Activation of Protein Kinase C in Vitro and in Intact Cells or Synaptosomes Determined by Acetic Acid Extraction of MARCKS

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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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 protein kinase C activity in neuronal differentiation induced by the N-ras oncogene in PC-12 cells.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2983 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2983-2990 ◽

Cited By ~ 16

Author(s):

J C Lacal ◽

A Cuadrado ◽

J E Jones ◽

R Trotta ◽

D E Burstein ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Neuronal Differentiation ◽

Phorbol Esters ◽

Ras Oncogene ◽

Intact Cells ◽

Pc 12 Cells ◽

Kinase C ◽

Ras P21

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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In situ determination of [Ca2+]i threshold for translocation of the alpha-protein kinase C isoform

AJP Cell Physiology ◽

10.1152/ajpcell.1994.266.6.c1544 ◽

1994 ◽

Vol 266 (6) ◽

pp. C1544-C1551 ◽

Cited By ~ 74

Author(s):

R. A. Khalil ◽

C. Lajoie ◽

K. G. Morgan

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Second Messengers ◽

In Vitro Assays ◽

Intact Cells ◽

Kinase C ◽

Biochemical Assays

Because of inherent difficulties in maintaining physiological conditions in biochemical assays, the intracellular free Ca2+ concentration ([Ca2+]i) required for activation of protein kinase C (PKC) in intact cells remains unclear. In the present study, [Ca2+]i was measured in freshly isolated vascular smooth muscle cells loaded with fura 2 while, in parallel, the distribution of the Ca(2+)-dependent alpha-PKC isoform was monitored using digital imaging microscopy. The [Ca2+]i alpha-PKC translocation threshold was determined by changing extracellular free Ca2+ concentration in steps while monitoring [Ca2+]i. In the absence of agonists, increasing [Ca2+]i caused < 25% of maximal translocation. In the presence of phenylephrine, maximum translocation occurred at [Ca2+]i > or = 198 nM. Phenylephrine augmented translocation of alpha-PKC primarily by increasing the slope of the [Ca2+]i-PKC translocation relationship. These results indicate that the [Ca2+]i threshold of alpha-PKC translocation in situ is less than that reported in most in vitro assays and are consistent with an effect of agonist-induced generation of other second messengers that cause cooperative interactions leading to translocation.

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Direct interaction between p47phox and protein kinase C: evidence for targeting of protein kinase C by p47phox in neutrophils

Biochemical Journal ◽

10.1042/bj3440859 ◽

1999 ◽

Vol 344 (3) ◽

pp. 859-866 ◽

Cited By ~ 36

Author(s):

Emer P. REEVES ◽

Lodewijk V. DEKKER ◽

Louisa V. FORBES ◽

Frans B. WIENTJES ◽

Ann GROGAN ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Gel Filtration ◽

Direct Interaction ◽

Subcellular Fractionation ◽

Intact Cells ◽

Kinase C ◽

C Terminus ◽

Time Courses

p47phox is an essential component of the NADPH oxidase, and phosphorylation of p47phox is associated with activation of the enzyme. Here we have used p47phox affinity chromatography to extract a p47phox kinase from neutrophil cytosol. The kinase activity was purified by gel filtration and Mini Q chromatography and shown to be indistinguishable from the catalytic fragments of protein kinase C (PKC)-βI, -βII and -∆. The C-terminus of p47phox represented the site of interaction with PKC. Co-immunoprecipitation experiments revealed that the interaction between PKC isotypes and p47phox takes place in intact cells. However PKC-β and -∆ showed different time courses of co-immunoprecipitation, suggesting that the interactions may serve different functions for the various PKC isotypes. Using cells lacking p47phox, we investigated the functional relevance of the interaction between PKC and p47phox. Subcellular fractionation revealed an abnormal recruitment of PKC-βI and -βII, but not PKC-∆, to particulate fractions in p47phox-deficient cells. Phosphorylation of cytosolic proteins was generally increased in stimulated p47phox-deficient neutrophils as compared with normal neutrophils. Furthermore, the cytoskeletal protein coronin was not phosphorylated upon stimulation of p47phox-deficient neutrophils. These findings were confirmed in an in vitro-reconstituted system using rat brain cytosol in which addition of p47phox affected phosphorylation by PKC/PKM (PKM is the catalytic fragment of PKC). These results indicate that p47phox can act as a regulator of PKC in neutrophils.

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Characterization of calreticulin as a protein interacting with protein kinase C

Biochemical Journal ◽

10.1042/bj3440469 ◽

1999 ◽

Vol 344 (2) ◽

pp. 469-475 ◽

Cited By ~ 9

Author(s):

Erika RENDÓN-HUERTA ◽

Guillermo MENDOZA-HERNÁNDEZ ◽

Martha ROBLES-FLORES

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Specific Inhibitor ◽

Rat Hepatocytes ◽

Kinase Assay ◽

Intact Cells ◽

Calculated Molecular Mass ◽

Kinase C

A protein kinase C (PKC)-binding protein was purified to homogeneity from the Triton-insoluble fraction from rat hepatocytes homogenates. The protein was identified as the mature calreticulin chain by N-terminal amino acid sequencing and by its immunoreactivity with anti-calreticulin antibody raised against the C-terminal KDEL (single-letter code) sequence. The calculated molecular mass was 46.6 kDa but the protein migrates in SDS/PAGE as a doublet with apparent molecular masses of 60 and 55 kDa. Studies in vitro with purified calreticulin with the use of an overlay assay approach demonstrated that it binds to activated PKC isoenzymes expressed in rat hepatocytes. Phosphorylation of purified calreticulin with a PKC isoenzyme-specific immune complex kinase assay showed that it is also a very good substrate for all PKC isoforms in vitro. The treatment of intact cells with phorbol ester or with adrenaline (epinephrine) plus propranolol increased calreticulin phosphorylation, which was blocked by the pretreatment of cells with the PKC-specific inhibitor Ro 31-8220. The analysis of calreticulin immunoprecipitates from control or treated cells indicated that PKCα, PKCβ, PKCθ, PKCζ and PKCμ, but not PKCδ or PKCϵ, co-immunoprecipitated with calreticulin. Taken together, our results indicate that PKC interacts in vivo with calreticulin and suggest that they can operate in common signalling pathways.

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