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

1989 ◽  
Vol 108 (2) ◽  
pp. 553-567 ◽  
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.

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

1990 ◽  
Vol 10 (6) ◽  
pp. 2983-2990
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.

scholarly journals Phorbol esters sensitize platelets to activation by physiological agonists

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1373-1381 ◽  
Author(s):  
W Siess ◽  
EG Lapetina
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phorbol Esters ◽  
Platelet Rich Plasma ◽  
Dense Granule ◽  
Myosin Light Chain Phosphorylation ◽  
Kinase C

Phorbol esters such as phorbol 12, 13-dibutyrate (PdBu; 40 to 200 nmol/L) or 12-O-tetradecanoyl phorbol 13-acetate (20 to 80 nmol/L) added to aspirinized platelet-rich plasma (PRP) 5 to 15 seconds prior to various platelet stimuli (epinephrine, ADP, prostaglandin endoperoxide analog U44069, collagen, PAF, or vasopressin) potentiate the rate and extent of aggregation and ATP secretion induced by those agonists. Platelet aggregation, but not secretion, is potentiated at low concentrations of agonists; platelet secretion is potentiated at higher concentrations of the platelet stimuli. Potentiation of platelet responses was also observed when the preincubation time with PdBu was extended to 12 minutes and also occurred in washed platelets. The potentiating effect of phorbol esters is not mediated by formation of arachidonate metabolites or by released ADP. The sensitizing effect of PdBu on platelet aggregation induced by epinephrine is unique, since in contrast to the other platelet stimuli it is also found at maximal concentrations of epinephrine and does not diminish with prolonged preincubation of platelets with PdBu. Activation of protein kinase C ranges from 20% to 80% over control after 1 to 10 minutes of platelet pretreatment with PdBu but dramatically increases after subsequent addition of a stimulus such as vasopressin. In contrast, agonist- induced myosin light chain phosphorylation is reduced after platelet pretreatment with PdBu. The results indicate that protein kinase C activation enhances platelet aggregation and dense granule secretion triggered by physiologic stimuli, although it desensitizes agonist- induced myosin light chain phosphorylation.

Phorbol ester-induced contraction in chemically skinned vascular smooth muscle

1986 ◽  
Vol 251 (3) ◽  
pp. C356-C361 ◽  
Author(s):  
M. Chatterjee ◽  
M. Tejada
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Phorbol Ester ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phorbol Esters ◽  
Myosin Light Chain Phosphorylation ◽  
Kinase C

We studied the contractile response to phorbol esters and its relationship to myosin light chain phosphorylation in intact and Triton X-100-skinned porcine carotid preparations. Muscle contraction was activated by phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD). Dose-dependent contractions to PDBu were obtained both in the intact and skinned preparations. The maximal values of stress in response to PDBu were 1.11 +/- 0.10 X 10(5) N/m2 (n = 7) in the intact and 5.72 +/- 0.59 X 10(4) N/m2 (n = 10) in the skinned muscles. The skinned tissues responded to PDD, which has been shown to activate protein kinase C, but not to the inactive isomer 4 alpha-PDD, thus ruling out nonspecific phorbol effects. The phorbol ester response exhibited a Ca2+ dependence. High stresses in the skinned muscles (5.53 +/- 0.69 X 10(4) N/m2, n = 8) were associated with low values of myosin light chain phosphorylation (0.18 +/- 0.01 mol Pi/mol light chain, n = 8). Thus phorbol esters can contract vascular smooth muscle by a mechanism that is not proportional to myosin light chain phosphorylation and that may involve activation of protein kinase C.

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

1990 ◽  
Vol 10 (6) ◽  
pp. 2983-2990 ◽  
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.

