Myristoylation-dependent and Electrostatic Interactions Exert Independent Effects on the Membrane Association of the Myristoylated Alanine-rich Protein Kinase C Substrate Protein in Intact Cells

Expression of the N-ras oncogene under the control of the glucocorticoid-responsive promoter in the pheochromocytoma cell line UR61, a subline of PC-12 cells, has been used to investigate the differentiation process to neuronal cells triggered by ras oncogenes (I. Guerrero, A. Pellicer, and D. E. Burstein, Biochem. Biophys. Res. Commun. 150:1185-1192, 1988). Using ras-inducible cell lines, we observed that expression of the oncogenic N-ras p21 protein interferes with the ability of phorbol esters to induce downregulation of protein kinase C. This effect was associated with the appearance of immunologically detectable protein kinase C as well as the activity of the enzyme as analyzed either by binding of [3H]phorbol-12,13-dibutyrate in intact cells or by in vitro kinase activity. These results indicate a relationship between ras p21 and protein kinase C in neuronal differentiation in this model system. Comparison to the murine fibroblast system suggests that this relationship may be functional.

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Protein kinase C mediates cholinergically regulated protein phosphorylation in a Cl(-)-secreting epithelium

AJP Cell Physiology ◽

10.1152/ajpcell.1990.258.2.c227 ◽

1990 ◽

Vol 258 (2) ◽

pp. C227-C233 ◽

Cited By ~ 29

Author(s):

J. A. Cohn

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Phosphorylation ◽

Cell Extract ◽

T84 Cells ◽

Intact Cells ◽

Cell Monolayers ◽

Kinase C ◽

Cell Fractions ◽

Prominent Response

T84 cell monolayers were used to study the cholinergic regulation of protein phosphorylation in epithelial cells. When T84 cell monolayers are labeled with 32Pi and stimulated with carbachol, six proteins exhibit altered phosphorylation. The most prominent response is a fivefold increase in labeling of p83, an acidic protein of Mr 83,000. Increasing labeling of p83 parallels stimulated secretion with respect to the onset of agonist action, agonist potency, and antagonism by atropine. However, the p83 and secretory responses differ in that the p83 response is more sustained. When T84 cell fractions are incubated with [gamma-32P]ATP, Ca2(+)-phospholipid stimulates p83 labeling. Phosphorylation of p83 also occurs when a T84 cell extract is incubated with purified protein kinase C and when intact cells are exposed to phorbol myristate acetate. p83 does not become phosphorylated in cell fractions incubated with adenosine 3',5'-cyclic monophosphate (cAMP) or in monolayers stimulated with agonists acting via cAMP. Thus carbachol stimulates the phosphorylation of an endogenous substrate for protein kinase C in T84 cells. The duration of this phosphorylation response suggests that protein kinase C may mediate a sustained response to carbachol, possibly acting to limit the duration of stimulated secretion.

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Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway.

The EMBO Journal ◽

10.1002/j.1460-2075.1996.tb01012.x ◽

1996 ◽

Vol 15 (22) ◽

pp. 6220-6230 ◽

Cited By ~ 195

Author(s):

J. L. Zugaza ◽

J. Sinnett-Smith ◽

J. Van Lint ◽

E. Rozengurt

Keyword(s):

Signal Transduction ◽

Protein Kinase C ◽

Protein Kinase ◽

Signal Transduction Pathway ◽

Protein Kinase D ◽

Transduction Pathway ◽

Intact Cells ◽

Kinase C ◽

Dependent Signal

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Mitogen stimulation of Na+-H+ exchange: differential involvement of protein kinase C

AJP Cell Physiology ◽

10.1152/ajpcell.1987.253.2.c219 ◽

1987 ◽

Vol 253 (2) ◽

pp. C219-C229 ◽

Cited By ~ 19

Author(s):

L. L. Muldoon ◽

G. A. Jamieson ◽

A. C. Kao ◽

H. C. Palfrey ◽

M. L. Villereal

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cell Types ◽

Intact Cells ◽

Kinase C ◽

Protein Kinase C Activity ◽

Human Fibroblast Strains ◽

Stimulation Of

The mitogen-induced activation of Na+-H+ exchange was investigated in two cultured human fibroblast strains (HSWP and WI-38 cells) that, based on previous studies, differed in their response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (L. M. Vincentini and M. L. Villereal, Proc. Natl. Acad. Sci. USA 82: 8053-8056, 1985). The role of protein kinase C in the activation of Na+-H+ exchange was investigated by comparing the effects of TPA on Na+ influx, in vitro phosphorylation, and in vivo phosphorylation in both cell types. Although both cell types have significant quantities of protein kinase C activity that can be activated by TPA in intact cells, the addition of TPA to intact cells stimulates Na+ influx in WI-38 cells but not in HSWP cells, indicating that in HSWP cells the stimulation of protein kinase C is not sufficient to activate the Na+-H+ exchanger. Cells were then depleted of protein kinase C activity by chronic treatment with high doses of TPA. Both HSWP and WI-38 cells were rendered protein kinase C deficient by this treatment as determined by in vitro and in vivo phosphorylation studies. Protein kinase C-deficient HSWP cells lose the ability for TPA to inhibit the serum-induced activation of Na+-H+ exchange, but there is no reduction in the stimulation of Na+ influx by serum, bradykinin, vasopressin, melittin, or vanadate, indicating that protein kinase C activity is not necessary for the mitogen-induced activation of Na+-H+ exchange in HSWP cells by agents known to stimulate phosphatidylinositol turnover (G. A. Jamieson and M. Villereal. Arch. Biochem. Biophys. 252: 478-486, 1987). In contrast, depletion of protein kinase C activity in WI-38 cells significantly reduces both the TPA- and the serum-induced activation of the Na+-H+ exchange system, suggesting that protein kinase C activity is necessary for at least a portion of the mitogen-induced activation of the Na+-H+ exchanger in WI-38 cells. These results indicate that the mechanisms for regulating Na+-H+ exchange can differ dramatically between different types of fibroblasts.

