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PubMed1999

Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation.

DNA polymerase alpha-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase alpha-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase alpha-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase alpha-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase alpha-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.

Animals↗

PubMed1997

Protein phosphatases maintain the organization and structural interactions of hepatic keratin intermediate filaments.

The importance of protein phosphatases in the maintenance of cytoskeletal structure is supported by the serious liver injury caused by microcystin-LR, a hepatotoxic inhibitor of type-1 and type-2A serine/threonine protein phosphatases. We used the microcystin-LR-induced cell injury as a model to study the roles of protein dephosphorylation in maintaining cytoskeletal structure and cellular interactions in primary rat hepatocyte cultures. Confocal microscopy revealed that the first visible effect of microcystin-LR is disruption of desmoplakin organization at the cell surface, indicating dissociation of desmosomes. This effect is followed by a dramatic reorganization of both the intermediate filament (keratins 8 and 18) and microfilament networks, resulting in a merged structure in which the intermediate filaments are organized around a condensed actin core. Keratin 8, keratin 18 and desmoplakin I/II are the major cytoskeleton-associated targets for microcystin-LR-induced phosphorylation. Hyperphosphorylation of keratin 8 and 18 is accompanied by an increased keratin solubility, which correlates with the observed morphological effects. Phosphopeptide mapping shows that four specific tryptic phosphopeptides are highly phosphorylated predominantly in the soluble pool of keratin 18, whereas keratin 8 shows no indications of such assembly state-specific sites. Phosphopeptide maps of keratins phosphorylated in vivo and in vitro indicate that Ca2+/calmodulin-dependent kinase may be involved in regulating the serine-specific phosphorylation of both keratin 8 and keratin 18, while cAMP-dependent protein kinase does not seem to play a major role in this context. Taken together, our results show that the interactions between keratin intermediate filaments and desmosomes as well as the assembly states of their main constituent proteins, are directly regulated by serine/threonine kinase/phosphatase equilibria.

Actin Cytoskeleton↗

PubMed2007

Identification of phosphorylated proteins.

Reversible protein phosphorylation is crucially involved in all aspects of plant cell physiology. The highly challenging task of revealing and characterizing the dynamic protein phosphorylation networks in plants has only recently begun to become feasible, owing to application of dedicated proteomics and mass spectrometry techniques. The experimental methodology that identified most of the presently known proteins phosphorylated in vivo is based on protein cleavage with trypsin, following chromatographic enrichment of phosphorylated peptides and mass spectrometric fragmentation and sequencing of these phosphopeptides. This procedure is most efficient when it is limited to the tryptic digestion of proteins in distinct isolated fractions or compartments of plant cells. Immobilized metal affinity chromatography (IMAC) is most useful for phosphopeptide enrichment after methylation of the peptides in the complex protein digests. The following tandem mass spectrometry of the isolated phosphopeptides results in both identification of phosphorylated proteins and mapping of the in vivo phosphorylation sites. The relative quantitation of the extent of phosphorylation at individual protein modification sites may be accomplished by either stable isotope labeling technique or dedicated liquid chromatography-mass spectrometry measurements.

Cell Fractionation↗

PubMed2000

Systematic analysis of the role of CD19 cytoplasmic tyrosines in enhancement of activation in Daudi human B cells: clustering of phospholipase C and Vav and of Grb2 and Sos with different CD19 tyrosines.

