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The Journal of Biological Chemistry Sep 1996Protein kinase C (PKC) isozymes alpha, delta, epsilon, and zeta, shown to be expressed in adult rat cardiomyocytes, displayed distinct substrate specificities in... (Comparative Study)
Comparative Study
Phosphorylation specificities of protein kinase C isozymes for bovine cardiac troponin I and troponin T and sites within these proteins and regulation of myofilament properties.
Protein kinase C (PKC) isozymes alpha, delta, epsilon, and zeta, shown to be expressed in adult rat cardiomyocytes, displayed distinct substrate specificities in phosphorylating troponin I and troponin T subunits in the bovine cardiac troponin complex. Thus, because they have different substrate affinities, PKC-alpha, -delta, and -epsilon phosphorylated troponin I more than troponin T, but PKC-zeta conversely phosphorylated the latter more than the former. Furthermore, PKC isozymes exhibited discrete specificities in phosphorylating distinct sites in these proteins as free subunits or in the troponin complex. Unlike other isozymes, PKC-delta was uniquely able to phosphorylate Ser-23/Ser-24 in troponin I, the bona fide phosphorylation sites for protein kinase A (PKA); and consequently, like PKA, it reduced Ca2+ sensitivity of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1. In addition, PKC-delta, like PKC-alpha, readily phosphorylated Ser-43/Ser-45 (sites common for all PKC isozymes) and reduced maximal activity of MgATPase. In this respect, PKC-delta functioned as a hybrid of PKC-alpha and PKA. In contrast to PKC-alpha, -delta, and -epsilon, PKC-zeta exclusively phosphorylated two previously unknown sites in troponin T. Phosphorylation of troponin T by PKC-alpha resulted in decreases in both Ca2+ sensitivity and maximal activity, whereas phosphorylation by PKC-zeta resulted in a slight increase of the Ca2+ sensitivity without affecting the maximal activity of MgATPase. Most of the in vitro phosphorylation sites in troponin I and troponin T were confirmed in situ in adult rat cardiomyocytes. The present study has demonstrated for the first time distinct specificities of PKC isozymes for phosphorylation of two physiological substrates in the myocardium, with functional consequences.
Topics: Actin Cytoskeleton; Animals; Ca(2+) Mg(2+)-ATPase; Cattle; Isoenzymes; Myocardium; Peptide Mapping; Phosphopeptides; Phosphorylation; Protein Kinase C; Rats; Substrate Specificity; Troponin; Troponin I; Troponin T
PubMed: 8798526
DOI: 10.1074/jbc.271.38.23277 -
Journal of Biochemistry Jun 1992In regenerating rat liver, nuclear protein histone H2A was shown to be phosphorylated on its amino-terminal serine residue [Sung et al. (1971) J. Biol. Chem. 246,...
In regenerating rat liver, nuclear protein histone H2A was shown to be phosphorylated on its amino-terminal serine residue [Sung et al. (1971) J. Biol. Chem. 246, 1358-1364], but the protein kinase which phosphorylates this residue has not been identified. To evaluate the possibility that protein kinase C can phosphorylate this residue, calf thymus histone H2A was 32P-labeled by incubation with [gamma-32P]ATP and highly purified protein kinase C from rat brain in the presence of calcium and phospholipid. About 1 mol of 32P was incorporated per mol of histone H2A and the Km and apparent Vmax of the reaction were calculated to be 2.1 microM and 0.35 mumol/min/mg, respectively. So histone H2A seemed to be a good substrate for protein kinase C. Further, the proteolytic phosphopeptides of 32P-labeled histone H2A were isolated by means of a series of column chromatographies and analyzed for their amino acid compositions. Comparison of the data with the known primary structure of histone H2A revealed their amino acid sequence as 1Ser-Gly-Arg. These data suggest that protein kinase C may be a candidate for the protein kinase which phosphorylates the amino-terminal serine residue of histone H2A during the regeneration of rat liver.
