.

Protein phosphorylation is a well-known mechanism by which extracellular molecules or factors transduce their signals into intracellular effects. In the context of myelin assembly, phosphorylation of major myelin proteins affects the electrostatic repulsion between adjacent proteins within myelin structure and therefore constitutes one of the mechanisms by which myelin stability is regulated. We report here that arachidonic acid (AA) decreases the phosphorylation of myelin basic protein (MBP) both in the absence and in the presence of phorbol esters in cultured rat oligodendrocytes (OLGs). The effect of AA on MBP phosphorylation is not mediated by cyclooxygenase products, though the possibility that leukotrienes or other epoxides may have a role cannot be excluded. AA did not act by inactivation of protein kinase C. Based on our findings from gadolinium and low K+ experiments, we conclude that inhibition of MBP phosphorylation is not dependent on AA-induced increases in OLG Ca(i), but rather on its depolarizing action. We have thus demonstrated that a brief exposure to AA, which either acts as a diffusible paracrine signal to OLGs or as a signal transducer, can trigger changes in protein phosphorylation in OLGs/myelin via ionic signaling events at the plasma membrane.

Animals↗

PubMed1984

Changes in protein phosphorylation in guinea pig polymorphonuclear leukocytes by treatment with membrane-perturbing agents which stimulate superoxide anion production.

Phosphorylation of proteins was examined in guinea pig polymorphonuclear leukocytes in relation to the effects of membrane-perturbing agents, which stimulate superoxide anion production, and their inhibitors. The phosphorylation was detected by 32P autoradiography after separation by two-dimensional electrophoresis of proteins phosphorylated in 32P-preloaded cells. Though phosphorylation of various proteins was stimulated by each of the membrane-perturbing agents, the stimulation was especially marked in six proteins. Phorbol myristate acetate and digitonin enhanced the phosphorylation of the six proteins, while myristate and concanavalin A increased the phosphorylation of five and three proteins, respectively, out of the six proteins. p-Bromophenacyl bromide, an inhibitor of phospholipase A2, inhibited the stimulatory effect of phorbol myristate acetate on both superoxide anion production and protein phosphorylation. Trifluoperazine, a calmodulin inhibitor, also inhibited the effect of phorbol myristate acetate on both, except for an increase in the phosphorylation of one out of the six proteins. alpha-Methylmannoside, an inhibitor of concanavalin A binding, inhibited the stimulation of the phosphorylation of the three proteins by concanavalin A. The results indicate that the activation of superoxide anion production by the membrane-perturbing agents in guinea pig polymorphonuclear leukocytes is accompanied by the phosphorylation of, at least some of, these six proteins.

Animals↗

PubMed1990

[Second messengers and protein phosphorylation in cellular signal transduction].

Protein phosphorylation has been recognized as a major mechanism by which cellular functions are controlled by neurotransmitters and hormones. In this review, applications of molecular biological techniques to the analyses of regulatory mechanisms of protein phosphorylation by four major second messengers, cAMP, cGMP, diacylglycerol, and Ca2+, are described. 1) Complementary DNA of the regulatory subunit of the cAMP-dependent protein kinase was cloned and expressed in E. coli. Point mutations were introduced in order to analyze functional domains of the subunit. 2) The soluble isoform of guanylate cyclase was purified, and a cDNA of its 70-KD subunit was cloned. Cyclic GMP binding to purified cGMP-dependent protein kinase was characterized using a rapid filtration assay. 3) Primary structure of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A) was determined and the presence of the second isoform of the enzyme was shown by the cDNA cloning technique. 4) The regulatory domain of the protein kinase C was expressed in E. coli. Analysis using site-directed mutagenesis revealed that a "zinc finger"-like structure is responsible for the binding of phorbol esters. In these studies, the molecular biological approach has proven to be useful for clarifying the molecular mechanisms of cellular signal transduction related to second messengers and protein phosphorylation.

Animals↗

PubMed1982

Insulin release and protein phosphorylation: possible role of calmodulin.