CD19 is a coreceptor on B cells that enhances the increase in cytoplasmic calcium and ERK2 activation when coligated with the B cell Ag receptor. Constructs containing point mutations and truncations were expressed in Daudi human B lymphoblastoid cells to systematically determine the requirement for individual CD19 cytoplasmic tyrosines in these responses. Evidence for activity was found for Y330, Y360, and Y421 as well as that previously published for Y391. Precipitates formed with phosphopeptides consisting of CD19 sequences flanking these residues were used to screen for cytoplasmic proteins that mediate signaling. Phosphopeptide Y330 precipitated Grb2 and Sos, whereas phosphopeptides Y391 and Y421 both precipitated Vav and phospholipase C-gamma2. These molecules also were found associated with native CD19. In mapping studies with altered constructs, CD19 Y330 and/or Y360 were necessary for binding Grb2 and Sos. Vav associated with CD19 constitutively in unstimulated cells by a tyrosine-independent mechanism requiring the portion of CD19 encoded by exons 9-12. After B cell Ag receptor stimulation, Vav association was tyrosine-dependent, but binding was influenced by multiple residues. However, when maximally phosphorylated by pervanadate, Y391 and, to a lesser extent, Y421 were sufficient. CD19 Y391 was also both necessary and sufficient for binding phospholipase C-gamma2. Thus, different tyrosines along the CD19 cytoplasmic domain provide scaffolding for the formation of complexes of different signaling molecules.

Adaptor Proteins, Signal Transducing↗

PubMed1975

Regulation of glycogen phosphorylase. Role of the peptide region surrounding the phosphoserine residue in determining enzyme properties.

A phosphopeptide which contains 14 residues including phosphoserine and which is derived from the NH2-terminal region of skeletal muscle glycogen phosphorylase (Nolan, C., Novoa, W. B., Krebs, E. G., and Fischer, E. H. (1964) Biochemistry 3, 542-551) has been shown to induce the enzymic properties of phosphorylase a in phosphorylase b and b'. When phosphorylase b is incubated with the phosphorylated tetradecapeptide, the following changes occur: (1) the enzyme becomes partially catalytically active in the absence of AMP; (2) the allosteric interactions of the enzyme are altered, as evidenced by the fact that phosphorylase b does not bind AMP cooperatively, and is no longer inhibited by glucose-6-P; and (3) the enzyme, normally present as a dimer, associates to a tetramer. Phosphorylase b' is a modified form of phosphorylase in which the phosphorylation site has been removed by limited tryptic attack. In the presence of phosphopeptide, 86% of the total enzyme activity can be induced in the absence of AMP. The properties of phosphorylases b and b' with phosphopeptide, cited above, are all characteristics of the phosphonenzyme, phosphorylase a. In addition, evidence is presented that these effects are specific. They are not the result of the polycationic nature of the peptide since they cannot be duplicated by spermine, and the phosphate group must also be present for the peptide to effect changes on the enzyme.

Adenosine Monophosphate↗

PubMed1992

Insulin-dependent formation of a complex containing an 85-kDa subunit of phosphatidylinositol 3-kinase and tyrosine-phosphorylated insulin receptor substrate 1.

Monoclonal antibodies raised against the 85-kDa subunit (p85) of bovine phosphatidylinositol (PI) 3-kinase were found to recognize uncomplexed p85 or p85 in the active PI 3-kinase. Immunoprecipitation studies of Chinese hamster ovary cells, which overexpress the human insulin receptor when treated with insulin, showed increased amounts of p85 and PI 3-kinase activity immunoprecipitable with monoclonal anti-p85 antibody and no increase in the tyrosine phosphorylation of p85. Insulin also induced an association of p85 with the tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and other phosphorylated proteins ranging in size from 100 to 170 kDa but not with the activated insulin receptor. In vitro reconstitution studies were used to show p85 in the active PI 3-kinase associated with the tyrosine-phosphorylated IRS-1 but not with the activated insulin receptor. Competition studies using synthetic phosphopeptides corresponding to potential tyrosine phosphorylation sites of IRS-1 revealed that phosphopeptides containing YMXM motifs inhibited this association with different potencies, whereas nonphosphorylated analogues and a phosphopeptide containing the EYYE motif had no effect. Src homology region 2 domains of p85 expressed as glutathione S-transferase fusion proteins also bound to tyrosine-phosphorylated IRS-1. These results suggest that insulin causes the association of PI 3-kinase with IRS-1 via phosphorylated YMXM motifs of IRS-1 and Src homology region 2 domains of p85.

Amino Acid Sequence↗

PubMed1991

Determination of the tyrosine phosphorylation sites of the nicotinic acetylcholine receptor.

