A238L Inhibits NF-ATc2, NF-κB, and c-Jun Activation through a Novel Mechanism Involving Protein Kinase C-θ-Mediated Up-Regulation of the Amino-Terminal Transactivation Domain of p300

Available data indicate that the liver is a target organ for parathyroid hormone (PTH) and that this effect is most likely mediated by PTH-induced calcium entry into hepatocytes. The present study examined the effects of both PTH-(1-84) and its amino-terminal fragment [PTH-(1-34)] on cytosolic calcium concentration ([Ca2+]i) of hepatocytes and explored the cellular pathways that mediate this potential action of PTH. Both moieties of PTH produced a dose-dependent rise in [Ca2+]i, but the effect of PTH-(1-84) was greater (P < 0.01) than an equimolar amount of PTH-(1-34). This effect required calcium in the medium and was totally [PTH-(1-34)] or partially [PTH-(1-84)] blocked by PTH antagonist ([Nle8,18,Tyr34]bPTH-(7-34)-NH2] and by verapamil or nifedipine. Sodium or chloride channel blockers did not modify this effect. 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), and G protein activator also produced a dose-dependent rise in [Ca2+]i. Staurosporine abolished the effect of TPA, and both staurosporine and calphostin C partially inhibited the effect of PTH. Staurosporine and verapamil together produced greater inhibition of PTH action than each alone. Rp-cAMP, a competitive inhibitor of cAMP binding to the R subunit of protein kinase A, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, blocked the effect of both DBcAMP and PTH, but the effect of these agents was greater (P < 0.01) on DBcAMP action. G protein inhibitor and pertussis toxin partially blocked the action of PTH. The data indicate that 1) PTH increases [Ca2+]i of hepatocytes; 2) this action of the hormone is receptor mediated; 3) the predominant pathway for this PTH action is the stimulation of a G protein-adenylate cyclase-cAMP system, which then leads to stimulation of a calcium transport system inhibitable by verapamil or nifedipine or activation of L-type calcium channels; 4) activation of protein kinase C is also involved; and 5) the PTH-induced rise in [Ca2+]i is due, in major parts, to movement of extracellular calcium into the cell.

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Yeast phenotype classifies mammalian protein kinase C cDNA mutants.

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4728 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4728-4735 ◽

Cited By ~ 9

Author(s):

H Riedel ◽

L Su ◽

H Hansen

Keyword(s):

Protein Kinase C ◽

Catalytic Activity ◽

Protein Kinase ◽

Phorbol Ester ◽

Colony Size ◽

Receptor Protein ◽

Amino Terminal ◽

Kinase C ◽

Carboxyl Terminal ◽

Pkc Alpha

The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of eukaryotic cellular signals. Molecular dissection of their mechanisms of action has been limited in part by the lack of random mutagenesis approaches and by the complexity of signaling pathways in mammalian cells which involve multiple PKC isoforms. Here we present a rapid screen which permits the quantification of mammalian PKC activity phenotypically in the yeast Saccharomyces cerevisiae. Bovine PKC alpha cDNA is functionally expressed in S. cerevisiae. This results in a phorbol ester response: a fourfold increase in the cell doubling time and a substantial decrease in yeast colony size on agar plates. We have expressed pools of bovine PKC alpha cDNAs mutagenized by Bal 31 deletion of internal, amino-terminal, or carboxyl-terminal sequences and have identified three classes of mutants on the basis of their distinct yeast phenotypes. Representatives of each class were analyzed. An internal deletion of amino acids (aa) 172 to 225 displayed ligand-dependent but reduced catalytic activity, an amino-terminal truncation of aa 1 to 153 displayed elevated and ligand-independent activity, and a carboxyl-terminal 26-aa truncation (aa 647 to 672) lacked activity under any conditions. Additional mutations confirmed the distinct functional characteristics of these classes. Our data show that deletion of the V1 and C1 regions results in elevated basal catalytic activity which is still Ca2+ responsive. Internal deletions in the V2 and C2 regions do not abolish phorbol ester or Ca2+ regulation of PKC activity, suggesting that most of the C2 domain is not essential for phorbol ester stimulation and most of the regulatory domain is dispensable for Ca2+ regulation of PKC activity. These distinct activities od the PKC mutants correlate with a specific and proportional yeast phenotype and are quantified on agar plates by yeast colony size. This provides a phenotypic screen which is suitable to identity rare, randomly altered but active mammalian PKC mutants. It quantifies their catalytic and biological activities in response to PKC activators or inhibitors for a systematic mapping of PKC structure and function or PKC-drug interaction.

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Protein Kinase C-Dependent Phosphorylation Regulates the Cell Cycle-Inhibitory Function of the p73 Carboxy Terminus Transactivation Domain

Molecular and Cellular Biology ◽

10.1128/mcb.00585-08 ◽

2009 ◽

Vol 29 (7) ◽

pp. 1814-1825 ◽

Cited By ~ 14

Author(s):

Ulrika Nyman ◽

Pinelopi Vlachos ◽

Anna Cascante ◽

Ola Hermanson ◽

Boris Zhivotovsky ◽

...

