Thrombin and phorbol esters cause the selective phosphorylation of a G protein other than Gi in human platelets.
Preincubation of human platelets with activators of protein kinase C such as phorbol 12-myristate 13-acetate (PMA) has been shown previously to attenuate the ability of agonists both to suppress formation of cAMP and to stimulate hydrolysis of phosphoinositides. In the present study, we have examined whether the attenuation caused by PMA can be attributed to the phosphorylation of the alpha subunit(s) of Gi, a GTP-binding regulatory protein implicated in several pathways of signal transduction. PMA was found to promote the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [gamma-32P]ATP and [32P]H3PO4, respectively. None of the phosphoproteins, however, was precipitated by either of two antisera containing antibodies differing in specificities for epitopes within Gi alpha, despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently described Gz alpha or both Gz alpha and Gi alpha, precipitated a 40-kDa phosphoprotein. Phosphorylation of this protein occurred not only in response to PMA, but to thrombin and the thromboxane A2 analog U46619. These data suggest that activators of protein kinase C lead to the phosphorylation within platelets of a select population of G alpha subunits. The identified phosphoprotein is not Gi alpha, but is similar or identical to Gz alpha. Because Gz alpha does not contain the consensus site for ADP-ribosylation by the Bordetella pertussis toxin islet-activating protein, the data also suggest that effects of PMA on processes otherwise sensitive to this toxin are not exerted at the level of G proteins responsible for transduction.
Carlson
K E
KE
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.
Brass
L F
LF
Manning
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002463:Cell Membrane Permeability; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera / Q000032:analysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011233:Precipitin Tests; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology*
D007106:Immune Sera; D010446:Peptide Fragments; D010750:Phosphoproteins; D013917:Thrombin; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate