SEQUESTRATION OF A G-PROTEIN BETA-GAMMA SUBUNIT OR ADP-RIBOSYLATION OF RHO CAN INHIBIT THROMBIN-INDUCED ACTIVATION OF PLATELET PHOSPHOINOSITIDE 3-KINASES

被引:99
作者
ZHANG, J
ZHANG, J
BENOVIC, JL
SUGAI, M
WETZKER, R
GOUT, I
RITTENHOUSE, SE
机构
[1] THOMAS JEFFERSON UNIV, JEFFERSON MED COLL, JEFFERSON CANC INST, DEPT PHARMACOL, PHILADELPHIA, PA 19107 USA
[2] THOMAS JEFFERSON UNIV, JEFFERSON MED COLL, CARDEZA FDN HEMATOL RES, PHILADELPHIA, PA 19107 USA
[3] HIROSHIMA UNIV, SCH DENT, DEPT MICROBIOL, MINAMI KU, HIROSHIMA 734, JAPAN
[4] UNIV JENA, MAX PLANCK RES GRP GROWTH FACTROR SIGNAL TRANSDUC, D-07747 JENA, GERMANY
[5] UCL, LUDWIG INST CANC RES, LONDON W1P 8BT, ENGLAND
关键词
D O I
10.1074/jbc.270.12.6589
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Stimulation of platelets by thrombin leads to an increased association of activated phosphoinositide 3-kinase (PI 3-K) with a membrane cytoskeletal fraction (CSK). Activation of PI 3-K is dependent upon GTP-binding protein(s), since PI 3-K in permeabilized platelets is stimulated by GTP gamma S (guanosine 5'-3-O-(thio)triphosphate), and stimulation of platelet cytosolic PI 3-K by GTP gamma S requires a functional small G protein, Rho. Recent reports indicate that cytosolic PI 3-Ks can also be activated by the beta gamma subunits of heterotrimeric G-proteins (G beta gamma). We now report that the activated PI 3-K that is associated with CSK can be inhibited by a recombinant protein containing the G beta gamma-binding pleckstrin homology domain of beta-adrenergic receptor kinase 1 (beta ARK-PH). Inhibition is blocked by G beta gamma. PI 3-K in nonactivated platelet CSK is activated by GTP gamma S but unaffected by beta ARK-PH or G beta gamma, Western blots indicate that activated platelet CSK contains a novel 110-kDa PI 3-K(gamma) that has been shown to be stimulated by G beta gamma and to lack binding sites for the 85-kDa subunit of conventional PI 3-K. PI 3-K in immunoprecipitates obtained via p85 subunit-directed antibodies can be activated by GTP gamma S but not by G beta gamma. PI 3-K that is stimulatable by G beta gamma remains soluble, as does PI 3-K(gamma), and is unaffected by Rho, In contrast, ADP-ribosylation of Rho present in p85 immunoprecipitates is inhibitory, Further, activation of PI 3-K in permeabilized platelets exposed to thrombin or GTP gamma S is inhibited by beta ARK-PH and/or Rho-specific ADP-ribosylating enzymes. We conclude that Rho and G beta gamma each, respectively, contributes to the activation of different PI 3-Ks (p85-containing heterodimer and PI3-K (gamma)) in thrombin-stimulated platelets.
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页码:6589 / 6594
页数:6
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