scholarly journals Ultrastructural localization of protein kinase C in human sperm.

1995 ◽  
Vol 43 (4) ◽  
pp. 439-445 ◽  
Author(s):  
M Kalina ◽  
R Socher ◽  
R Rotem ◽  
Z Naor
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Human Sperm ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Sperm Head ◽  
Structural Elements ◽  
Kinase C ◽  
Outer Dense Fibers ◽  
Pkc Alpha

We localized protein kinase C (PKC) in human sperm cells at the ultrastructural level by the immunogold technique. The sperm head PKC was localized in the acrosome, equatorial segment, and post-acrosomal region. In the flagellum, PKC was associated with the segmented column of the neck and was distributed along the mid, principal, and end pieces. Immunoreactive sites were observed in patches along the axoneme and outer dense fibers and were evenly distributed between these regions. Pre-absorption of the antibody used with rat brain PKC (alpha and beta) eliminated gold labeling of the sperm head but only reduced labeling of the sperm tail. The co-localization of PKC with various cytoskeletal and other structural elements suggests that the proteins involved are potential substrates for sperm PKC subspecies. The localization of PKC in distinct structures of the human sperm (head, neck, and tail) strongly suggests a role for this enzyme in various aspects of sperm physiology.

Protein kinase C(alpha) actively downregulates through caveolae-dependent traffic to an endosomal compartment

2000 ◽  
Vol 113 (14) ◽  
pp. 2575-2584
Author(s):  
C. Prevostel ◽  
V. Alice ◽  
D. Joubert ◽  
P.J. Parker
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Initial Step ◽  
Receptor Internalization ◽  
Similar Process ◽  
Receptor Desensitization ◽  
Kinase C ◽  
Annexin I ◽  
Pkc Alpha

Receptor desensitization occurs through receptor internalization and targeting to endosomes, a prerequisite for sorting and degradation. Such trafficking processes may not be restricted to membrane associated receptors but may also play an important role in the downregulation of cytoplasmic transducers such as protein kinase C (PKC). It is demonstrated here that acute TPA exposure induces the transport of activated PKC(alpha) from the plasma membrane to endosomes. This process requires PKC activity and catalytically competent PKC can even promote a similar process for a truncated regulatory domain PKC(α) protein. It is established that PKC(α) is targeted to the endosome compartment as an active kinase, where it colocalizes with annexin I, a substrate of PKC. Thus, PKC(alpha) downregulation shares features with plasma membrane associated receptor sorting and degradation. However, it is shown that PKC(α) delivery to the endosome compartment is not a Rab5 mediated process in contrast to the well characterised internalisation of the transferrin receptor. An alternative route for PKC(alpha) is evidenced by the finding that the cholesterol binding drugs nystatin and filipin, known to inhibit caveolae mediated trafficking, are able to block PKC(alpha) traffic and down regulation. Consistent with this, the endosomes where PKC(alpha) is found also contain caveolin. It is concluded that the initial step in desensitisation of PKC(alpha) involves active delivery to endosomes via a caveolae mediated process.

Dephosphorylation of activated protein kinase C contributes to downregulation by bryostatin

1996 ◽  
Vol 271 (1) ◽  
pp. C304-C311 ◽  
Author(s):  
H. W. Lee ◽  
L. Smith ◽  
G. R. Pettit ◽  
J. Bingham Smith
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Synthesis ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Bryostatin 1 ◽  
Rapid Loss ◽  
Renal Epithelial Cells ◽  
Kinase C ◽  
Phosphatase Treatment ◽  
Pkc Alpha

We show that bryostatin 1 (Bryo) rapidly produces an inactive, incompetent 76-kDa form of protein kinase C-alpha (PKC-alpha) in the LLC-MK2 line of renal epithelial cells. Bryo, like phorbol 12-myristate 13-acetate (PMA), acutely activated PKC, as indicated by autophosphorylation and translocation of PKC-alpha, the predominant PMA-sensitive isoform expressed by the cells. Bryo concomitantly increased the 32P labeling of 80-kDa PKC-alpha by autophosphorylation and produced a 76-kDa form of PKC-alpha that lacked detectable 32P. The 76-kDa form was in the particulate rather than the cytosolic fraction, which suggests that it was produced from activated kinase. Alkaline phosphatase treatment of immunoprecipitated PKC-alpha converted the 80-kDa form to 76 kDa, but it had no effect on the mobility of the 76-kDa form, suggesting that it was not phosphorylated. Pulse-chase labeling of PKC-alpha with [35S]Met/Cys indicated that there is a precursor-product relationship between the 80- and 76-kDa forms, respectively. Inhibition of protein synthesis had no effect on the production of 76-kDa PKC-alpha by Bryo. PMA also produced 76-kDa PKC-alpha but was less potent and efficacious than Bryo. Bryo produced a more rapid loss of 80-kDa PKC-alpha protein and total Ca(2+)- and phospholipid-dependent PKC activity than PMA. The 76-kDa form is inactive and incompetent because it lacked detectable 32P under conditions that strongly autophosphorylated the 80-kDa form. We suggest that dephosphorylation predisposes PKC to proteolysis, and greater production of the 76-kDa form explains the more efficient downregulation of the kinase by Bryo vs. PMA.

scholarly journals Association of protein kinase-C-alpha with cytoplasmic vesicles in melanoma cells.