The peripheral nicotinic acetylcholine receptor (nAChR) is phosphorylated on tyrosine residues in vivo and in vitro at a high stoichiometry. We have previously reported that this tyrosine phosphorylation occurs on the beta, gamma, and delta subunits of the receptor and is implicated in both the modulation of the function of the receptor and localization of the receptor at the synapse. The specific tyrosine residue of each subunit which is phosphorylated is now identified. The endogenously phosphorylated nAChR from the electric organ of Torpedo californica was phosphorylated to maximal stoichiometry in vitro exclusively on tyrosine residues as indicated by phosphoamino acid analysis. Two-dimensional phosphopeptide maps of thermolysin limit digests of the isolated phosphorylated subunits indicated that each subunit is phosphorylated at a single site. To determine the site of tyrosine phosphorylation of the beta, gamma, and delta subunits, phosphorylated subunits were isolated and digested with trypsin. A single phosphotyrosine containing peptide from each subunit was purified by antiphosphotyrosine antibody affinity chromatography and reverse phase high performance liquid chromatography. The purified phosphopeptides were subjected to sequential Edman degradation and sequence analysis. Comparison of the phosphopeptide sequence data with the deduced amino acid sequence of each subunit indicated that Tyr-355 of beta, Tyr-364 of gamma, and Tyr-372 of delta are the sites of in vitro and in vivo tyrosine phosphorylation of the nAChR. Identification of these sites should facilitate further studies of the role of tyrosine phosphorylation in the regulation of receptor function.

Amino Acid Sequence↗

PubMed1990

Synthetic peptide analogs of DARPP-32 (Mr 32,000 dopamine- and cAMP-regulated phosphoprotein), an inhibitor of protein phosphatase-1. Phosphorylation, dephosphorylation, and inhibitory activity.

Synthetic peptides based on the threonine phosphorylation site and proposed inhibitory site of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were prepared and analyzed as substrates for cAMP-dependent protein kinase and protein phosphatases-1c, -2Ac (the catalytic subunits of protein phosphatase-1 and 2A, respectively) and -2B, and as inhibitors of protein phosphatase-1c. Studies of the kinetics of phosphorylation of the peptides by cAMP-dependent protein kinase indicated an important role in facilitating phosphorylation for the region COOH-terminal to the phosphorylatable threonyl residue. Studies of the dephosphorylation of the phosphopeptides demonstrated that they were effectively dephosphorylated by protein phosphatase-2A and -2B and poorly dephosphorylated by protein phosphatase-1. The active inhibitory region of phospho-DARPP-32 was analyzed by determining the effects of synthetic phosphopeptides on the activity of protein phosphatase-1c. Phospho-D32-(8-48) and phospho-D32-(8-38) inhibited protein phosphatase-1c with IC50 values of 2 x 10(-8) and 4 x 10(-8) M, respectively, compared with an IC50 of 8 x 10(-9) M for intact phospho-DARPP-32. Phospho-D32-(9-38) was equipotent with phospho-D32-(8-38); however, further NH2-terminal deletions resulted in marked reductions in IC50 values. An analog of an active DARPP-32 phosphopeptide containing a phosphoseryl residue in place of the phosphothreonyl residue also exhibited a much reduced IC50. These data identify the essential inhibitory region of phospho-DARPP-32 as residues 9-38, which contains the phosphorylation site (Thr34). This region exhibits extensive amino acid sequence identity with phosphatase inhibitor-1, a distinct inhibitor of protein phosphatase-1. Kinetic studies of the inhibition of protein phosphatase-1c by phospho-D32-(9-38), a potent inhibitor, as well as by phospho-D32-(10-38), a weak inhibitor, indicated a mixed competitive/noncompetitive mechanism of inhibition, as has been previously found for both intact phospho-DARPP-32 and intact phospho-inhibitor-1. These findings support the hypothesis that a 30-amino acid domain in the NH2-terminal region of phospho-DARPP-32 is sufficient for the inhibition of protein phosphatase-1.