Keyword(s):

Cell Cycle ◽

Protein Kinase C ◽

Protein Kinase ◽

Cell Cycle Arrest ◽

Cell Cycle Progression ◽

Transactivation Domain ◽

Carboxy Terminus ◽

Cycle Progression ◽

Cycle Arrest ◽

Kinase C

ABSTRACT The transcription factor p73, a member of the p53 family of proteins, is involved in the regulation of cell cycle progression and apoptosis. However, the regulatory mechanisms controlling the distinct roles for p73 in these two processes have remained unclear. Here, we report that p73 is able to induce cell cycle arrest independently of its amino-terminal transactivation domain, whereas this domain is crucial for p73 proapoptotic functions. We also characterized a second transactivation domain in the carboxy terminus of p73 within amino acid residues 381 to 399. This carboxy terminus transactivation domain was found to preferentially regulate genes involved in cell cycle progression. Moreover, its activity is regulated throughout the cell cycle and modified by protein kinase C-dependent phosphorylation at serine residue 388. Our results suggest that this novel posttranslational modification within the p73 carboxy terminus transactivation domain is involved in the context-specific guidance of p73 toward the selective induction of cell cycle arrest.

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Normal cellular and transformation-associated abl proteins share common sites for protein kinase C phosphorylation.

Molecular and Cellular Biology ◽

10.1128/mcb.7.12.4280 ◽

1987 ◽

Vol 7 (12) ◽

pp. 4280-4289 ◽

Cited By ~ 29

Author(s):

A M Pendergast ◽

J A Traugh ◽

O N Witte

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Growth Factor Receptor ◽

Kinase Domain ◽

Kinase Assay ◽

Amino Terminal ◽

Kinase C ◽

Carboxy Terminal

Viral transduction and chromosomal translocations of the c-abl gene result in the synthesis of abl proteins with structurally altered amino termini. These altered forms of the abl protein, but not the c-abl proteins, are detectably phosphorylated on tyrosine in vivo. In contrast, all forms of the abl protein are phosphorylated on serine following in vivo labeling with Pi. Treatment of NIH-3T3 cells with protein kinase C activators resulted in a four- to eightfold increase in the phosphorylation of murine c-abl due to modification of two serines on the c-abl protein. Purified protein kinase C phosphorylated all abl proteins at the same two sites. Both sites are precisely conserved in murine and human abl proteins. The sites on the abl proteins were found near the carboxy terminus. In contrast, for the epidermal growth factor receptor (T. Hunter, N. Ling, and J. A. Cooper, Nature [London] 311:480-483, 1984) and pp60src (K. L. Gould, J. R. Woodgett, J. A. Cooper, J. E. Buss, D. Shalloway, and T. Hunter, Cell 42:849-857, 1985), the sites of protein kinase C phosphorylation are amino-terminal to the kinase domain. The abl carboxy-terminal region is not necessary for the tyrosine kinase activity or transformation potential of the viral abl protein and may represent a regulatory domain. Using an in vitro immune complex kinase assay, we were not able to correlate reproducible changes in c-abl activity with phosphorylation by protein kinase C. However, the high degree of conservation of the phosphorylation sites for protein kinase C between human and mouse abl proteins suggests an important functional role.

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Schizosaccharomyces pombe protein kinase C homologues, pck1p and pck2p, are targets of rho1p and rho2p and differentially regulate cell integrity

Journal of Cell Science ◽

10.1242/jcs.112.20.3569 ◽

1999 ◽

Vol 112 (20) ◽

pp. 3569-3578 ◽

Cited By ~ 3

Author(s):

M. Arellano ◽

M.H. Valdivieso ◽

T.M. Calonge ◽

P.M. Coll ◽

A. Duran ◽

...

Keyword(s):

Cell Wall ◽

Protein Kinase C ◽

Protein Kinase ◽

Schizosaccharomyces Pombe ◽

Cell Integrity ◽

Beta Glucan ◽

Functional Link ◽

Glucan Synthase ◽

Amino Terminal ◽

Kinase C

Schizosaccharomyces pombe rho1(+) is required for maintenance of cell integrity and polarization of the actin cytoskeleton. However, no other effector besides the (1,3)beta-D-glucan synthase enzyme has been identified in S. pombe. We have further investigated if rho1(+)signalling could be also mediated by the two protein kinase C homologues, pck1p and pck2p. We show in this study that both kinases interact with rho1p and rho2p only when bound to GTP, as most GTPase effectors do. Interestingly, the interaction was mapped in a different part of the proteins than in Saccharomyces cerevisiae Pkc1p. Thus, active rho1p binds to the amino-terminal region of the pcks where two HR1 motifs are located, and binding to the GTPase dramatically stabilizes the kinases. Detailed biochemical analysis suggests that pck2p is more important in the regulation of the enzyme (1–3)beta-D-glucan synthase. Thus, overexpression of pck2(+), but not pck1(+), caused a general increase in cell wall biosynthesis, mainly in beta-glucan, and (1–3)beta-D-glucan synthase activity was considerably augmented. When this activity was separated into soluble and membrane fractions and reconstituted, the increase caused by pck2(+) overexpression was exclusively detected in the membrane component. We also show that both protein kinase C homologues are required for the maintenance of cell integrity. pck1delta and pck2delta strains present a number of defects related to the cell wall, indicating that this structure might be co-ordinately regulated by both kinases. In addition, pck2p, but not pck1p, seems to be involved in keeping cell polarity. Genetic evidence indicates that both pck1(+) and pck2(+) interact with cps1(+) and gls2(+), two genes similar to S. cerevisiae FKS1 and FKS2 that encode membrane subunits of the (1–3)beta-D-glucan synthase. pck1(+)also showed a genetic interaction with ras1(+) and ral1(+) suggesting the existence of a functional link between both signalling pathways.

