Showing papers on "Phosphorylation published in 1989"
TL;DR: Four major serine/threonine-specific protein phosphatase catalytic subunits are present in the cytoplasm of animal cells and have broad and overlapping specificities in vitro, and account for virtually all measurable activity in tissue extracts toward a variety of phosphoproteins that regulate metabolism, muscle contractility, and other processes.
Abstract: Four major serine/threonine-specific protein phosphatase catalytic subunits are present in the cytoplasm of animal cells. Three of these enzymes, PP-1, PP-2A, and PP-2B, are members of the same gene family, while PP-2C appears to be distinct. PP-1, PP-2A, and PP-2B are complexed to other subunits in vivo, whereas PP-2C has only been isolated as a monomeric protein. PP-1, PP-2A, and PP-2C have broad and overlapping specificities in vitro, and account for virtually all measurable activity in tissue extracts toward a variety of phosphoproteins that regulate metabolism, muscle contractility, and other processes. Their precise functions in vivo are unknown, although important clues to the physiological roles of PP-1 and PP-2A are provided by the effects of okadaic acid and by the subcellular localization of PP-1. The active forms of PP-1 are largely particulate, and their association with subcellular structures is mediated by "targetting subunits" that direct PP-1 to particular locations, enhance its activity toward certain substrates, and confer important regulatory properties upon it. This concept is best established for the glycogen-bound enzymes in skeletal muscle and liver (PP-1G) and the myofibrillar form (PP-1M) in skeletal muscle. The activities of PP-1 and PP-2B are controlled by the second messengers cyclic AMP and calcium. The activity of PP-2B is dependent on calcium and calmodulin, while PP-1 is controlled in a variety of ways that depend on the form of the enzyme and the tissue. PP-1 can be inhibited by cyclic AMP in a variety of cells through the A-kinase-catalyzed phosphorylation of inhibitor-1 and its isoforms. Phosphorylation of the glycogen-binding subunit of PP-1G by A-kinase promotes translocation of the catalytic subunit from glycogen particles to cytosol in skeletal muscle, inhibiting the dephosphorylation of glycogen-metabolizing enzymes. Allosteric inhibition of hepatic PP-1G by phosphorylase a occurs in response to signals that elevate cyclic AMP or calcium, and prevents the activation of glycogen synthase in liver. PP-1 can also be activated indirectly by calcium through the ability of PP-2B to dephosphorylate inhibitor-1. This control mechanism may operate in dopaminoceptive neurones of the brain and other cells. The inactive cytosolic form of PP-1 (PP-1I) can be activated in vitro through the glycogen synthase kinase-3-catalyzed phosphorylation of its inhibitory subunit (inhibitor-2), but the physiological significance is unclear.(ABSTRACT TRUNCATED AT 400 WORDS)
2,585 citations
TL;DR: Results suggest that phosphorylation of CREB may stimulate transcription by a mechanism other than by simply providing negative charge, as CREB mutants containing acidic residues in place of the Ser-133 phosphoacceptor were also transcriptionally inactive.
2,585 citations
TL;DR: It is concluded that low or high concentrations of agonist elicit phosphorylation of beta 2AR on distinct domains, with different implications for the functional coupling of the receptors with effector molecules.
456 citations
TL;DR: Findings indicate an important role for the CD45 PTPase in pp56lck activation, which could be mediated by direct dephosphorylation of a regulatory tyrosine residue in pp 56lck.