Amino Acid Sequence↗

PubMed1990

Protein kinase A phosphorylates retinal phosducin on serine 73 in situ.

Photoreceptors of vertebrate retinas contain a 33,000-dalton phosphoprotein, phosducin, which complexes with the beta, gamma subunits of the photoreceptor G-protein (guanine nucleotide-binding protein), transducin. In situ, the retinal content of phosphorylated phosducin is modulated by light in conjunction with light-triggered changes in intracellular cyclic nucleotide concentration. In vitro, phosducin is phosphorylated by either exogenous or endogenous protein kinase A. 32P-Labeled rat retina phosducin was isolated by immunoprecipitation either after phosphorylation by protein kinase A in the presence of [gamma-32P]ATP or after incubation of retinas in darkness with 32Pi. In either case, phosphoamino acid analysis showed that greater than 98% of 32P was linked to serine, with less than 2% to threonine. Two-dimensional peptide mapping showed that [32P]phosphoserine was associated with the same characteristic set of tryptic peptides. Furthermore, Cleveland peptide analysis using four different proteases showed that either sample exhibited identical patterns of phosphopeptides which were characteristic of the protease used. Identical phosphopeptide maps were also obtained from 32P-labeled bovine retina phosducin, indicating that the serine phosphorylation site for protein kinase A is conserved between rat and bovine. Edman degradation of phosphopeptides derived from 32P-labeled bovine phosducin showed that radioactive phosphate was incorporated into serine residue 73 which is located within a consensus phosphorylation sequence for protein kinase A (-R-K-M-S73(P)-). These observations are uniformly in agreement with protein kinase A being the endogenous kinase that phosphorylates phosducin in vivo.

Adenosine Triphosphate↗

PubMed1985

Nerve growth factor and other agents mediate phosphorylation and activation of tyrosine hydroxylase. A convergence of multiple kinase activities.

A rapid phosphorylation of tyrosine hydroxylase occurs in the PC12 nerve-like clonal cell line in response to nerve growth factor (NGF), epidermal growth factor (EGF), dibutyryl-cAMP, cholera toxin, phorbol- 12-myristate-13-acetate (PMA), or potassium depolarization in the presence of calcium ions. Complete tryptic digestion and two-dimensional peptide mapping reveals four available sites of phosphorylation in the enzyme. Phosphoamino acid analysis demonstrates that serine is the amino acid residue phosphorylated in each peptide. Specific phosphorylation of each of the four sites is achieved by different subsets of the above agents. One peptide site is phosphorylated in response to EGF alone. A second site is phosphorylated only in response to NGF, cholera toxin or dibutyryl-cAMP. A third site is phosphorylated only in response to potassium depolarization and requires the presence of extracellular Ca2+. The fourth site is the only site phosphorylated in response to PMA. These data indicate that at least 4 distinct kinase systems can act to phosphorylate tyrosine hydroxylase in PC12 cells. The PMA-stimulated peptide site is also phosphorylated in response to every one of the other agents. Further proteolytic digestions and phosphopeptide mapping of this common peptide, using Staphylococcus V8 protease and thermolysin, did not generate different phosphopeptides resulting from the different agents. These data suggest that the phosphorylation of this common peptide in response to all of the agents may be mediated by a common kinase, and, hence, that tyrosine hydroxylase phosphorylation by some agents may be mediated by two kinases. Although phosphopeptide maps of tyrosine hydroxylase resulting from cAMP elevation or NGF are qualitatively similar, quantitative differences exist, suggesting differential regulation of the same kinases by these agents. Tyrosine hydroxylase was found to be activated 2--4-fold in response to each phosphorylating agent. Thus, NGF and EGF present novel, natural means of regulating the activation state of tyrosine hydroxylase in responsive neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Chemoattractant-elicited increases in Dictyostelium myosin phosphorylation are due to changes in myosin localization and increases in kinase activity.

