Phosphopleckstrin inhibits gbetagamma-activable platelet phosphatidylinositol-4,5-bisphosphate 3-kinase.
Pleckstrin, the prototypic protein containing two copies of the pleckstrin homology domain, is a prominent substrate of protein kinase C in platelets and neutrophils. Both cell types have p85 subunit-containing phosphoinositide 3-kinase (p85/PI3K) and non-p85-containing PI3K (PI3Kgamma) that is activated by betagamma subunits of heterotrimeric GTP-binding proteins. We have shown that a PI3K product, phosphatidylinositol (PI) 3,4,5-trisphosphate, promotes pleckstrin phosphorylation in platelets. Since pleckstrin homology domains are thought to interact with Gbetagamma heterodimers and/or PI(4,5)P2, we have examined the effects of recombinant pleckstrins on platelet PI3Kgamma and p85/PI3K activities. Depending upon its phosphorylation/charged state, pleckstrin inhibits PI3Kgamma, but not p85/PI3K. Pleckstrin-mediated inhibition of PI3Kgamma is overcome by excess Gbetagamma and is restricted to PI(4,5)P2 as substrate, i.e. pleckstrin does not inhibit phosphorylation of PI()P or PI. Consistent with this, activation of protein kinase C by exposure of platelets to beta-phorbol diester (to increase endogenous pleckstrin phosphorylation) prior to platelet lysis causes inhibition of Gbetagamma-stimulatable PI3K activity only with respect to PI(4,5)P2 substrate. This phosphopleckstrin-mediated inhibition is overcome by increasing concentrations of Gbetagamma. We propose that phosphorylation of pleckstrin may constitute an important inhibitory mechanism for PI3Kgamma-mediated cell signaling.
Abrams
C S
CS
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Zhang
J
J
Downes
C P
CP
Tang
X w
Xw
Zhao
W
W
Rittenhouse
S E
SE
D000595:Amino Acid Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology*; D001798:Blood Proteins / Q000378:metabolism* / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008954:Models, Biological; D010446:Peptide Fragments / Q000737:chemistry; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D010750:Phosphoproteins; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000097:blood*
D001798:Blood Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D019269:Phosphatidylinositol 4,5-Diphosphate; D010750:Phosphoproteins; C039077:platelet protein P47; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D017853:Phosphotransferases (Alcohol Group Acceptor); C068961:phosphatidylinositol 4,5-biphosphate kinase; D019204:GTP-Binding Proteins