Topics: Amino Acid Sequence; Animals; Binding Sites; Brain; Cattle; Histones; In Vitro Techniques; Kinetics; Molecular Sequence Data; Phosphorylation; Protein Kinase C; Rats; Rats, Inbred Strains
PubMed: 1500420
DOI: 10.1093/oxfordjournals.jbchem.a123837 -
The Journal of Biological Chemistry May 2013Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2...
Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2 interacts with tyrosine phosphopeptide, and the interaction with the tyrosine phosphopeptide affects the pyruvate kinase activity of PKM2. Our experiments suggest that PKM2 is also an active protein kinase (Gao, X., Wang, H., Yang, J. J., Liu, X., and Liu, Z. R. (2012) Mol. Cell 45, 598-609). We report here that growth signals reciprocally regulate the pyruvate kinase and protein kinase activities of PKM2 by different mechanisms. On the one hand, growth signals induce protein tyrosine phosphorylations. The tyrosine-phosphorylated protein(s) regulates the conversion of pyruvate kinase and protein kinase of PKM2 by directly interacting with PKM2. Binding of the tyrosyl-phosphorylated proteins at the fructose 1,6-bisphosphate-binding site converts the tetrameric PKM2 to a dimer. On the other hand, growth stimulations also lead to PKM2 phosphorylation, which consequently regulates the conversion of protein kinase and pyruvate kinase activities. Growth factor stimulations significantly increase the dimer/tetramer PKM2 ratio in cells and consequently activate the protein kinase activity of PKM2. Our study suggests that the conversion between the pyruvate kinase and protein kinase activities of PKM2 may be an important mechanism mediating the effects of growth signals in promoting cell proliferation.
Topics: Binding Sites; Cell Line; Cell Proliferation; Humans; Phosphorylation; Protein Multimerization; Protein-Tyrosine Kinases; Pyruvate Kinase; Signal Transduction
PubMed: 23576436
DOI: 10.1074/jbc.M112.448753 -
The Journal of Biological Chemistry Jul 2014Yeast Pah1p is the phosphatidate phosphatase that catalyzes the penultimate step in triacylglycerol synthesis and plays a role in the transcriptional regulation of...
Yeast Pah1p is the phosphatidate phosphatase that catalyzes the penultimate step in triacylglycerol synthesis and plays a role in the transcriptional regulation of phospholipid synthesis genes. The enzyme is multiply phosphorylated, some of which is mediated by Pho85p-Pho80p, Cdc28p-cyclin B, and protein kinase A. Here, we showed that Pah1p is a bona fide substrate of protein kinase C; the phosphorylation reaction was time- and dose-dependent and dependent on the concentrations of ATP (Km = 4.5 μm) and Pah1p (Km = 0.75 μm). The stoichiometry of the reaction was 0.8 mol of phosphate/mol of Pah1p. By combining mass spectrometry, truncation analysis, site-directed mutagenesis, and phosphopeptide mapping, we identified Ser-677, Ser-769, Ser-773, and Ser-788 as major sites of phosphorylation. Analysis of Pah1p phosphorylations by different protein kinases showed that prephosphorylation with protein kinase C reduces its subsequent phosphorylation with protein kinase A and vice versa. Prephosphorylation with Pho85p-Pho80p had an inhibitory effect on its subsequent phosphorylation with protein kinase C; however, prephosphorylation with protein kinase C had no effect on the subsequent phosphorylation with Pho85p-Pho80p. Unlike its phosphorylations by Pho85p-Pho80p and protein kinase A, which cause a significant reduction in phosphatidate phosphatase activity, the phosphorylation of Pah1p by protein kinase C had a small stimulatory effect on the enzyme activity. Analysis of phosphorylation-deficient forms of Pah1p indicated that protein kinase C does not have a major effect on its location or its function in triacylglycerol synthesis, but instead, the phosphorylation favors loss of Pah1p abundance when it is not phosphorylated with Pho85p-Pho80p.