Abstract: T lymphocytes express a tyrosine protein kinase (TPK; protein-tyrosine kinase; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112), pp56lck that is encoded by the lck protooncogene. This TPK was recently found to be associated with the intracellular domain of the T-cell surface glycoproteins, CD4 and CD8, suggesting that it plays an important role in T-cell development and activation. We have studied the regulation of pp56lck and found that this kinase can be rapidly activated by an endogenous mechanism present in T-lymphocyte membranes. This activation was sensitive to sodium orthovanadate and O-phosphotyrosine, consistent with the involvement of a phosphotyrosine phosphatase (PTPase; protein-tyrosine-phosphatase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) in pp56lck activation. Based on a recent report demonstrating that CD45, the leukocyte common antigen, is a membrane-bound PTPase, we analyzed its role in pp56lck activation. CD45 was found to be the major (greater than 90%) PTPase in membranes of the murine T-lymphoma line BW5147. Moreover, activation of pp56lck was undetectable in a mutant BW5147 line lacking CD45 expression (and the associated PTPase activity). In contrast, activation of pp56lck was readily detected in the wild-type lymphoma line. More important, when immunoprecipitated CD45 was added to pp56lck, the TPK activity of the latter increased greater than 2-fold within minutes. This effect of CD45 was completely blocked by sodium orthovanadate. These findings indicate an important role for the CD45 PTPase in pp56lck activation. This role could be mediated by direct dephosphorylation of a regulatory tyrosine residue in pp56lck.
441 citations
TL;DR: EGF rapidly and reversibly stimulated the anti-phosphotyrosine recovery of increased PLC activity when cells were treated with growth factor at 3 degrees C, indicating that receptor internalization is not required and that the phosphorylation event occurs prior to formation of inositol 1,4,5-trisphosphate.
Abstract: Epidermal growth factor (EGF) rapidly stimulates the formation of inositol 1,4,5-trisphosphate in a variety of cell types. Previously we have found that in intact cells stimulation of phospholipase C (PLC) activity by EGF is correlated with the retention of increased amounts of PLC activity by a phosphotyrosine immunoaffinity matrix, suggesting that the EGF-receptor tyrosine kinase phosphorylates PLC. We now define parameters of the mechanism by which EGF addition to A-431 cells stimulates phosphotyrosine immunoisolation of PLC activity and demonstrate that EGF addition to A-431 cells increases tyrosine phosphorylation of PLC. EGF rapidly and reversibly stimulated the anti-phosphotyrosine recovery of increased PLC activity when cells were treated with growth factor at 3 degrees C, indicating that receptor internalization is not required and that the phosphorylation event occurs prior to formation of inositol 1,4,5-trisphosphate. Also, the EGF stimulation of anti-phosphotyrosine recovery of PLC activity occurred in the absence of extracellular Ca2+. Stimulation of PLC activity in intact cells by other agonists, such as bradykinin or ATP, did not result in increased anti-phosphotyrosine recovery of PLC activity, suggesting two separate mechanisms exist in A-431 cells for hormone-stimulated formation of inositol phosphates. Finally, using monoclonal antibodies that specifically recognize three distinct PLC isozymes, we show that an approximately 145-kDa PLC isozyme (PLC-II) is present in A-431 cells and that EGF treatment of A-431 cells stimulates phosphorylation of PLC-II on both tyrosine and serine residues.
386 citations
TL;DR: The purposes of the present chapter are to discuss Ca2+ -dependent mechanisms other than myosin light chain phosphorylation by which contractile force in smooth muscle may be reg ulated, and other second messenger mechanisms that regulate contractile elements or the myOSin kinase/phosphatase system.
Abstract: In smooth muscle cells, development of force results from MgA TP-dependent cyclic interactions of myosin in thick filaments with actin in thin filaments. The force of contraction, in turn, is regulated by the concentration of free Ca2+ surrounding these myofilaments. The purposes of the present chapter are to discuss (a) Ca2+ -dependent mechanisms other than myosin light chain phosphorylation by which contractile force in smooth muscle may be reg ulated, and (b) other second messenger mechanisms that regulate contractile elements or the myosin kinase/phosphatase system . Since the discovery that phosphorylation of the 20-kDa light chains of myosin by Ca2+ /calmodulin-dependent myosin light chain kinase allows smooth muscle myosin MgATPase to be activated by actin, many in vestigators have studied the relationship between light chain phosphorylation and active force in smooth muscle (reviewed in 42; Hai & Murphy, this volume). It is generally accepted that myosin phosphorylation and de phosphorylation are sufficient to regulate contraction. While myosin phos phorylatiOn/dephosphorylation may be the dominant regulatory pathway, the coexistence of thin-filament linked regulation is not ruled out (51).
