Selective Phosphorylation of a G Protein within Permeabilized and Intact Platelets Caused by Activators of Protein Kinase C

Abstract

Phosphorylation of G_i by the phospholipid- and Ca²⁺-dependent enzyme protein kinase C has been proposed as a mechanism by which phorbol esters and certain agonists either attenuate inhibition of adenylyl cyclase or impair stimulation of phospholipase C (Jakobs, et al., 1985; Orellana et al., 1987; Smith et al., 1987; Pyne et al., 1989). Support for the phosphorylation of G_iα is provided by the occurrence of this modification for one or more forms of G_iα in solution with partially purified protein kinase C (Katada et al., 1985). Indeed, phosphorylation of the purified subunit(s) can also be accomplished with cAMP dependent and insulin receptor kinases (O’Brien et al., 1987; Krupinski et al., 1988; Watanabe et al., 1988). The occurrence of phosphorylation within the cell itself, however, has not been fully documented, nor have functional correlates of phosphorylation been established. In unstimulated rat hepatocytes incubated with [³²P] H₃PO₄, a 41-kDa phosphoprotein can be immunoprecipitated with a G_iα-directed antiserum (Rothenberg and Kahn, 1988; Pyne et al., 1989). Treatment of these cells with phorbol 12-myristate 13- acetate (PMA) results in a modest (i.e., 70%) increase in incorporated phosphate, while treatment with insulin has no effect. Incorporation of radiolabel into proteins having isoelectric or immunologic properties similar to G_iα has also been reported for PMA-treated human platelets (Halenda et al., 1986; Crouch and Lapetina, 1988). The identities of these proteins as G_iα, however, have not been rigorously established.