Protein kinase C mediation of Ca2+-independent contractions of vascular smooth muscle

1996 ◽  
Vol 74 (4) ◽  
pp. 485-502 ◽  
Author(s):  
Michael P. Walsh ◽  
Odile Clément-Chomienne ◽  
Jacquelyn E. Andrea ◽  
Bruce G. Allen ◽  
Arie Horowitz ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Phorbol Esters ◽  
Contractile Response ◽  
Cross Bridge ◽  
Kinase C

Tumour-promoting phorbol esters induce slow, sustained contractions of vascular smooth muscle, suggesting that protein kinase C (PKC) may play a role in the regulation of smooth muscle contractility. In some cases, e.g., ferret aortic smooth muscle, phorbol ester induced contractions occur without a change in [Ca2+]i or myosin phosphorylation. Direct evidence for the involvement of PKC came from the use of single saponin-permeabilized ferret aortic cells. A constitutively active catalytic fragment of PKC induced a slow, sustained contraction similar to that triggered by phenylephrine. Both responses were abolished by a peptide inhibitor of PKC. Contractions of similar magnitude occurred even when the [Ca2+] was reduced to close to zero, implicating a Ca2+-independent isoenzyme of PKC. Of the two Ca2+-independent PKC isoenzymes, ε and ζ, identified in ferret aorta, PKCε is more likely to mediate the contractile response because (i) PKCε, but not PKCζ, is responsive to phorbol esters; (ii) upon stimulation with phenylephrine, PKCε translocates from the sarcoplasm to the sarcolemma, whereas PKCζ translocates from a perinuclear localization to the interior of the nucleus; and (iii) when added to permeabilized single cells of the ferret aorta at pCa 9, PKCε, but not PKCζ, induced a contractile response similar to that induced by phenylephrine. A possible substrate of PKCε is the smooth muscle specific, thin filament associated protein, calponin. Calponin is phosphorylated in intact smooth muscle strips in response to carbachol, endothelin-1, phorbol esters, or okadaic acid. Phosphorylation of calponin in vitro by PKC (a mixture of α, β, and γ isoenzymes) dramatically reduces its affinity for F-actin and alleviates its inhibition of the cross-bridge cycling rate. Calponin is phosphorylated in vitro by PKCε but is a very poor substrate of PKCζ. A signal transduction pathway is proposed to explain Ca2+-independent contraction of ferret aorta whereby extracellular signals trigger diacylglycerol production without a Ca2+ transient. The consequent activation of PKCε would result in calponin phosphorylation, its release from the thin filaments, and alleviation of inhibition of cross-bridge cycling. Slow, sustained contraction then results from a slow rate of cross-bridge cycling because of the basal level of myosin light chain phosphorylation (≈0.1 mol Pi/mol light chain). We also suggest that signal transduction through PKCε is a component of contractile responses triggered by agonists that activate phosphoinositide turnover; this may explain why smooth muscles often develop more force in response, e.g., to α1-adrenergic agonists than to K+.Key words: smooth muscle, protein kinase C, calponin.

scholarly journals Phorbol esters sensitize platelets to activation by physiological agonists

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1373-1381 ◽  
Author(s):  
W Siess ◽  
EG Lapetina
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phorbol Esters ◽  
Platelet Rich Plasma ◽  
Dense Granule ◽  
Myosin Light Chain Phosphorylation ◽  
Kinase C

Abstract Phorbol esters such as phorbol 12, 13-dibutyrate (PdBu; 40 to 200 nmol/L) or 12-O-tetradecanoyl phorbol 13-acetate (20 to 80 nmol/L) added to aspirinized platelet-rich plasma (PRP) 5 to 15 seconds prior to various platelet stimuli (epinephrine, ADP, prostaglandin endoperoxide analog U44069, collagen, PAF, or vasopressin) potentiate the rate and extent of aggregation and ATP secretion induced by those agonists. Platelet aggregation, but not secretion, is potentiated at low concentrations of agonists; platelet secretion is potentiated at higher concentrations of the platelet stimuli. Potentiation of platelet responses was also observed when the preincubation time with PdBu was extended to 12 minutes and also occurred in washed platelets. The potentiating effect of phorbol esters is not mediated by formation of arachidonate metabolites or by released ADP. The sensitizing effect of PdBu on platelet aggregation induced by epinephrine is unique, since in contrast to the other platelet stimuli it is also found at maximal concentrations of epinephrine and does not diminish with prolonged preincubation of platelets with PdBu. Activation of protein kinase C ranges from 20% to 80% over control after 1 to 10 minutes of platelet pretreatment with PdBu but dramatically increases after subsequent addition of a stimulus such as vasopressin. In contrast, agonist- induced myosin light chain phosphorylation is reduced after platelet pretreatment with PdBu. The results indicate that protein kinase C activation enhances platelet aggregation and dense granule secretion triggered by physiologic stimuli, although it desensitizes agonist- induced myosin light chain phosphorylation.