369 citations
TL;DR: The observation of a 120,000-Mr protein whose phosphorylation on tyrosine correlates with the induction of morphological transformation is reported, consistent with the observation that transforming src proteins are membrane associated.
Abstract: We used myristylated and nonmyristylated c-src-based variants and phosphotyrosine-specific antibodies to reevaluate the role of tyrosine phosphorylation in cellular transformation by pp60src. Prior methods used to detect tyrosine-phosphorylated proteins failed to discriminate predicted differences in tyrosine phosphorylation which are clearly observed with phosphotyrosine-specific antibodies and Western blotting (immunoblotting). Here we report the observation of a 120,000-Mr protein whose phosphorylation on tyrosine correlates with the induction of morphological transformation. p120 was not observed in cells overexpressing the regulated, nononcogenic pp60c-src, whereas phosphorylation of p120 was greatly enhanced in cells expressing activated, oncogenic pp60527F. Furthermore, phosphorylation of p120 was not induced by expression of the activated but nonmyristylated src variant pp602A/527F, which is transformation defective. p120 partitioned preferentially with cellular membranes, consistent with the observation that transforming src proteins are membrane associated. Although a number of additional putative substrates were identified and partially characterized with respect to intracellular localization, tyrosine phosphorylation of these proteins was not tightly linked to transformation.
367 citations
TL;DR: Findings suggest that p13 controls the activation of the cdc2 kinase, and that tyrosine dephosphorylation is an important step in this process.
318 citations
TL;DR: The results suggest that overexpressed chicken pp60c-src is one of the targets for MPF action, which may account in part for the pleiotropic changes in protein phosphorylation and cellular architecture that occur during mitosis.
317 citations
TL;DR: Results clearly indicate that the carboxyl-terminal sequence of rat pp60c-src is identical to that of chicken pp60 c-src, and a tyrosine residue corresponding to chicken Tyr527 is the phosphorylation site, which supports the previous proposal that the novel tyrosinesine kinase acts as a specific regulator of pp60C-src in cells.
308 citations
TL;DR: The results, along with previous studies on myc deletion mutants, show that Myc is phosphorylated by CK‐II, or a kinase with similar specificity, in regions of functional importance, and postulate that CK‐ II mediated phosphorylation of Myc plays a role in signal transduction to the nucleus.
Abstract: Casein kinase II (CK-II) is a ubiquitous protein kinase, localized to both nucleus and cytoplasm, with strong specificity for serine residues positioned within clusters of acidic amino acids. We have found that a number of nuclear oncoproteins share a CK-II phosphorylation sequence motif, including Myc, Myb, Fos, E1a and SV40 T antigen. In this paper we show that cellular myc-encoded proteins, derived from avian and human cells, can serve as substrates for phosphorylation by purified CK-II in vitro and that this phosphorylation is reversible. One- and two-dimensional mapping experiments demonstrate that the major phosphopeptides from in vivo phosphorylated Myc correspond to the phosphopeptides produced from Myc phosphorylated in vitro by CK-II. In addition, synthetic peptides with sequences corresponding to putative CK-II phosphorylation sites in Myc are subject to multiple, highly efficient phosphorylations by CK-II, and can act as competitive inhibitors of CK-II phosphorylation of Myc in vitro. We have used such peptides to map the phosphorylated regions in Myc and have located major CK-II phosphorylations within the central highly acidic domain and within a region proximal to the C terminus. Our results, along with previous studies on myc deletion mutants, show that Myc is phosphorylated by CK-II, or a kinase with similar specificity, in regions of functional importance. Since CK-II can be rapidly activated after mitogen treatment we postulate that CK-II mediated phosphorylation of Myc plays a role in signal transduction to the nucleus.
TL;DR: It is proposed that phosphorylated PhoB protein activates transcription from the promoters of the phosphate regulon, and that the role of PhoR is to catalyze the formation and breakdown of phosphorylations in response to phosphate concentrations in the medium.
TL;DR: Results suggest that mitogenic signaling by PDGF is coincident with tyrosine phosphorylation of PLC-gamma, a key enzyme of the phosphoinositide pathway believed to be an important site for hormonal regulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces the intracellular second-messenger molecules inositol 1,2-diacylglycerol.
