scholarly journals Differential spatial and temporal phosphorylation of the visual receptor, rhodopsin, at two primary phosphorylation sites in mice exposed to light

2003 ◽  
Vol 374 (2) ◽  
pp. 537-543 ◽  
Author(s):  
Ryan A. ADAMS ◽  
Xinran LIU ◽  
David S. WILLIAMS ◽  
Alexandra C. NEWTON
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Outer Segment ◽  
Phorbol Esters ◽  
Phosphorylation Sites ◽  
Rod Outer Segment ◽  
Kinase C ◽  
Rhodopsin Kinase

Phosphorylation of rhodopsin critically controls the visual transduction cascade by uncoupling it from the G-protein transducin. The kinase primarily responsible for this phosphorylation is rhodopsin kinase, a substrate-regulated kinase that phosphorylates light-activated rhodopsin. Protein kinase C has been implicated in controlling the phosphorylation of both light-activated and dark-adapted rhodopsin. Two of the major rhodopsin phosphorylation sites in vivo, Ser334 and Ser338, are effective protein kinase C phosphorylation sites in vitro, while the latter is preferentially phosphorylated by rhodopsin kinase in vitro. Using phosphospecific antibodies against each of these two sites, we show that both sites are under differential spatial and temporal regulation. Exposure of mice to light results in rapid phosphorylation of Ser338 that is evenly distributed along the rod outer segment. Phosphorylation of Ser334 is considerably slower, begins at the base of the rod outer segment, and spreads to the top of the photoreceptor over time. In addition, we show that phosphorylation of both sites is abolished in rhodopsin kinase−/− mice, revealing an absolute requirement for rhodopsin kinase to phosphorylate rhodopsin. This requirement may reflect the need for priming phosphorylations at rhodopsin kinase sites allowing for subsequent phosphorylation by protein kinase C at Ser334. In this regard, treatment of mouse retinas with phorbol esters results in a 4-fold increase in phosphorylation on Ser334, with no significant effect on the phosphorylation of Ser338. Our results are consistent with light triggering rapid priming phosphorylations of rhodopsin by rhodopsin kinase, followed by a slower phosphorylation on Ser334, which is regulated by protein kinase C.

scholarly journals Phosphorylation of human pleckstrin on Ser-113 and Ser-117 by protein kinase C

1996 ◽  
Vol 314 (3) ◽  
pp. 937-942 ◽  
Author(s):  
Karen L. CRAIG ◽  
Calvin B. HARLEY
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Cos Cells ◽  
Phospholipid Hydrolysis ◽  
Kinase C

During platelet activation, receptor-coupled phospholipid hydrolysis stimulates protein kinase C (PKC) and results in the phosphorylation of several proteins, the most prominent being pleckstrin. Pleckstrin is composed of two repeated domains, now called pleckstrin homology (PH) domains, separated by a spacer region that contains several consensus PKC phosphorylation sites. To determine the role of PKC-dependent phosphorylation in pleckstrin function, we mapped the phosphorylation sites in vivo of wild-type and site-directed mutants of pleckstrin expressed in COS cells. Phosphorylation was found to occur almost exclusively on Ser-113 and Ser-117 within the sequence 108-KFARKS*TRRS*IRL-120. Phosphorylation of these sites was confirmed by phosphorylation of the corresponding wild-type and mutant synthetic peptides in vitro.

scholarly journals Effects of insulin on the translocation of protein kinase C-θ and other protein kinase C isoforms in rat skeletal muscles

1995 ◽  
Vol 308 (1) ◽  
pp. 177-180 ◽  
Author(s):  
K Yamada ◽  
A Avignon ◽  
M L Standaert ◽  
D R Cooper ◽  
B Spencer ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Skeletal Muscles ◽  
Phorbol Esters ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Isoforms ◽  
Pkc Alpha

Protein kinase C (PKC)-theta is a newly recognized major PKC isoform in skeletal muscle. In this study we found that insulin provoked rapid biphasic increases in membrane-associated immunoreactive PKC-theta, as well as PKC-alpha, PKC-beta and PKC-epsilon, in rat soleus muscles incubated in vitro. Effects of insulin on PKC isoforms in the soleus were comparable in magnitude with those of phorbol esters. Increases in membrane-associated PKC-theta, PKC-alpha, PKC-beta and PKC-epsilon were also observed in rat gastrocnemius muscles after insulin treatment in vivo. Our findings suggest that PKC-theta, like other diacylglycerol-sensitive PKC isoforms (alpha, beta and epsilon), may play a role in insulin action in skeletal muscles.

Calcitonin causes a sustained inhibition of protein kinase C-stimulated bone resorption in contrast to the transient inhibition of parathyroid hormone-induced bone resorption

1990 ◽  
Vol 123 (3) ◽  
pp. 251-256 ◽  
Author(s):  
Maria Ransjö ◽  
Ulf H. Lerner
Keyword(s):  
Protein Kinase C ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Bone Resorption ◽  
Phorbol Esters ◽  
Inhibitory Effect ◽  
Prostaglandin E ◽  
Kinase C

Abstract. Calcitonin is a well known inhibitor of osteoclastic bone resorption, both in vivo and in vitro. However, it is also known that calcitonin has only a transient inhibitory effect on bone resorption. The mechanism for this so-called "escape from inhibition" phenomenon is not clear. In the present study, the inhibitory effect of calcitonin on phorbol ester-induced bone resorption was examined in cultured neonatal mouse calvaria. Bone resorption was assessed as the release of radioactivity from bones prelabelled in vivo with 45Ca. Two protein kinase C-activating phorbol esters, phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate, both stimulated 45Ca release in 120-h cultures at a concentration of 10 nmol/l. Calcitonin (30 nmol/l) inhibited phorbol esterstimulated bone resorption without any "escape from inhibition". This was in contrast to the transient inhibitory effect of calcitonin on bone resorption stimulated by parathyroid hormone (10 nmol/l), prostaglandin E2 (2 μmol/l), and bradykinin (1 μmol/l). Our results suggest that activation of protein kinase C produces a sustained inhibitory effect of calcitonin on bone resorption.

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 Protein Kinase C Signaling Mediates a Program of Cell Cycle Withdrawal in the Intestinal Epithelium

2000 ◽  
Vol 151 (4) ◽  
pp. 763-778 ◽  
Author(s):  
Mark R. Frey ◽  
Jennifer A. Clark ◽  
Olga Leontieva ◽  
Joshua M. Uronis ◽  
Adrian R. Black ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Intestinal Epithelium ◽  
Molecular Mechanisms ◽  
Model Systems ◽  
Intestinal Epithelial ◽  
Kinase C

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G0. PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21waf1/cip1 and p27kip1, thus targeting all of the major G1/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G0 as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCα alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt–villus axis revealed that PKCα activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit–specific events in situ. Together, these data point to PKCα as a key regulator of cell cycle withdrawal in the intestinal epithelium.

scholarly journals Phosphorylation sites of protein kinase C   in H2O2-treated cells and its activation by tyrosine kinase in vitro

2001 ◽  
Vol 98 (12) ◽  
pp. 6587-6592 ◽  
Author(s):  
H. Konishi ◽  
E. Yamauchi ◽  
H. Taniguchi ◽  
T. Yamamoto ◽  
H. Matsuzaki ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Phosphorylation Sites ◽  
Kinase C

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

1987 ◽  
Vol 253 (2) ◽  
pp. C219-C229 ◽  
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.

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.

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