Both Ca2+ and cyclic AMP (cAMP) are implicated in the regulation of insulin release in the pancreatic beta cell. In hamster insulinoma cells used in our laboratory to study the mechanism of insulin release, Ca2+ and cAMP trigger secretion independently. Concomitant with stimulation of the secretory apparatus both cAMP and Ca2+ promote phosphorylation of distinct insulinoma cell proteins. Calmodulin may be involved in the stimulation of insulin release and protein phosphorylation induced by Ca2+ influx. The Ca2+-dependent protein kinase of the insulinoma cell is activated by exogenous calmodulin and blocked by trifluoperazine, and inhibitor of calmodulin action. This drug also inhibits glucose-induced insulin release in pancreatic islets. In insulinoma cells trifluoperazine blocks Ca2+ influx-mediated insulin release and protein phosphorylation with no effect on basal or cAMP-mediated insulin release and protein phosphorylation with no effect on basal or cAMP-mediated secretion. Inhibition of Ca2+ influx-mediated insulin release and protein phosphorylation occurs with nearly identical dose dependence. Inasmuch as trifluoperazine affects voltage-dependent Ca2+ uptake in insulinoma cells, an involvement of calmodulin cannot be directly inferred. The evidence suggests that protein phosphorylation may be involved in the activation of the secretory apparatus by both cAMP and Ca2+. It is proposed that stimulation of insulin release by cAMP and Ca2+ is mediated by cAMP-dependent protein kinase and calmodulin-dependent protein kinase, respectively.

Animals↗

PubMed1990

Functional relationship between cyclic AMP-dependent protein phosphorylation and platelet inhibition.

Exposure of human platelets to prostacyclin (PGI2), iloprost or prostaglandin E1 (PGE1) elicits the cyclic AMP-dependent phosphorylation of proteins of 22, 24, 30, 39, 50, 60 and 250 kDa (P22, P24 etc.). P22 was recently identified as rap 1B, a ras-like protein, and P24 was shown to be the beta-chain of glycoprotein Ib. We found that cyclic AMP-dependent phosphorylation of all proteins except P22 was maximal 1 min after exposure of platelets to PGI2, iloprost or PGE1; maximal phosphorylation of P22 occurred after 45 min of incubation. Inhibition of thrombin-induced platelet activation required only a 30 s incubation with PGI2 or iloprost; at this time phosphorylation of P22 was only slightly increased. Although at maximal concentrations PGI2 was more potent than PGE1 in inhibiting thrombin-induced platelet activation, no difference in the degree and the kinetics of cyclic AMP-dependent protein phosphorylation was found. Platelets that had been preincubated and washed in the presence of PGE1 and later resuspended in the absence of PGE1 responded fully to activation by thrombin despite maximal phosphorylation of P22 and P24. Furthermore, addition of PGI2 to PGE1-washed platelets prevented thrombin-induced platelet activation, but did not evoke further phosphorylation of P22 or P24. Phosphorylation of P39 and P50 correlated better with PGI2-induced inhibition of platelet activation. In experiments in which PGE1-induced inhibition of platelet activation was overcome by the addition of thrombin, no dephosphorylation of proteins phosphorylated by cyclic AMP-dependent kinases was observed. These experiments indicate that: (a) phosphorylation of rap 1B and glycoprotein Ib is not related to platelet inhibition by cyclic AMP; (b) phosphorylation of other proteins such as P39 and P50 probably plays a role in mediating cyclic AMP-dependent platelet inhibition; (c) reactions other than cyclic AMP-dependent protein phosphorylation may participate in platelet inhibition by cyclic AMP.

Alprostadil↗

PubMed1992

A proposed link between nitrogen and carbon metabolism involving protein phosphorylation in bacteria.

We demonstrate that certain phosphoryl transfer proteins of the bacterial phosphotransferase system (PTS), the fructose- and mannitol-specific IIA proteins or domains, are homologous to a class of proteins, one of which is known to affect transcription of some of the nitrogen-regulatory sigma 54-dependent operons in Klebsiella pneumoniae. The phosphorylatable histidyl residue in the homologous PTS proteins and the consensus sequence in the vicinity of the active-site histidine are fully conserved in all members that comprise this family of proteins. A phylogenetic tree of the eight protein members of this family was constructed, and a "signature" sequence that can serve for the identification of new protein members of this family is proposed. These observations suggest that PTS-catalyzed protein phosphorylation may provide a regulatory link between carbon and nitrogen assimilation in bacteria.

Amino Acid Sequence↗

PubMed1986

Protein phosphorylation and synaptic transmission: receptor mediated modulation of protein kinase C in a rat brain fraction enriched in synaptosomes.