Abstract: Platelet-derived growth factor (PDGF) stimulates the proliferation of quiescent fibroblasts through a series of events initiated by activation of tyrosine kinase activity of the PDGF receptor at the cell surface. Physiologically significant substrates for this or other growth factor receptor or oncogene tyrosine kinases have been difficult to identify. Phospholipase C (PLC), a key enzyme of the phosphoinositide pathway, is believed to be an important site for hormonal regulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces the intracellular second-messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Treatment of BALB/c 3T3 cells with PDGF led to a rapid (within 1 min) and significant (greater than 50-fold) increase in PLC activity, as detected in eluates of proteins from a phosphotyrosine immunoaffinity matrix. This PDGF-stimulated increase in phosphotyrosine-immunopurified PLC activity occurred for up to 12 h after addition of growth factor to quiescent cells. Interestingly, the PDGF stimulation occurred at 3 as well as 37 degrees C and in the absence or presence of extracellular Ca2+. Immunoprecipitation of cellular proteins with monoclonal antibodies specific for three distinct cytosolic PLC isozymes demonstrated the presence of a 145-kilodalton isozyme, PLC-gamma (formerly PLC-II), in BALB/c 3T3 cells. Furthermore, these immunoprecipitation studies showed that PLC-gamma is rapidly phosphorylated on tyrosine residues after PDGF stimulation. The results suggest that mitogenic signaling by PDGF is coincident with tyrosine phosphorylation of PLC-gamma.
TL;DR: All colon carcinoma cell lines with elevated pp60c-src in vitro kinase activity showed increased phosphorylation of proteins on tyrosine in vivo, suggesting the presence of an activated protein-tyrosine kinase(s).
Abstract: We measured the in vitro protein-tyrosine kinase activity of pp60c-src from human colon carcinoma cell lines and tumors. The activity of pp60c-src from six of nine carcinoma cell lines was higher (on average, fivefold as measured by enolase phosphorylation, or eightfold as measured by autophosphorylation) than that of pp60c-src from normal colonic mucosal cells, or human or rodent fibroblasts. Similarly, the activity of pp60c-src from 13 of 21 primary colon carcinomas was five- or sevenfold higher than that of pp60c-src from normal colonic mucosa adjacent to the tumor. The increased pp60c-src activity did not result solely from an increase in the level of pp60c-src protein, suggesting the specific activity of the pp60c-src kinase is elevated in the tumor cells. pp60c-src from colon carcinoma cells and normal colonic mucosal cells was phosphorylated at similar sites. We used immunoblotting with antibodies to phosphotyrosine to identify substrates of protein-tyrosine kinases in colonic cells. Three phosphotyrosine-containing proteins were detected at significantly higher levels in most colon carcinoma cell lines than in normal colonic mucosal cells or human or rat fibroblasts. All colon carcinoma cell lines with elevated pp60c-src in vitro kinase activity, showed increased phosphorylation of proteins on tyrosine in vivo, suggesting the presence of an activated protein-tyrosine kinase(s).
TL;DR: The results of studies in which antibodies to B-50 were introduced into rat cortical synaptosomes that were permeabilized with streptolysin-O (SL-O) found that the release of [3H]noradrenaline, induced by increasing the Ca2+ concentration in the buffer, is inhibited completely by the antibodies.
Abstract: Protein kinase C (PKC) is believed to have a crucial role in synaptic transmitter release and long-term potentiation. An important substrate of PKC in the brain is the neuron-specific presynaptically localized protein B-50 (also termed GAP-43, F1, pp46 or P-57). B-50 has been implicated in the regulation of polyphosphoinositide metabolism and calmodulin binding, and in the mechanisms of neurite outgrowth, long-term potentiation and transmitter release. It is still unknown, however, whether B-50 (and/or its phosphorylation) is essential to any of these processes. Here we report the results of studies in which antibodies to B-50, which interfere with B-50 phosphorylation, were introduced into rat cortical synaptosomes that were permeabilized with streptolysin-O (SL-O). We found that the release of [3H]noradrenaline, induced by increasing the Ca2+ concentration in the buffer, is inhibited completely by the antibodies. These results provide the first demonstration of a causal relationship between the PKC substrate B-50 and the release of neurotransmitter.