Topics: Binding Sites; Cyclin-Dependent Kinases; Cyclins; Mutation; Phosphatidate Phosphatase; Phosphorylation; Protein Kinase C; Proteolysis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Serine
PubMed: 24876385
DOI: 10.1074/jbc.M114.581462 -
International Journal of Molecular... Feb 2021Developmental growth and patterning are regulated by an interconnected signalling network of several pathways. In , the Warts (Wts) kinase, a component of the Hippo...
Developmental growth and patterning are regulated by an interconnected signalling network of several pathways. In , the Warts (Wts) kinase, a component of the Hippo signalling pathway, plays an essential role in regulating transcription and growth by phosphorylating its substrate Yorkie (Yki). The phosphorylation of Yki critically influences its localisation and activity as a transcriptional coactivator. In this study, we identified the homeodomain-interacting protein kinase (Hipk) as another kinase that phosphorylates Yki and mapped several sites of Yki phosphorylated by Hipk, using in vitro analysis: Ser168, Ser169/Ser172 and Ser255. These sites might provide auxiliary input for Yki regulation in vivo, as transgenic flies with mutations in these show prominent phenotypes; Hipk, therefore, represents an additional upstream regulator of Yki that works in concert with Wts.
Topics: Animals; Drosophila Proteins; Drosophila melanogaster; Intracellular Signaling Peptides and Proteins; Nuclear Proteins; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Signal Transduction; Trans-Activators; YAP-Signaling Proteins
PubMed: 33668437
DOI: 10.3390/ijms22041862 -
Molecular Endocrinology (Baltimore, Md.) May 2015FAM20C is a secretory kinase responsible for the phosphorylation of multiple secreted proteins in mammalian cells; it has been shown to phosphorylate serine residues...
FAM20C is a secretory kinase responsible for the phosphorylation of multiple secreted proteins in mammalian cells; it has been shown to phosphorylate serine residues within a variety of different bone proteins. In this work we demonstrate that FAM20C also phosphorylates threonines, specifically those within the N-terminal domain of the neuroendocrine chaperone 7B2. Analysis of the primary sequence of 7B2 revealed that three threonine residues in its N-terminal domain are located within FAM20C consensus motifs: Thr73, Thr99, and Thr111. The individual substitution of Thr73 and Thr111 residues by neutral alanines caused a marked decrease in the total phosphorylation of 7B2. Furthermore, the phosphomimetic substitution of Thr111 by Glu clearly diminished the ability of 7B2 to activate pro-prohormone convertase 2 (PC2) in 7B2-lacking SK-N-MC neuroblastoma cells, suggesting that the phosphorylation of this residue critically impacts the 7B2-proPC2 interaction. However, the phosphomimetic mutation did not alter 7B2's ability to function as an antiaggregant for human islet amyloid polypeptide. FAM20C-mediated phosphorylation of a common alternatively spliced variant of human 7B2 that lacks Ala100 (thus eliminating the Thr99 phosphorylation consensus site) was similar to the Ala-containing protein, but this variant did not activate proPC2 as efficiently as the Ala-containing protein. Although threonines within 7B2 were phosphorylated efficiently, FAM20C was incapable of performing the well-known regulatory threonine phosphorylation of the molecular chaperone binding immunoglobulin protein. Taken together, these results indicate that FAM20C plays a role in 7B2-mediated proPC2 activation by phosphorylating residue Thr111; and that 7B2 function is regulated by alternative splicing.