Aspects of protein phosphorylation related to events occurring during synaptic transmission were briefly reviewed. High resolution two-dimensional electrophoresis was used to study protein phosphorylation catalysed by protein kinase C in a fraction from rat brain enriched in synaptosomes. Incubation of 32P-labelled synaptosomes with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate resulted in an increase in the phosphorylation of a 45 K polypeptide (generally known as B-50) and an 82 K polypeptide; other major phosphoproteins in the preparation were unaffected by this treatment. It appears therefore that the 45 K and 82 K polypeptides are the only significant substrates for protein kinase C in synaptosomes. Depolarisation of labelled synaptosomes by high K+ increased the phosphorylation of the 82 K polypeptide, synapsin I and several unknown phosphoproteins. Incubation of labelled synaptosomes with the cholinergic agonist carbachol resulted in a modest, but statistically significant, increase in the phosphorylation of the 45 K (B-50) and 82 K polypeptides. This effect was blocked by atropine. The results are discussed in relation to a possible role for the B-50 phosphoprotein in regulating the resynthesis of polyphosphoinositides following cholinergic stimulation.

Animals↗

PubMedDate not supplied

Role of protein phosphorylation in the inhibition of protein synthesis caused by hypoxia in rat hepatocytes.

Hypoxia causes a rapid and reversible inhibition of translation in freshly isolated rat hepatocytes. This inhibition is neither due to an ATP loss nor to an increase in cell death. Because protein synthesis is mainly regulated by reversible phosphorylation of initiation and/or elongation factors, we investigated whether translation inhibition by hypoxia may be related to changes in the phosphorylation status of proteins. Whatever the incubation conditions, three phosphoreactive bands (molecular weights 220, 129, and 83 kDa) were detected by antiphosphotyrosine antibodies. The phosphorylation in the 129- and 83-kDa bands, however, was significantly and progressively decreased under hypoxia. Although this time-dependent decrease was sensitive to changes in oxygen tension, it occurred after the early protein synthesis inhibition caused by hypoxia. Moreover, sodium orthovanadate prevented tyrosine dephosphorylation in hypoxic cells, but did not restore the depressed protein synthesis caused by hypoxia. Under aerobic conditions, orthovanadate inhibited the synthesis of proteins, confirming that protein phosphorylation is a major mechanism involved in translational regulation. Once again, this inhibitory effect occurred only after 90 minutes of incubation whereas hypoxia inhibits the protein synthesis at the beginning of the incubation. Labeling cells with [33-32P]-ortho-phosphoric acid allowed detection of several phosphorylated proteins that appeared under hypoxia. Because they were not recognized by the phosphotyrosine antibodies, we suggest that serine/threonine residues of key proteins may be the putative hypoxic targets.

Adenosine Triphosphate↗

PubMed1986

Calcium channel agonists and antagonists regulate protein phosphorylation in intact synaptosomes.

Protein phosphorylation in intact synaptosomes is highly sensitive to alterations in calcium fluxes and was used to probe the possible mechanism of action of the calcium channel agonist BAY K 8644 and antagonists verapamil and nifedipine. These agents (at 1 microM) all increased the basal phosphorylation of a specific set of 4 synaptosomal phosphoproteins termed P139, P124, P96 and P60, but did not alter depolarization-dependent protein phosphorylation. The increases could not be explained by a direct stimulation of protein kinases and appears unrelated to the known effects of these drugs on K+-stimulated neurotransmitter release. This finding may reveal a possible new mechanism of action for drugs which interact with calcium channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2003

Protein phosphorylation in mammalian spermatozoa.

Spermatozoa undergo a series of changes before and during egg binding to acquire the ability to fuse with the oocyte. These priming events are regulated by the activation of compartmentalized intracellular signalling pathways, which control the phosphorylation status of sperm proteins. Increased protein tyrosine phosphorylation is associated with capacitation, hyperactivated motility, zona pellucida binding, acrosome reaction and sperm-oocyte binding and fusion. The main tyrosine phosphorylated proteins during the course of capacitation and fertilization are localized to the flagellum, although tyrosine phosphorylation of less abundant proteins may also be regulated in the sperm head. Spermatozoa bound to the zona pellucida and fusing with the oocyte plasma membrane are characterized by a tyrosine phosphorylated flagellum. Protein phosphorylation in the flagellum is linked to hyperactivated motility in spermatozoa, but may also regulate additional functions involved in sperm-oocyte fusion. Factors involved in the appearance of phosphorylation more likely arise from the milieu surrounding the spermatozoa, but their uptake and processing are likely to be regulated differentially at specific steps within the female genital tract and during penetration of the egg vestments. One of these factors is glucose, the metabolic products of which (ATP and NADPH) appear to participate in signalling pathways by supporting a precise onset of tyrosine phosphorylation in the sperm flagellum leading to successful fertilization.

Acrosome Reaction↗