TL;DR: The enhanced cell adhesion observed upon activation of leukocytes is associated with increased surface membrane expression of CD11/CD18, as well as a qualitative upregulation ofCD11/ CD18 functions, which may regulate the adhesion functions mediated by the CD11-CD18 family of molecules.
Abstract: The leukocyte CD11/CD18 adhesion molecules (beta 2 integrins) are a family of three heterodimeric glycoproteins each with a distinct alpha subunit (CD11a, b, or c) and a common beta subunit (CD18). CD11/CD18 mediate crucial leukocyte adhesion functions such as chemotaxis, phagocytosis, adhesion to endothelium, aggregation, and cell-mediated cytotoxicity. The enhanced cell adhesion observed upon activation of leukocytes is associated with increased surface membrane expression of CD11/CD18, as well as a qualitative upregulation of CD11/CD18 functions. To elucidate the nature of the qualitative modifications that occur, we examined the phosphorylation status of these molecules in resting human leukocytes and upon activation with PMA or with the chemotactic peptide F-met-leu-phe (FMLP). In unstimulated cells, all three CD11 subunits were found to be constitutively phosphorylated. In contrast, phosphorylation of the common CD18 subunit was minimal. PMA induced rapid and sustained phosphorylation of CD18 that occurred at high stoichiometry, but had only minimal effects on phosphorylation of the associated CD11 subunits. FMLP also induced rapid phosphorylation of CD18, but the effect was of short duration. FMLP-induced phosphorylation of CD18 was not related to its Ca++-mobilizing effect, as CD18 phosphorylation was not observed upon treatment of leukocytes with the Ca++ ionophore, ionomycin. Phosphoamino acid analysis of CD11/CD18 in PMA- or FMLP-treated monocytes revealed a predominance of phosphoserine residues in all CD11/CD18 subunits. A small component of phosphothreonine was present in CD11c and CD18 and a minor component of phosphotyrosine was also detected in CD18 upon leukocyte activation may regulate the adhesion functions mediated by the CD11/CD18 family of molecules.
TL;DR: In this paper, the major phosphorylation sites that affect carboxylase activity and the specific protein kinases responsible for activating or dephosphorylation of different sites have been identified.
Abstract: Acetyl-CoA carboxylase, the rate-limiting enzyme in the biogenesis of long-chain fatty acids, is regulated by phosphorylation and dephosphorylation. The major phosphorylation sites that affect carboxylase activity and the specific protein kinases responsible for phosphorylation of different sites have been identified. A form of acetyl-CoA carboxylase that is independent of citrate for activity occurs in vivo. This active form of carboxylase becomes citrate-dependent upon phosphorylation under conditions of reduced lipogenesis. Therefore, phosphorylation-dephosphorylation of acetyl-CoA carboxylase is the enzyme's primary short-term regulatory mechanism; this control mechanism together with cellular metabolites such as CoA, citrate, and palmitoyl-CoA serves to fine-tune the synthesis of long-chain fatty acids under different physiological conditions.
TL;DR: A large number of phosphorylation changes are induced by severe heat stress and occur with kinetics similar to the inhibition of protein synthesis, but, under mild heat stress conditions, these initiation factor phosphorylated changes do not occur.
