Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets.
Human platelets undergo a rapid, major reorganization of the cytoskeletal matrix upon exposure to thrombin, and accumulate 3-phosphorylated phosphoinositides in a protein kinase C (PKC)-dependent manner. These phosphoinositides have been suggested to be involved in actin polymerization/depolymerization. We reasoned that, if newly generated 3-phosphorylated phosphoinositide modulates cytoskeletal reorganization, a prerequisite for such action would be generation near cytoskeletal proteins. We have found that, after platelet activation, phosphatidylinositol 3-kinase and phosphatidylinositol(4)P 3-kinase activities, antibody-detectable phosphoinositide 3-kinase, and PKC become markedly and specifically enriched in a Triton X-100-insoluble cytoskeletal fraction that contains GPIIb/IIIa (integrin) and pp60c-src. The cytoskeletal fraction then accounts for up to 70% of total phosphoinositide 3-kinase activity, a function of recruited activated enzyme. These proteins are not occluded or directly associated with newly polymerized actin, since blockage by cytochalasin D of actin polymerization, and consequent inhibition of accumulation of about 40% of incremental protein and actin in this fraction, has no effect on its content of phosphoinositide 3-kinase, GPIIb/IIIa, pp60c-src, or PKC. Depolymerization of actin with DNase I, or inhibition of ligand binding to GPIIb/IIIa by RGDS, however, in combination with cytochalasin D, further depletes actin and significantly decreases sedimentability of GPIIb/IIIa as well as phosphoinositide 3-kinase, pp60c-src, and PKC, without inhibiting total 3-kinase activity. Our results suggest that, as a function of platelet activation, enzymes that regulate the synthesis of 3-phosphorylated phosphoinositides rapidly associate with the membrane skeleton and that skeletally associated phosphoinositide 3-kinase is more active than the Triton-soluble form.
Zhang
J
J
Department of Biochemistry, University of Vermont, Burlington 05405.
Fry
M J
MJ
Waterfield
M D
MD
Jaken
S
S
Liao
L
L
Fox
J E
JE
Rittenhouse
S E
SE
D019870:1-Phosphatidylinositol 4-Kinase; D000199:Actins / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002855:Chromatography, Thin Layer; D015638:Cytochalasin D / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D010770:Phosphotransferases / Q000378:metabolism*; D015539:Platelet Activation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D016608:Talin / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D016596:Vinculin / Q000378:metabolism
D000199:Actins; D010980:Platelet Membrane Glycoproteins; D016608:Talin; D016596:Vinculin; D015638:Cytochalasin D; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; D011493:Protein Kinase C; D013917:Thrombin