Receptor- and phorbol-ester-mediated redistribution of protein kinase C in human platelets. Evidence that aggregation promotes degradation of protein kinase C

Abstract

Translocation of Ca2+/phospholipid-dependent protein kinase (PKC) activity from cytosolic to membrane fractions was assessed in washed human platelet suspensions. Phorbol myristate acetate (PMA) induced a rapid loss of PKC activity from the cytosolic compartment in stirred platelets, which was not accompanied by measurable increases in membrane-associated activity, but was paralleled by a decrease in total cellular enzyme activity (cytosol plus membrane). When platelet aggregation was prevented by not stirring, (i) cytosolic activity was decreased by PMA, (ii) significant and maintained (1-15 min with PMA) increases in membrane-bound PKC were detected, and (iii) the decline in total enzyme activity was markedly slower. In stirred platelets, total and specific inhibition of PMA-induced aggregation by a fibrinogen-derived peptide (RGDS, i.e. Arg-Gly-Asp-Ser) promoted maximal increases in membrane-associated PKC in the presence of PMA and completely prevented the loss in cellular activity. Thrombin and collagen both induced a decrease in cytosolic PKC and a loss of total activity, but a significant rise in membrane activity was seen only with collagen; ADP had no detectable effect on enzyme distribution. These results demonstrate an agonist-induced redistribution of PKC and indicate that platelet aggregation may play an important role in the proteolysis, and hence persistence, of membrane-associated PKC. This observation has implications for the potency and duration of PKC-mediated responses induced by agonists and exogenous PKC activators.

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

“…The reasons for this difference are currently unclear but may relate to the lower diacylglycerol concentrations observed in platelets stimulated with ADP compared with thrombin [39,40]. The reduced translocation of PKC to the plasma membrane might limit the ability of PKC to significantly alter the activity of Na + /K + -ATPase in ADP-stimulated platelets [41,42]. Future work examining the role of modulating diacylglycerol levels in ADP-stimulated platelets might help delineate this pathway further [39].…”

Protein tyrosine kinases regulate agonist-stimulated prostacyclin release but not von Willebrand factor secretion from human umbilical vein endothelial cells

“…Columns were washed and the enzyme was eluted with 150 µl of buffer A containing 120 mM NaCl. The enzyme activity in 10 µl aliquots of column effluent was assessed in the absence or presence of various kinase inhibitors by measuring the incorporation of $#P from [γ-$#P]ATP into histone IIIS substrate [39]. PKC activity was calculated from the difference in $#P incorporation measured in the presence and absence of phosphatidylserine and diolein.…”

Identification of 14-3-3 proteins in human platelets: effects of synthetic peptides on protein kinase C activation

“…Incubations were terminated by rapid centrifugation (1500 g for 5 min at 4 mC), the supernatant was discarded and the platelet pellet was resuspended in 100 µl of a buffer consisting of 20 mM Tris\HCl, 2 mM EDTA, 0.5 mM EGTA, 50 µg\ml leupeptin, 1 mM PMSF and 1 % (v\v) 2mercaptoethanol. Particulate and cytosolic platelet fractions were prepared and PKC was partly purified by ion-exchange chromatography as described [25]. Column eluates were assayed for PKC activity by measuring the incorporation of $#P from [γ-$#P]ATP into histone IIIS in the absence and presence of added phospholipids as previously reported [25].…”