Abstract: Incubating cells at elevated temperatures causes an inhibition of protein synthesis. Mild heat stress at 41-42 degrees C inhibits the fraction of active, polysomal ribosomes from greater than 60% (preheating) to less than 30%. A return to 37 degrees C leads to an increase in protein synthesis, termed "recovery." Continuous incubation at 41-42 degrees C also leads to a gradual restoration of protein synthesis (greater than 70% of ribosomes reactivated by 2-4 h), termed "adaptation". Protein synthesis inhibition and reactivation is prestressed, recovered cells that contain elevated levels of the heat stress proteins occur to the same extent and at the same rate as in "naive" cells. The adaptation response requires transcription of new RNA whereas recovery does not. A large number of phosphorylation changes are induced by severe heat stress and occur with kinetics similar to the inhibition of protein synthesis. These include phosphorylation of eukaryotic protein synthesis initiation factor (eIF)-2 alpha and dephosphorylation of eIF-4B and eIF-4Fp25 (eIF-4E). However, the extent to which the modification occurs is proportional to the severity of the stress, and, under mild (41-42 degrees C) heat stress conditions, these initiation factor phosphorylation changes do not occur. Similarly, under conditions of severe heat stress eIF-2 alpha and eIF-4B frequently recover to their prestress phosphorylation state before the recovery of protein synthesis. eIF-4E dephosphorylation likewise does not occur under mild heat stress conditions. Therefore, these changes in phosphorylation states, which are thought to be sufficient cause, are not necessary for the inhibition of protein synthesis observed.
TL;DR: It is demonstrated that the DNA-binding ability of AT-1, from nuclear extracts of pea, can be reversibly modulated by phosphorylation.
Abstract: There have been numerous recent reports documenting phosphorylation of DNA-binding proteins [Montminy and Bilezikjian (1987); Sorger, Lewis, and Pelham (1987); Hoeffler, Kovelman, and Roeder (1988); Jones et al. (1988); Prywes et al. (1988); Sorger and Pelham (1988); Yamamoto et al. (1988)], and the transcriptional regulatory activity of at least one of these proteins appears to be modulated by this modification [Montminy and Bilezikjian (1987); Yamamoto et al. (1988)]. We report here on a plant nuclear protein, the DNA-binding activity of which is strongly affected by phosphorylation. This protein, AT-1, binds to specific AT-rich elements (the AT-1 box) within promoters of certain nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the polypeptide components of the light-harvesting chlorophyll a/b protein complex. A consensus sequence of AATATTTTTATT was derived for the AT-1 box. We demonstrate that the DNA-binding ability of AT-1, from nuclear extracts of pea, can be reversibly modulated by phosphorylation. AT-1 is active in the nonphosphorylated form and loses all DNA-binding ability as a result of phosphorylation. The kinase that phosphorylates AT-1 uses both Mg-ATP and Mg-GTP as a substrate and is inhibited by heparin and spermine, indicative of an NII-type casein kinase.
TL;DR: It appears that the receptor(s) for aFGF is related to the FLG gene product, because acidic fibroblast growth factor stimulated tyrosine kinase activity of FLG in vitro and in living cells, suggesting that FLG encodes the membrane receptor for a fms-like gene.
Abstract: We have previously isolated a human gene from an endothelial cell cDNA library encoding a putative tyrosine kinase; we have designated this gene the fms-like gene (FLG). To analyze the gene product(s) of FLG, we have generated rabbit polyclonal antibodies directed against a synthetic peptide from FLG and used it to immunoprecipitate biosynthetically labeled FLG protein from a variety of human cell lines. These antibodies specifically recognized glycoprotein(s) of 100, 120, and 135 kDa with protein cores of 90 and 110 kDa. Acidic fibroblast growth factor (aFGF) stimulated tyrosine kinase activity of FLG in vitro and in living cells, suggesting that FLG encodes the membrane receptor for aFGF. Further supporting evidence came from cross-linking experiments on intact cells with the covalent cross-linking agent disuccinimidyl suberate and 125I-labeled aFGF as a specific probe. The cross-linked 125I-labeled aFGF-aFGF receptor complex was specifically immunoprecipitated with FLG antipeptide antibodies. It appears, therefore, that the receptor(s) for aFGF is related to the FLG gene product.
TL;DR: It is proposed that autophosphorylation of solubilized EGFR is mediated by intermolecular cross-phosphorylated, probably facilitated by receptor oligomerization.