We previously reported (Berlot, C. H., Spudich, J. A., and Devreotes, P. N. (1985) Cell 43, 307-314) that cAMP stimulation of chemotactically competent Dictyostelium amoebae causes transient increases in phosphorylation of the myosin heavy chain and 18,000-dalton light chain in vivo and in vitro. In this report we investigate the mechanisms involved in these changes in phosphorylation. In the case of heavy chain phosphorylation, the amount of substrate available for phosphorylation appears to be the major factor regulating the in vitro phosphorylation rate. Almost all heavy chain kinase activity is insoluble in Triton X-100, and the increase in the heavy chain phosphorylation rate in vitro parallels an increase in Triton insolubility of myosin. Changes in heavy chain phosphatase activity are not involved in the changes in the in vitro phosphorylation rate. In the case of light chain phosphorylation, increases in the vitro phosphorylation rate occur under conditions where the amount of substrate available for phosphorylation is constant and phosphatase activity is undetectable, implicating light chain kinase activation as the means of regulation. The specificity of the myosin kinases operating in vivo and in vitro was explored using phosphoamino acid and chymotryptic phosphopeptide analysis. The light chain is phosphorylated on serine both in vivo and in vitro, and phosphopeptide maps of the light chain phosphorylated in vivo and in vitro are indistinguishable. In the case of the heavy chain, both serine and threonine are phosphorylated in vivo and in vitro, although the cAMP-stimulated increases in phosphorylation occur primarily on threonine. Phosphopeptide maps of the heavy chain show that the peptides phosphorylated in vitro represent a major subset of those phosphorylated in vivo. The kinetics of the transient increases in myosin phosphorylation rates observed in vitro can be predicted quantitatively from the in vivo myosin phosphorylation data assuming that there is a constant phosphatase activity.

Amino Acids↗

PubMed1983

Protease-activated kinase II as the potential mediator of insulin-stimulated phosphorylation of ribosomal protein S6.

One of the earliest responses to insulin in target cells is stimulation of the phosphorylation of ribosomal protein S6. When exponentially growing 3T3-L1 cells are serum-starved, little phosphorylation of S6 is observed; however, following addition of insulin (10(-7) M), up to 5 phosphoryl groups are incorporated into S6. An enzyme mediating the insulin-stimulated phosphorylation of S6 has been identified as protease-activated kinase II. Two-dimensional peptide maps of tryptic digests of S6 from insulin-treated 3T3-L1 cells contain 5 phosphopeptides; the same 5 phosphopeptides are observed with tryptic digests of 40 S ribosomal subunits phosphorylated in vitro by protease-activated kinase II from rabbit reticulocytes. Protease-activated kinase II has also been identified and partially purified from the postribosomal supernatant of serum-starved and insulin-treated 3T3-L1 cells. The enzyme is present in the inactive proenzyme form in serum-starved cells; following insulin treatment, approximately 50% of the enzyme is in an activated form. Identical tryptic phosphopeptide maps are observed with these enzymes.

Animals↗

PubMed1984

Peptide mapping of phosphorylated vimentin. Evidence for a site-specific alteration in mitotic cells.

Vimentin, the subunit protein of one type of intermediate filament, has been isolated from 32Pi-labeled nonmitotic and mitotic mouse L-929 cells. Analysis of tryptic phosphopeptides by two-dimensional maps indicates that vimentin is phosphorylated at multiple sites in mitotic cells. Comparison of nonmitotic and mitotic vimentin phosphotryptic peptides indicated that in addition to the 6-7 major phosphorylated tryptic peptides found in nonmitotic cells, vimentin isolated from mitotic cells contained an additional 2 distinct phosphorylated peptides following trypsin digestion. Partial acid hydrolysis and one-dimensional phosphoamino acid analysis indicates that phosphoserine is present in all 9 major phosphopeptides. Treatment of L-929 cells with 8-bromo-cAMP did not result in a qualitative change in the phosphopeptide map of vimentin isolated from a normal cell population. These results suggest that the reorganization of vimentin filaments during mitosis is accompanied by a site-specific change in phosphorylation.