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Increased Secretion of the Amino-Terminal Fragment of Amyloid Precursor Protein in Brains of Rats with a Constitutive Up-Regulation of Protein Kinase C

Journal of Neurochemistry ◽

10.1046/j.1471-4159.1997.68062523.x ◽

2002 ◽

Vol 68 (6) ◽

pp. 2523-2529 ◽

Cited By ~ 34

Author(s):

A. Caputi ◽

S. Barindelli ◽

L. Pastorino ◽

M. Cimino ◽

J. D. Buxbaum ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Amyloid Precursor Protein ◽

Precursor Protein ◽

Amino Terminal ◽

Kinase C ◽

Terminal Fragment ◽

Amino Terminal Fragment

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Protein kinase C mu is located at the Golgi compartment.

The Journal of Cell Biology ◽

10.1083/jcb.134.6.1401 ◽

1996 ◽

Vol 134 (6) ◽

pp. 1401-1410 ◽

Cited By ~ 80

Author(s):

J Prestle ◽

K Pfizenmaier ◽

J Brenner ◽

F J Johannes

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Transport Processes ◽

Laser Scanning Microscopy ◽

Leader Peptide ◽

Pleckstrin Homology Domain ◽

Amino Terminal ◽

Kinase C ◽

Long Term Treatment ◽

Golgi Compartment

Protein kinase C mu (PKC mu) displays unusual structural features like a pleckstrin homology domain and an amino-terminal hydrophobic region with a putative leader peptide and transmembrane sequence. As a discrete location often is a direct clue to the potential biological function of a kinase, antibodies directed against unique amino- and carboxy-terminal domains of PKC mu were used to localize the protein within intracellular compartments in immunofluorescence and subcellular fractionation studies. Confocal laser scanning microscopy showed colocalization of PKC mu with the resident Golgi marker protein beta 1,4 galactosyltransferase in PKC mu transfectants and in the human hepatocellular carcinoma cell line HepG2, expressing endogenous PKC mu. Long-term treatment of cells with brefeldin A, which disintegrates the Golgi apparatus, disrupted PKC mu-specific staining. Cosegregation of PKC mu with beta 1,4 galactosyltransferase, but not with the endosomal marker rab5, upon density gradient fractionation and Western blot analysis of HepG2 cell extracts, provides independent evidence for a Golgi localization of PKC mu. Moreover, cellular sulfate uptake and Golgi-specific glycosaminoglycan sulfation was enhanced in PKC mu transfectants. Together, these data suggest that PKC mu is a resident protein kinase of the core Golgi compartment and is involved in basal transport processes.

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Normal cellular and transformation-associated abl proteins share common sites for protein kinase C phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.7.12.4280-4289.1987 ◽

1987 ◽

Vol 7 (12) ◽

pp. 4280-4289

Author(s):

A M Pendergast ◽

J A Traugh ◽

O N Witte

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Growth Factor Receptor ◽

Kinase Domain ◽

Kinase Assay ◽

Amino Terminal ◽

Kinase C ◽

Carboxy Terminal

Viral transduction and chromosomal translocations of the c-abl gene result in the synthesis of abl proteins with structurally altered amino termini. These altered forms of the abl protein, but not the c-abl proteins, are detectably phosphorylated on tyrosine in vivo. In contrast, all forms of the abl protein are phosphorylated on serine following in vivo labeling with Pi. Treatment of NIH-3T3 cells with protein kinase C activators resulted in a four- to eightfold increase in the phosphorylation of murine c-abl due to modification of two serines on the c-abl protein. Purified protein kinase C phosphorylated all abl proteins at the same two sites. Both sites are precisely conserved in murine and human abl proteins. The sites on the abl proteins were found near the carboxy terminus. In contrast, for the epidermal growth factor receptor (T. Hunter, N. Ling, and J. A. Cooper, Nature [London] 311:480-483, 1984) and pp60src (K. L. Gould, J. R. Woodgett, J. A. Cooper, J. E. Buss, D. Shalloway, and T. Hunter, Cell 42:849-857, 1985), the sites of protein kinase C phosphorylation are amino-terminal to the kinase domain. The abl carboxy-terminal region is not necessary for the tyrosine kinase activity or transformation potential of the viral abl protein and may represent a regulatory domain. Using an in vitro immune complex kinase assay, we were not able to correlate reproducible changes in c-abl activity with phosphorylation by protein kinase C. However, the high degree of conservation of the phosphorylation sites for protein kinase C between human and mouse abl proteins suggests an important functional role.

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