Abstract: Structurally distinguishable mutants of human epidermal growth factor receptor (EGFR) were used to investigate the mechanism of EGFR autophosphorylation. Mutant receptors generated by site-directed mutagenesis were expressed in transfected NIH 3T3 cells lacking endogenous receptors. After coincubation of cell lysates in the presence or absence of EGF, receptor immunoprecipitates were incubated with [gamma-32P]ATP. A kinase-negative mutant EGFR (K721A), in which Lys-721 in the ATp binding site was replaced by an alanine residue, was shown to be phosphorylated in an EGF-dependent manner by an enzymatically active EGFR deletion mutant lacking two autophosphorylation sites. A mutant EGFR lacking the EGF-binding domain as well as the phosphorylation sites also phosphorylated the kinase-negative mutant. In both cases the kinase-negative mutant K721A was phosphorylated on sites virtually identical to the sites that are autophosphorylated by wild-type recombinant or native human EGFRs. With four different site-specific anti-EGFR antibodies, it was shown that deletion mutants devoid of epitopes recognized by the antibodies were coimmunoprecipitated together with wild-type or mutant receptors recognized by the antibodies. This indicates that EGFR oligomers were preserved during immunoprecipitation. On the basis of these results, we propose that autophosphorylation of solubilized EGFR is mediated by intermolecular cross-phosphorylation, probably facilitated by receptor oligomerization.
TL;DR: The data suggest that activators of protein kinase C lead to the phosphorylation within platelets of a select population of G alpha subunits, which would suggest that effects of PMA on processes otherwise sensitive to this toxin are not exerted at the level of G proteins responsible for transduction.
TL;DR: Amino-terminal protein sequence analysis revealed that exponentially growing human HeLa cells at 37 degrees C express two closely related 90-kDa "heat shock" proteins (hsp 90) in nearly equal amounts.
TL;DR: Kinetic analyses indicated that inhibition of a completely Ca2+/CaM-independent form of CaM-kinase II by CaMK-(281-309) was noncompetitive with respect to peptide substrate (syntide-2) but was competitive withrespect to ATP.
Journal Article•
TL;DR: In the presence of MgCl2, KCl, and inosine-5'-monophosphate as phosphate donor, purified cytosolic 5'-nucleotidase catalyzed the phosphorylation of ddlno, yielding antiviral activity in human lymphoid cells.
Abstract: 29,39-Dideoxyinosine (ddlno) is a potent and selective inhibitor of human immunodeficiency virus in human lymphoid cells and monocytes/macrophages. Earlier studies [J. Biol. Chem. 263:15354 (1988)] showed that anabolism of ddlno in human lymphoid cells is mediated via an initial step of phosphorylation and subsequent amination to dideoxy-AMP via adenylosuccinate synthetase/lyase. Evidence was obtained that neither adenosine kinase nor deoxycytidine kinase is involved in the phosphorylation of this compound in human lymphoid cells. We now find that, in the presence of MgCl2, KCl, and inosine-59-monophosphate as phosphate donor, purified cytosolic 59-nucleotidase catalyzed the phosphorylation of ddlno. Although not phosphate donors, ATP, diadenosine tetraphosphate, and glycerate-2,3-bisphosphate stimulate this phosphorylation by the nucleotidase 4-5-fold. In addition to ddlno, the antiviral nucleoside analogs 29,39-dideoxyguanosine and carbovir were substrates for this enzyme. The relative phosphorylation of these compounds varied with the concentration of the phosphate donor IMP. Approximate Km values of the nucleotidase for inosine, ddlno, dideoxyguanosine, and carbovir were, respectively, 3.4, 0.5, 0.9, and 1.7 mM. Although the substrate activity of dideoxynucleosides is inefficient, it appears likely that this nucleotidase is responsible for the metabolism of these compounds to their active nucleotides, yielding antiviral activity in human lymphoid cells.
TL;DR: It is concluded that in rat hippocampal slices B‐50 may mediate the action of PKC in neurotransmitter release, and Polymyxin B, an inhibitor ofPKC and neurotransmitterRelease, decreases concentration dependently the depolarization‐induced stimulation of B‐ 50 phosphorylation.