Differential Regulation of Myosin Regulatory Light Chain Phosphorylation by Protein Kinase C Isozymes in Human Uterine Myocytes

2018 ◽  
Vol 26 (7) ◽  
pp. 988-996
Author(s):  
Bryan F. Mitchell ◽  
Mei Chi ◽  
Elle Surgent ◽  
Bailey M. Sorochan ◽  
Curtis N. Tracey ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Ex Vivo ◽  
Neonatal Morbidity ◽  
The Novel ◽  
Uterine Contractility ◽  
Uterine Activity ◽  
Kinase C

Background: Preterm birth is the most common cause of neonatal morbidity and mortality and a common precedent to lifelong disability. Current treatment has minimal efficacy. Objective: We assessed the role of isozymes of the protein kinase C (PKC) family in regulating the phosphorylation of myosin regulatory light chains (RLCs), which regulate uterine contractility. We also explored the mechanisms through which these isozymes function. Study Design: We used a previously characterized and validated quantitative in-cell Western (ICW) assay to measure site-specific phosphorylations on myosin RLC and CPI-17. Cultures of human uterine myocytes (hUM) were treated with the potent contractile stimulant oxytocin to induce uterine contractility or a pharmacological mimic of diacyl-glycerol to stimulate the conventional and novel isozymes of the PKC family. Combinations of isozyme-selective inhibitors were used to determine the effects of the conventional and novel classes of isozymes. Results: Stimulation of PKC using phospho-dibutyrate caused immediate, concentration-dependent inhibition of uterine activity ex vivo. Using the ICW assay with hUM, the oxytocin-stimulated increase in the pro-contractile phosphorylations of myosin RLCs at serine19 and threonine18 was completely inhibited by prior treatment with phorbol-12-myristate-13-acetate, which stimulates both convention and novel classes of isozymes. Our results suggest that the conventional class of isozymes cause a reduction in phosphorylations at serine19 and threonine18 by reducing activity of myosin light chain kinase. The novel class of isozymes has 2 mechanisms of action: the first is activation of CPI-17 through phosphorylation at threonine38, which results in reduced activity of myosin light chain phosphatase and increased levels of activated myosin RLC; the second is increased phosphorylation of the N-terminal region of myosin RLC. Conclusions: Specific agonists for the conventional isozymes or inhibitors of the novel isozymes of the PKC family could be useful pharmacological agents for regulation of uterine activity.

Isolation and characterization of PKC-L, a new member of the protein kinase C-related gene family specifically expressed in lung, skin, and heart

1991 ◽  
Vol 11 (1) ◽  
pp. 126-133 ◽  
Author(s):  
N Bacher ◽  
Y Zisman ◽  
E Berent ◽  
E Livneh
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Gene Family ◽  
Phorbol Esters ◽  
Structural Features ◽  
Cos Cells ◽  
Isolation And Characterization ◽  
Kinase C ◽  
Human Epidermoid Carcinoma

We have isolated and characterized a new human cDNA, coding for a protein kinase, related to the protein kinase C (PKC) gene family. Although this protein kinase shares some homologous sequences and structural features with the four members of the PKC family initially isolated (alpha, beta I, beta II, and gamma), it shows more homology with the recently described PKC-related subfamily, encoded by the cDNAs delta, epsilon, and zeta. The transcript for this gene product, termed PKC-L, is most abundant in lung tissue, less expressed in heart and skin tissue, and exhibited very low expression in brain tissue. Thus, its tissue distribution is different from that described for other mammalian members of the PKC gene family, their expression being enriched in brain tissues. PKC-L is also expressed in several human cell lines, including the human epidermoid carcinoma line A431. The ability of phorbol esters to bind to and stimulate the kinase activity of PKC-L was revealed by introducing the cDNA into COS cells.

Sign in / Sign up

Export Citation Format

Share Document