Topics: Alternative Splicing; Amino Acid Sequence; Casein Kinase I; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Activation; Extracellular Matrix Proteins; HEK293 Cells; Heat-Shock Proteins; Humans; Molecular Sequence Data; Neuroendocrine Secretory Protein 7B2; Phosphorylation; Proprotein Convertase 2; Protein Processing, Post-Translational
PubMed: 25811241
DOI: 10.1210/me.2014-1394 -
European Journal of Biochemistry Nov 1987The dihydropyridine receptor purified from rabbit skeletal muscle contains three proteins of 165, 55 and 32 kDa. cAMP kinase and protein kinase C phosphorylate the... (Comparative Study)
Comparative Study
The dihydropyridine receptor purified from rabbit skeletal muscle contains three proteins of 165, 55 and 32 kDa. cAMP kinase and protein kinase C phosphorylate the 165-kDa and the 55-kDa proteins. At identical concentrations of each protein kinase, cAMP kinase phosphorylates the 165-kDa protein faster than the 55-kDa protein. Protein kinase C phosphorylates preferentially the 55-kDa protein. cAMP kinase incorporates up to 1.6 mol phosphate/mol protein into the 165-kDa protein and 1 mol/mol into the 55-kDa protein upon prolonged incubation. At a physiological concentration of cAMP kinase 1 mol phosphate is incorporated/mol 165-kDa protein within 10 min, suggesting a physiological role of this phosphorylation. Protein kinase C incorporates up to 1 mol phosphate/mol into the 55-kDa protein and less than 1 mol/mol into the 165-kDa protein. Tryptic phosphopeptide analysis reveals that cAMP kinase phosphorylates two distinct peptides in the 165-kDa protein, whereas protein kinase C phosphorylates a single peptide in the 165-kDa protein. cAMP kinase and protein kinase C phosphorylate three and two peptides in the 55-kDa protein, respectively. Mixtures of the tryptic phosphopeptides derived from the 165-kDa and 55-kDa proteins elute according to the composite of the two elution profiles. These results suggest that the 165-kDa protein, which contains the binding sites for each class of calcium channel blockers and the basic calcium-conducting structure, is a specific substrate for cAMP kinase. The 55-kDa protein apparently contains sites preferentially phosphorylated by protein kinase C.
Topics: Animals; Calcium Channels; Cattle; Chromatography, Gel; Chromatography, High Pressure Liquid; Cyclic AMP; Kinetics; Microsomes; Muscles; Peptide Fragments; Phosphorylation; Protein Kinase C; Protein Kinases; Rabbits; Rats; Receptors, Nicotinic; Substrate Specificity; Trypsin
PubMed: 2824197
DOI: 10.1111/j.1432-1033.1987.tb13590.x -
Current Biology : CB Jul 2008Studies show that high phosphotidylinositol 3,4,5-trisphosphate (PIP(3)) promotes cytoskeletal rearrangements and alters cell motility and chemotaxis, possibly through...
BACKGROUND
Studies show that high phosphotidylinositol 3,4,5-trisphosphate (PIP(3)) promotes cytoskeletal rearrangements and alters cell motility and chemotaxis, possibly through activation of protein kinase Bs (PKBs). However, chemotaxis can still occur in the absence of PIP(3), and the identities of the PIP(3)-independent pathways remain unknown.
RESULTS
Here, we outline a PIP(3)-independent pathway linking temporal and spatial activation of PKBs by Tor complex 2 (TorC2) to the chemotactic response. Within seconds of stimulating Dictyostelium cells with chemoattractant, two PKB homologs, PKBA and PKBR1, mediate transient phosphorylation of at least eight proteins, including Talin, PI4P 5-kinase, two Ras GEFs, and a RhoGap. Surprisingly, all of the substrates are phosphorylated with normal kinetics in cells lacking PI 3-kinase activity. Cells deficient in TorC2 or PKB activity show reduced phosphorylation of the endogenous substrates and are impaired in chemotaxis. The PKBs are activated through phosphorylation of their hydrophobic motifs via TorC2 and subsequent phosphorylation of their activation loops. These chemoattractant-inducible events are restricted to the cell's leading edge even in the absence of PIP(3). Activation of TorC2 depends on heterotrimeric G protein function and intermediate G proteins, including Ras GTPases.
CONCLUSIONS
The data lead to a model where cytosolic TorC2, encountering locally activated small G protein(s) at the leading edge of the cell, becomes activated and phosphorylates PKBs. These in turn phosphorylate a series of signaling and cytoskeletal proteins, thereby regulating directed migration.