Abstract: Recent studies have demonstrated that phorbol diesters enhance the release of various neurotransmitters. It is generally accepted that activation of protein kinase C (PKC) is the mechanism by which phorbol diesters act on neurotransmitter release. The action of PKC in neurotransmitter release is very likely mediated by phosphorylation of substrate proteins localized in the presynaptic nerve terminal. An important presynaptic substrate of PKC is B-50. To investigate whether B-50 mediates the actions of PKC in neurotransmitter release, we have studied B-50 phosphorylation in intact rat hippocampal slices under conditions that stimulate or inhibit PKC and neurotransmitter release. The slices were labelled with [32P]orthophosphate. After treatment, the slices were homogenized, B-50 was immunoprecipitated from the slice homogenate, and the incorporation of 32P into B-50 was determined. Chemical depolarization (30 μM K+) and the presence of phorbol diesters, conditions that stimulate neurotransmitter release, separately and in combination, also enhance B-50 phosphorylation. Polymyxin B, an inhibitor of PKC and neurotransmitter release, decreases concentration dependently the depolarization-induced stimulation of B-50 phosphorylation. The effects of depolarization are not detectable at low extracellular Ca2+ concentrations. It is concluded that in rat hippocampal slices B-50 may mediate the action of PKC in neurotransmitter release.
TL;DR: Comparison of the dose‐response curves of OA on maturation, isolated enzymes and phosphatase activities in crude oocyte preparations suggests that inhibition of both polycation‐stimulated (PCS) and ATP,Mg‐dependent (AMD) phosphatases is sufficient but requires that a critical phosphorylation level is attained of one or several of their substrates, resulting in the formation of active MPF and meiotic maturation.
TL;DR: Phosphorylation of mT by pp60c-src in vivo is therefore a critical event for binding of Ptdlns 3-kinase and for expression of the full tumorigenic potential of the virus.
TL;DR: It is suggested that this proteins "7" and "8" might be an ubiquitous regulatory phosphoprotein playing the role of an intracellular "relay" for extracellular signals, after their binding to specific membrane receptors and the generation of second messengers.
TL;DR: Analysis by limited proteolysis, tryptic fingerprinting and of phosphoamino acids established that the purified protein is identical with the 50-kDa protein phosphorylation was increased both by cAMP- and cGMP-elevating vasodilators in intact platelets and by endogenous cAMP/gMP-dependent protein kinase in platelet membranes.
Abstract: Cyclic-nucleotide-elevating vasodilators such as prostaglandin E1, prostacyclin, sodium nitroprusside and endothelium-derived relaxing factor inhibit both contraction of vascular smooth muscle cells and the aggregation of platelets at an early step of the activation cascade.
Previous studies from this laboratory [Waldmann, R., Nieberding, M. and Walter, U. (1987) Eur. J. Biochem. 167, 441–448) established that in human platelets cyclic-nucleotide-elevating vasodilators stimulated a pattern of protein phosphorylation which was mediated by both cAMP- and cGMP-dependent protein kinases. Of particular interest was a membrane-bound 50-kDa protein whose phosphorylation was increased both by cAMP-and cGMP-elevating vasodilators in intact platelets and by endogenous cAMP- and cGMP-dependent protein kinase in platelet membranes.
Since the molecular mechanism of action of cyclic-nucleotide-elevating vasodilators is unknown, this 50-kDa phosphoprotein from human platelets was purified to apparent homogeneity by salt extraction, anion, cation and dye-ligand chromatography. The purified protein migrated as a 46-kDa protein in SDS/PAGE, was an excellent substrate for both cAMP- and cGMP-dependent protein kinases and migrated in SDS/PAGE as a 50-kDa protein after phosphorylation by these protein kinases. Analysis by limited proteolysis, tryptic fingerprinting and of phosphoamino acids established that the purified protein is identical with the 50-kDa protein phosphorylated by both cAMP- and cGMP-dependent protein kinases in platelet membranes and in response to cAMP- and cGMP-elevating vasodilators with intact platelets. Evidence is presented that the purified protein contains at least two phosphorylation sites, each of which is preferentially phosphorylated by either cAMP- or cGMP-dependent protein kinase. The availability of this vasodilator-regulated phosphoprotein as a purified protein should now allow new approaches for investigating the function of this protein and its possible role in the mechanism of action of cyclic-nucleotide-elevating vasodilators.