1996 ◽  
Vol 44 (2) ◽  
pp. 177-182 ◽  
Author(s):  
J Timar ◽  
B Liu ◽  
R Bazaz ◽  
K V Honn
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Fluid Phase ◽  
Subcellular Fractionation ◽  
Melanoma Cells ◽  
Kinase C ◽  
Cytoplasmic Vesicles ◽  
Protein Kinase C Alpha ◽  
Pkc Alpha

In B16a melanoma cells, protein kinase-C-alpha (PKC alpha) is immunomorphologically associated with cytoplasmic vesicles in addition to the previously observed locations (plasma membrane, cytoskeleton, nucleus), as detected with monoclonal antibody (MAb) MC3a. Subcellular fractionation indicated that the authentic 80-KD protein as well as PKC activity can be detected in several particulate fractions except for L2, which contains dense lysosomes. The highest PKC activity is associated with the cytosol-ultralight vesicles and the L1 fraction (containing plasma membrane, endosomes, and the Golgi apparatus). Both of these fractions contained the fluid-phase endocytosis marker peroxidase, indicating that PKC alpha, in addition to other subcellular structures, is most probably associated with endosomal membranes in B16a melanoma cells.

PDGF and IL-1 induce and activate specific protein kinase C isoforms in mesangial cells

1996 ◽  
Vol 271 (1) ◽  
pp. F108-F113 ◽  
Author(s):  
M. B. Ganz ◽  
B. Saksa ◽  
R. Saxena ◽  
K. Hawkins ◽  
J. R. Sedor
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
De Novo ◽  
Mesangial Cells ◽  
De Novo Synthesis ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Alpha Beta ◽  
Pkc Alpha

In vitro and in vivo data suggest a remarkable plasticity in the differentiated phenotype of intrinsic glomerular cells, which after injury express new structures and functions. We have shown that a protein kinase C (PKC) isoform, beta II, is expressed in diseased but not normal glomeruli. Since intrarenal cytokine synthesis has been implicated in the pathogenesis of progressive glomerular injury, we have hypothesized that these mediators induce a change in isoform profile. To test this hypothesis in vitro, we have determined whether platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) alter the expression or activation of PKC isoforms in cultured mesangial cells (MCs). By immunoblot and ribonuclease (RNase) protection assays, both PDGF and IL-1 induce as early as 2 h de novo synthesis of PKC-beta II. Since MCs constitutively express PKC-alpha, -beta I, and -zeta, we also determined whether IL-1 or PDGF alter the activity of these isoforms. PDGF maximally induced translocation of PKC-alpha (10 min), -beta I (90 min), -epsilon (120 min), and -zeta (120 min) from the cytosolic to the membrane fraction. IL-1, in contrast, did not alter the distribution of alpha, beta I, or epsilon at any time measured but did induce PKC-zeta translocation. These data suggest inflammatory mediators regulate PKC isoform activity in diseased glomeruli both by de novo synthesis of unexpressed isoforms and by activation of constitutively expressed PKC isoforms.

scholarly journals Protein kinase C-mediated phospholipase A2 activation, platelet-activating factor generation and prostacyclin release in spontaneously beating rat cardiomyocytes

1993 ◽  
Vol 290 (2) ◽  
pp. 477-482 ◽  
Author(s):  
D J Church ◽  
S Braconi ◽  
M B Vallotton ◽  
U Lang
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Platelet Activating Factor ◽  
Eicosatetraenoic Acid ◽  
Induced Response ◽  
Rat Cardiomyocytes ◽  
Kinase C ◽  
Eicosanoid Release ◽  
Pkc Alpha

The expression of protein kinase C (PKC) isoenzymes and the effects of PKC activation on myocardial phospholipase A2 (PLA2) activity, platelet-activating factor (PAF) generation and eicosanoid release were studied in spontaneously beating cultured rat cardiomyocytes. Western blotting analysis indicated that these cells contain PKC alpha, beta, delta and zeta, but not PKC gamma or epsilon. Stimulation of cardiomyocytes with 4 beta-phorbol 12-myristate 13-acetate (PMA) led to a rapid increase in particulate-bound PKC activity, a response attributed to the activation of alpha-, delta- and zeta- type PKCs but not beta-type PKC. Translocation of PKC alpha, delta and zeta was accompanied by simultaneous increases in cellular lysophosphatidylcholine (lyso-PC), PAF, 15(S)-hydroxy-5,8,-11,13-eicosatetraenoic acid (15-HETE), prostaglandin E2 (PGE2) and prostacyclin (PGI2) generation, suggesting that one or more of these isoenzymes directly or indirectly activates a PLA2 in these cells. Confirming this, 4 beta-phorbol 12-monoacetate and 4 alpha-phorbol had no effect on cellular eicosanoid formation, while the PMA-induced response was fully abolished both in the presence of the PKC inhibitors staurosporine and CGP 41251 and in PKC-down-regulated cells. PKC alpha, delta and/or zeta therefore appear to play an important role in the PMA-mediated activation of cardiomyocyte PLA2, an event leading to subsequent production of PGI2, PGE2, 15-HETE, lyso-PC and PAF in this tissue.

scholarly journals Hormonal regulation of caveolae internalization.