Animals↗

PubMed1980

Site-specific phosphorylation if initiation factor 2 by three cyclic nucleotide-independent protein kinases.

The site-specific phosphorylation of initiation factor 2 (eIF-2) by three different cyclic nucleotide-independent protein kinases from rabbit reticulocytes was examined. The hemin-controlled repressor modified serine in the alpha subunit (Mr = 38,000), while casein kinase II and protease-activated kinase II phosphorylated serine residues of the beta subunit (Mr = 53,000). Under conditions of maximal phosphorylation, 1 mol of phosphate was incorporated into eIF-2 by each of the protein kinases. However, following treatment of eIF-2 with alkaline phosphatase, 2 mol of phosphate were added by casein kinase II, indicating residual phosphate was present on the beta subunit of purified eIF-2. The tryptic and chymotryptic peptides of the phosphorylated subunits were analyzed by two-dimensional peptide mapping involving thin layer electrophoresis followed by ascending chromatography. When eIF-2 was phosphorylated by the hemin-controlled repressor, three major chymotryptic and four tryptic phosphopeptides with molecular weights ranging from approximately 600 to 3000 were identified. When phosphorylation of eIF-2 beta was examined, two tryptic and two chymotryptic phosphopeptides were obtained after phosphorylation with casein kinase II and were different from the phosphopeptides observed following phosphorylation with protease-activated kinase II. This indicates that three distinct sites in the beta subunit were phosphorylated, two by casein kinase II and one by protease-activated kinase II.

Animals↗

PubMed1980

Structural characterization of the phosphorylation sites of human erythrocyte spectrin.

The phosphorylation sites of spectrin dimer were characterized before and after incubation of intact human red cells with [32P]orthophosphate. Phosphate measurements of unlabeled spectrin dimer show it contains 4.0 +/- 0.3 covalent protein phosphates, all located on band 2. Quantitation of the number of exchangeable phosphorylation sites in erythrocytes labeled with [32P]orthophosphate yields 3.9 +/- 0.3 phosphates/spectrin dimer, indicating that all four spectrin phosphorylation sites are metabolically active. Tryptic and chymotryptic peptide mapping reveals that the dimer contains three unique 32P-labeled tryptic peptides of approximately 4,600 (A1), 3,500 (A2), and 2,400 (B) daltons. Peptide A1 contains both phosphoserine and phosphothreonine while peptides A2 and B possess only phosphoserine. Peptides A1 and A2 remain associated after trypsinization of spectrin dimer and are only separable in detergents. All three 32P-labeled tryptic peptides are contained within a 20,000 dalton cyanogen bromide fragment which is within a 60,000 dalton staphylococcal protease phosphopeptide. All these fragments are found within a 90,000 dalton nitrothiocyanobenzoic acid phosphopeptide. The purified 20,000 dalton fragment contains no homoserine or homoserine lactone and is the COOH-terminal cyanogen bromide peptide of band 2. The isolated tryptic peptide B possesses no lysine or arginine and is presumably the COOH-terminal tryptic peptide of band 2 and the most distal phosphopeptide. Thus, the four phosphorylation sites of spectrin dimer are clustered at the extreme COOH-terminal end of band 2.

Adenosine Triphosphate↗

PubMed1994

Insulin-dependent phosphorylation of calmodulin in rat hepatocytes.

Insulin-stimulated phosphorylation of calmodulin in vivo was examined using a highly specific anti-calmodulin monoclonal antibody combined with high resolution two-dimensional gel electrophoresis. The two major isoforms of calmodulin immunoprecipitated from insulin-treated hepatocytes migrated on two-dimensional gel electrophoresis to the same position as nonphosphorylated calmodulin and calmodulin phosphorylated in vitro. Immunoblotting verified the identity of calmodulin. Insulin enhanced the phosphorylation of calmodulin 3.1 +/- 0.4-fold (mean +/- S.E., n = 10), with a stoichiometry in insulin-treated hepatocytes of 0.47 +/- 0.06 (mean +/- S.E., n = 3) mol of phosphate/mol of calmodulin. Two-dimensional phosphopeptide mapping of calmodulin immunoprecipitated from rat hepatocytes and calmodulin phosphorylated in vitro by the insulin receptor kinase or casein kinase II revealed several common phosphopeptides. The common phosphopeptides that appeared insulin-sensitive in intact cells comprised 61 and 40% of casein kinase II- and insulin receptor-catalyzed 32P incorporation into calmodulin in vitro, respectively. This suggests that casein kinase II and the insulin receptor kinase are, at least in part, responsible for insulin-stimulated phosphorylation of calmodulin in rat hepatocytes. These data indicate that phosphorylation of calmodulin in intact hepatocytes is significantly enhanced by insulin, supporting a critical role for calmodulin in insulin signal transduction.

