Phosphatidylinositol (3,4,5)-trisphosphate stimulates phosphorylation of pleckstrin in human platelets.
We have reported that platelets exposed to thrombin or thrombin receptor-directed ligand activate phospholipase C and rapidly accumulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol (3,4)-bisphosphate (PtdIns(3,4)P2) as a function of the activation of phosphoinositide (PI) 3-kinases in a GTP-binding protein-dependent manner. In such platelets, serine- and threonine-directed phosphorylation of pleckstrin also occurs and has been attributed to protein kinase C activation. We now report that the phosphorylation of pleckstrin is partially dependent upon PI 3-kinase. Pleckstrin phosphorylation in response to thrombin receptor stimulation is progressively susceptible to inhibition by wortmannin, a potent and specific inhibitor of platelet PI 3-kinases. PI 3-kinase thus seems to play a gradually increasing role in promoting pleckstrin phosphorylation. The IC50 for wortmannin in inhibiting SFLLRN-stimulated 3-phosphorylated phosphoinositide accumulation is 10 nM, and that (i.e. 50% of maximum inhibition) for inhibiting pleckstrin phosphorylation is 15 nM. Synthetic PtdIns(3,4,5)P3, when added to saponin-permeabilized (but not intact) platelets, causes wortmannin-insensitive phosphorylation of pleckstrin. PtdIns(3,4,5)P3 also overcomes the inhibition by wortmannin of thrombin- or guanosine 5'-3-O-(thio)trisphosphate-stimulated pleckstrin phosphorylation. In contrast, PtdIns(4,5)P2 or inositol (1,3,4,5)-tetrakisphosphate are ineffective in these respects. The pattern of phosphorylation of pleckstrin activated by PtdIns(3,4,5)P3 is not distinguishable from that of pleckstrin phosphorylated in intact platelets exposed to protein kinase C-activating beta-phorbol myristate acetate, mimicking diacylglycerol. Activation of protein kinase(s) by PtdIns(3,4,5)P3 thus offers a route for pleckstrin phosphorylation in vivo that is an alternative to activation of phospholipase C-->diacylglycerol-->protein kinase C.
Zhang
J
J
Jefferson Cancer Institute, Philadelphia, Pennsylvania, USA.
Falck
J R
JR
Reddy
K K
KK
Abrams
C S
CS
Zhao
W
W
Rittenhouse
S E
SE
D000730:Androstadienes / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001798:Blood Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000097:blood; D010750:Phosphoproteins / Q000097:blood / Q000302:isolation & purification; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000097:blood; D011493:Protein Kinase C / Q000378:metabolism; D018179:Receptors, Thrombin / Q000502:physiology; D000077191:Wortmannin
D000730:Androstadienes; D001798:Blood Proteins; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D018179:Receptors, Thrombin; C060974:phosphatidylinositol 3,4,5-triphosphate; C039077:platelet protein P47; C082835:thrombin receptor peptide (42-47); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011493:Protein Kinase C; D000077191:Wortmannin