1995 ◽  
Vol 131 (4) ◽  
pp. 929-938 ◽  
Author(s):  
E J Smart ◽  
Y S Ying ◽  
R G Anderson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Protein Phosphatase ◽  
Hormonal Regulation ◽  
Cyclic Transition ◽  
Present Evidence ◽  
Kinase C ◽  
Pkc Alpha ◽  
Histamine H1 Receptors

Caveolae undergo a cyclic transition from a flat segment of membrane to a vesicle that then returns to the cell surface. Here we present evidence that this cycle depends on a population of protein kinase C-alpha (PKC-alpha) molecules that reside in the caveolae membrane where they phosphorylate a 90-kD protein. This cycle can be interrupted by treatment of the cells with phorbol-12,13-dibutyrate or agents that raise the concentration of diacylglycerol in the cell. Each of these conditions displaces PKC-alpha from caveolae, inhibits the phosphorylation of the 90-kD protein, and prevents internalization. Caveolae also contain a protein phosphatase that dephosphorylates the 90-kD once PKC-alpha is gone. A similar dissociation of PKC-alpha from caveolae and inhibition of invagination was observed when cells were treated with histamine. This effect was blocked by pyrilamine but not cimetidine, indicating the involvement of histamine H1 receptors. These findings suggest that the caveolae internalization cycle is hormonally regulated.

scholarly journals Phosphorylation of p90 and p52 in response to phorbol-esters in Swiss/3T3 cells overexpressing protein kinase C-alpha.

1992 ◽  
Vol 3 (9) ◽  
pp. 1049-1056 ◽  
Author(s):  
H Eldar ◽  
E Livneh
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Cell Lines ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Tumor Promotion ◽  
3T3 Cells ◽  
Kinase C ◽  
Pkc Alpha

Cell lines stably overexpressing protein kinase C (PKC)-alpha were previously described by us. These cell lines were generated by the introduction of the full length cDNA coding for PKC-alpha into Swiss/3T3 cells. Here we show that activation of PKC-alpha by phorbol-esters induced in these cells specific phosphorylation of two cellular proteins p90 and p52. Phosphorylation of p80 (MARCKS protein), previously identified as a substrate for PKC, was also enhanced. Phosphorylated p90 and p52 proteins were associated with particulate membrane-enriched fractions and were extractable with the use of nonionic detergents. Time course analysis of phorbol-ester induced phosphorylation of p90 and p52 revealed maximal stimulation of phosphorylation after 15-30 min. Phosphamino acid analysis showed that phosphorylation of p90 and p52 occurred mainly on serine residues. Phosphorylation of p52 was also on threonine residues. Whereas, phorbol ester activation induced phosphorylation of both p90 and p52, the mitogens platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) enhanced phosphorylation of p90, but not p52. Thus, our studies showed the involvement of PKC-alpha in the regulation of p90 and p52 phosphorylation and provided direct evidence for the role of PKC-alpha in cellular signaling by PDGF and FGF. Moreover, the fact that phosphorylation of p52 was specific to phorbol ester activation may suggest its involvement in tumor promotion. Characterization of p90 and p52 will enable us to reveal the phosphorylation cascade activated downstream to PKC-alpha and to determine their role in mitogenic signaling and tumor promotion.

scholarly journals Nerve growth factor activates calcium-insensitive protein kinase C-ɛ in PC-12 rat pheochromocytoma cells

1993 ◽  
Vol 295 (3) ◽  
pp. 767-772 ◽  
Author(s):  
M Ohmichi ◽  
G Zhu ◽  
A R Saltiel
Keyword(s):  
Protein Kinase C ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Pheochromocytoma Cells ◽  
Pc 12 Cells ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Alpha

Protein kinase C (PKC) family members were examined in PC-12 rat pheochromocytoma cells to evaluate their role in the action of nerve growth factor (NGF). Immunoblot analysis of whole cell lysates using antibodies against various PKC isoforms revealed that PC-12 cells contained PKC-alpha, -delta, -epsilon and zeta. Assay of the protein kinase activity in these different anti-PKC immunoprecipitates demonstrated that NGF stimulated the kinase activity of PKC-epsilon, but not PKC-alpha, -delta and -zeta. Both histone phosphorylation and autophosphorylation of PKC-epsilon were increased by treatment of PC-12 cells with NGF. This increased phosphorylation observed in vitro is rapid, occurring maximally at 2.5 min and declining thereafter. Moreover, this effect of NGF is dose-dependent over physiological concentrations of the growth factor. Although the mechanistic basis for this specificity in PKC activation is not clear, NGF acutely stimulated the production of diacylglycerol without causing corresponding changes in intracellular Ca2+ concentrations. These results suggest that NGF may selectively stimulate the Ca(2+)-insensitive epsilon isoform of PKC by a phosphatidylinositol-independent mechanism.

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