Topics: Amino Acid Motifs; Animals; Chemotaxis; Dictyostelium; Enzyme Activation; GTP-Binding Proteins; Hydrophobic and Hydrophilic Interactions; Models, Biological; Phosphatidylinositol Phosphates; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Protozoan Proteins; Signal Transduction; Substrate Specificity
PubMed: 18635356
DOI: 10.1016/j.cub.2008.06.068 -
Journal of Neurochemistry Dec 2010The D(1) dopamine receptor (D(1) DAR) is robustly phosphorylated by multiple protein kinases, yet the phosphorylation sites and functional consequences of these... (Comparative Study)
Comparative Study
The D(1) dopamine receptor (D(1) DAR) is robustly phosphorylated by multiple protein kinases, yet the phosphorylation sites and functional consequences of these modifications are not fully understood. Here, we report that the D(1) DAR is phosphorylated by protein kinase C (PKC) in the absence of agonist stimulation. Phosphorylation of the D(1) DAR by PKC is constitutive in nature, can be induced by phorbol ester treatment or through activation of Gq-mediated signal transduction pathways, and is abolished by PKC inhibitors. We demonstrate that most, but not all, isoforms of PKC are capable of phosphorylating the receptor. To directly assess the functional role of PKC phosphorylation of the D(1) DAR, a site-directed mutagenesis approach was used to identify the PKC sites within the receptor. Five serine residues were found to mediate the PKC phosphorylation. Replacement of these residues had no effect on D(1) DAR expression or agonist-induced desensitization; however, G protein coupling and cAMP accumulation were significantly enhanced in PKC-null D(1) DAR. Thus, constitutive or heterologous PKC phosphorylation of the D(1) DAR dampens dopamine activation of the receptor, most likely occurring in a context-specific manner, mediated by the repertoire of PKC isozymes within the cell.
Topics: Amino Acid Sequence; Animals; Dopamine; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Mice; Molecular Sequence Data; Phosphorylation; Protein Kinase C; Rats; Receptors, Dopamine D1; Signal Transduction
PubMed: 20969574
DOI: 10.1111/j.1471-4159.2010.07074.x -
Molecular and Cellular Biology Dec 2008Fidelity in DNA replication and repair requires adequate and balanced deoxyribonucleotide pools that are maintained primarily by regulation of ribonucleotide reductase...
Fidelity in DNA replication and repair requires adequate and balanced deoxyribonucleotide pools that are maintained primarily by regulation of ribonucleotide reductase (RNR). RNR is controlled via transcription, protein inhibitor association, and subcellular localization of its two subunits, R1 and R2. Saccharomyces cerevisiae Sml1 binds R1 and inhibits its activity, while Schizosaccharomyces pombe Spd1 impedes RNR holoenzyme formation by sequestering R2 in the nucleus away from the cytoplasmic R1. Here we report the identification and characterization of S. cerevisiae Dif1, a regulator of R2 nuclear localization and member of a new family of proteins sharing separate homologous domains with Spd1 and Sml1. Dif1 is localized in the cytoplasm and acts in a pathway different from the nuclear R2-anchoring protein Wtm1. Like Sml1 and Spd1, Dif1 is phosphorylated and degraded in cells encountering DNA damage, thereby relieving inhibition of RNR. A shared domain between Sml1 and Dif1 controls checkpoint kinase-mediated phosphorylation and degradation of the two proteins. Abolishing Dif1 phosphorylation stabilizes the protein and delays damage-induced nucleus-to-cytoplasm redistribution of R2. This study suggests that Dif1 is required for nuclear import of the R2 subunit and plays an essential role in regulating the dynamic RNR subcellular localization.
Topics: Active Transport, Cell Nucleus; Cytoplasm; DNA Damage; Phosphorylation; Protein Subunits; Ribonucleotide Reductases; Saccharomyces cerevisiae Proteins
PubMed: 18838542
DOI: 10.1128/MCB.01388-08