No abstract

(1994) J. Biol. Chem. 269, 32358 -32367). Activation of platelet PKC in response to TRAP is detected by the phosphorylation of the major PKC substrate in platelets, the p47 phosphoprotein, also known as pleckstrin. Here we provide evidence for two phases of pleckstrin phosphorylation in response to TRAP. A rapid phase of pleckstrin phosphorylation (<1 min) precedes the peak of PtdIns-3,4-P 2 production and is unaffected by concentrations of wortmannin (10 -100 nM) that block production of this lipid. However prolonged phosphorylation of pleckstrin (>2 min) is inhibited by wortmannin concentrations that block PtdIns-3,4-P 2 production. Phorbol ester-mediated pleckstrin phosphorylation was not affected by wortmannin and wortmannin had no effect on purified platelet PKC activity. Phosphorylation of pleckstrin could be induced using permeabilized platelets supplied with exogenous ␥-32 P[ATP] and synthetic dipalmitoyl PtdIns-3,4,5-P 3 and dipalmitoyl PtdIns-3,4-P 2 micelles, but not with dipalmitoyl phosphatidylinositol 3-phosphate or phosphatidylinositol 4,5-bisphosphate. These results suggest two modes of stimulating pleckstrin phosphorylation: a rapid activation of PKC (via diacylglycerol and calcium) followed by a slower activation of calcium-independent PKCs via PtdIns-3,4-P 2 .The activation of phosphoinositide 3-kinase (PI 3-K) 1 in agonist-stimulated cells results in the rapid formation of the two novel polyphosphoinositides PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 . A critical requirement for PI 3-K activation in a variety of cellular functions has been established. These include growth factor dependent mitogenesis, chemotaxis, receptor down-regulation, insulin-induced glucose transport, and actin-filament rearrangements leading to membrane ruffling (for a review, see Ref. 1). Despite these correlations, the direct targets for these polyphosphoinositides remain undescribed. PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 have been proposed to act as second messengers as they are not hydrolyzed by any known phospholipase type C enzymes (2, 3). The other product of PI 3-K activity, PtdIns-3-P, does not increase in agonist-stimulated cells and may be important in intracellular protein sorting mechanisms (4). Two enzymes, pp70S6 kinase (5) and the serine-threonine protein kinase Akt (6, 7) have been shown to be downstream of activated PI 3-K, but there is no evidence that these are the immediate targets of PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 . Recent data from our laboratory points to the calcium-independent isoforms of PKC (␦, ⑀, and ) as direct targets of these lipids (8), although in vivo evidence for this is still lacking. The diacylglycerol-insensitive isoform PKC may also be a target for these phosphoinositides (9).Thrombin activation of platelets results in the rapid activation of PI 3-K (10, 11). The major product of PI 3-K in activated platelets is PtdIns-3,4-P 2 , which peaks at 2-3 min following stimulation. A small peak of PtdIns-3,4,5-P 3 at 30 s to 1 min has also been reported (11). Although the exact function of t...

During activation of platelets by agonists, a number of proteins become phosphorylated at tyrosine residues. Using immunoblotting with a monoclonal anti-phosphotyrosine antibody, we have compared the different phosphotyrosine-protein (PTP) profiles of platelets stimulated with thrombin, collagen, ADP, arachidonic acid, phorbol myristate acetate and P256, an anti-glycoprotein-IIb-IIIa (GPIIb-IIIa) monoclonal antibody (mAb). Only a few PTPs were observed in resting platelets, of molecular masses 130, 64, 56-60 and 36 kDa. After stimulation by different agonists these proteins were more intensely phosphorylated and additional PTPs appeared with molecular masses of 170, 150, 140, 120, 105/97 (doublet), 85, 80, 75 and 45 kDa. The kinetics of phosphorylation differed from one agonist to another, but no significant differences in the overall patterns were detected, except in presence of ADP and P256-F(ab')2, which induced only the additional tyrosine phosphorylation of the 64 kDa protein and to a lesser extent that of a 75 kDa protein. The use of various agonists and the inhibitors (staurosporine, ajoene and RGDS) permitted a better characterization of the relationship between the different steps of activation and phosphorylation on tyrosine residues. The studies suggest the following conclusions: (i) stimulation of tyrosine phosphorylation occurs after activation of protein kinase C; (ii) there is a relationship between ligand binding to GPIIb-IIIa and the tyrosine phosphorylation of the 64 kDa protein; and (iii) there is a close relationship between PTP formation and the intensity of platelet activation and aggregation.