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Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton.

The Journal of Cell Biology ◽

10.1083/jcb.108.2.553 ◽

1989 ◽

Vol 108 (2) ◽

pp. 553-567 ◽

Cited By ~ 39

Author(s):

V Papadopoulos ◽

P F Hall

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Light Chain ◽

Myosin Light Chain ◽

Phorbol Esters ◽

Intact Cells ◽

Adrenal Cells ◽

Isolation And Characterization ◽

Kinase C

The cytoskeletons of Y-1 mouse adrenal tumor cells contain a calcium and phospholipid-dependent protein kinase (protein kinase C) that is bound sufficiently tight to resist extraction by 0.5% Triton but not by 1.0% Triton. The enzyme has been purified to near homogeneity from cytoskeleton and cytosol. It shows features typical of this type of kinase, namely a requirement for Ca2+ and phospholipid, stimulation by tumor promoters but not by nontumor-promoting phorbol esters, and inhibition by trifluoperazine. The enzyme shows specificity for four substrates found in the cytoskeleton, namely 80, 33, 20, and 18 kD. The first three substrates are phosphorylated by the enzyme; the fourth is dephosphorylated and is therefore affected by the kinase indirectly. The 80-kD protein is the kinase enzyme itself which is autophosphorylated in vitro and in the cytoskeleton. The 20-kD protein is myosin light chain. The 33- and 18-kD proteins are unidentified. The same substrates were phosphorylated when Y-1 cells were permeabilized with digitonin and incubated with [gamma-32P]ATP and phorbol-12-myristate-13-acetate. Partly purified protein kinase C changes the extent of phosphorylation of the same substrates when added to cytoskeletons previously extracted to remove endogenous protein kinase C. Addition of Ca2+, phosphatidylserine, and phorbol-12-myristate-13-acetate to cytoskeletons, and addition of these three agents plus protein kinase C to extracted cytoskeletons, causes these structures to undergo a rapid and extensive rounding. A similar change is induced in intact cells by addition of phorbol ester. It is concluded that protein kinase C is capable of changing the shape of adrenal cells by an action that involves autophosphorylation and phosphorylation of myosin light chain. This response may in turn be related to the steroidogenic responses to ACTH and cyclic AMP.

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Regulation of cation channels in liver cells by intracellular calcium and protein kinase C

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1994.266.4.g677 ◽

1994 ◽

Vol 266 (4) ◽

pp. G677-G684 ◽

Cited By ~ 2

Author(s):

J. G. Fitz ◽

A. H. Sostman ◽

J. P. Middleton

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Resting Potential ◽

Cation Channels ◽

Nucleotide Receptors ◽

Direct Role ◽

Intact Cells ◽

Kinase C ◽

Channel Opening ◽

Gamma S

The regulation of Ca(2+)-permeant cation channels in HTC hepatoma cells was investigated using patch clamp and fluorescence techniques. In intact cells, exposure to nucleotide analogues ATP, uridine 5'-triphosphate (UTP), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) caused transient opening of channels with linear conductances of approximately 18 and approximately 28 pS. Channels were permeable to Na+, K+, and Ca2+ and carried inward (depolarizing) current at the resting potential. Exposure to thapsigargin to increase cytosolic Ca2+ concentration ([Ca2+]i) opened similar channels, suggesting that opening is stimulated by a rise in [Ca2+]i. In subconfluent monolayers, ATP increased [Ca2+]i with half-maximal effects at approximately 7.4 microM; at 10(-4) M, the peak increase in [Ca2+]i was ATP > UTP > ATP gamma S >> 2-methylthioadenosine 5'-triphosphate, alpha,beta-methyleneadenosine 5'-triphosphate, and adenosine. The relative potency suggests that the effects are mediated by 5'-nucleotide receptors. In excised inside-out patches, channels were not activated by myo-inositol 1,4,5-trisphosphate (50-100 microM) or myo-inositol 1,3,4,5-trisphosphate (20 microM) but opened after increases in Ca2+ to greater than approximately 250 nM, consistent with a direct role for Ca2+ in channel opening. In intact cells, channel opening was followed by a prolonged refractory period. Protein kinase C appears to contribute by inhibition of the ATP-stimulated [Ca2+]i response and by direct inhibitory effects on the channel. These findings indicate that extracellular ATP leads to modulation of liver cell cation channels through activation of 5'-nucleotide receptors and are consistent with a model in which transient opening of channels is stimulated by a rise in [Ca2+]i and subsequent closure is mediated by protein kinase C-dependent pathways.

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