Animals↗

PubMed1996

Phosphoinositide-dependent in vitro phosphorylation of profilin by protein kinase C. Phospholipid specificity and localization of the phosphorylation site.

Phosphoinositides bind to profilin and regulate actin-based cytoskeletal protein assembly. We report here that profilin is phosphorylated in vitro by protein kinase C (PKC) in the presence of phosphoinositides and micromolar concentrations of calcium. PKC-mediated phosphorylation of profilin was observed only in the presence of phosphoinositides; phosphatidylserine and diacylglycerol (known activators of PKC) and other lipids, including phosphatidic acid and phosphatidylglycerol phosphate, did not activate the phosphorylation. The activation of PKC-mediated phosphorylation of profilin by phosphoinositides was as follows: phosphatidylinositol (PI) 4-phosphate (K(m) = 18 microM) > PI 4,5-bisphosphate (K(m) = 30 microM) > PI (no activation). About 0.5 mol phosphate was incorporated per mol of profilin. Phosphorylation of profilin by PKC was not affected by the presence of various concentrations of actin. Phospho-amino acid analysis showed serine to be the only amino acid phosphorylated. The amino acid sequence of a phosphopeptide from CNBr-digested profilin corresponded to the COOH-terminal peptide of profilin (Ala-Ser-His-Leu-Arg-Ser-Gln-Tyr). Further digestion of this phosphopeptide by trypsin generated two phosphopeptides (Arg-Ser-Gln-Tyr and Ser-Gln-Tyr), thereby confirming that the phosphorylation site was the antepenultimate Ser (Ala-Ser-His-Leu-Arg-Arg-Ser(P)-Gln-Tyr).

Actins↗

PubMed1999

Phosphorylation-dependent protein kinase D activation.

The novel mouse serine-threonine kinase protein kinase D (PKD) is activated in intact Swiss 3T3 cells stimulated by phorbol esters, cell permeant diacylglycerols, bryostatin, neuropeptides and growth factors via a phosphorylation-dependent mechanism requiring protein kinase C (PKC) activity. Structural comparison of the PKD catalytic domain with other kinases reveals a close similarity with MEK family kinases, which are activated upon phosphorylation of key serine and threonine residues in a region termed the activation loop. To study the regulation of PKD, we transfected mutant PKD cDNAs in which putative activation loop serine residues 744 and 748 were mutated to either alanine or glutamic acid into COS-7 cells. Replacement of serines 744 and 748 with alanine prevented activation of the overexpressed PKD form upon phorbol ester treatment of cells, whereas replacement with glutamic acid results in full constitutive activation. Single serine to glutamic acid replacement mutants were partially activated. In vivo 32P-labeling and two-dimensional phosphopeptide mapping of PKD and catalytically inactive PKD mutants at serine 744, 748 or at both residues revealed that phorbol ester-sensitive phosphopeptides could be selectively eliminated from patterns observed as a result of these mutations. Treatment of cells with the PKC inhibitor GFI also prevented the appearance of phosphopeptide spots occuring in response to phorbol ester stimulation. These results provide direct evidence that PKD becomes activated in vivo as a consequence of PKC-mediated phosphorylation of serines 744 and 748. These results support our view of PKD as an important clement in PKC signal transduction.

Amino Acid Sequence↗