MULTISITE PHOSPHORYLATION OF THE INHIBITORY GUANINE-NUCLEOTIDE REGULATORY PROTEIN G(I)-2 OCCURS IN INTACT RAT HEPATOCYTES

A phosphorylated form of alpha-G(1)-2 (the alpha-subunit of G(1)-2), i mmunoprecipitated from hepatocytes under basal conditions, migrated as a single species of pI similar to 5.7, the labelling of which increas ed similar to 2-fold in cells challenged with either vasopressin or ph orbol 12-myristate 13-acetate (PMA); agents which activate protein kin ase C. In contrast, treatment of hepatocytes with 8-bromo-cyclic AMP p roduced a more acidic species of phosphorylated alpha-G(1)-2 having a pI of similar to 5.4 and whose labelling was increased similar to 3-fo ld. Trypsin digestion of labelled alpha-G(1)-2 isolated from hepatocyt es under basal conditions identified, on two-dimensional peptide analy ses, three positively charged phosphoserine-containing peptides (C1, C 2 and C3), with only peptides C1 and C2 being evident upon less extens ive digestion with trypsin. These are suggested to reflect a single si te of phosphorylation, with proteolysis by trypsin being incomplete, a nd where C2 is larger than C1, which is larger than C3. An identical p attern of tryptic phosphopeptides was seen in hepatocytes treated with either vasopressin or PMA, although labelling of this group of peptid es was increased by similar to 2-fold compared with the basal state. I n contrast, treatment of hepatocytes with glucagon, 8-bromo-cyclic AMP or forskolin not only resulted in increased labelling of the 'basal' sites similar to 3-fold, but identified a novel positively charged try ptic phosphoserine-containing peptide (AN). All four tryptic peptides were susceptible to proteolysis by V8 protease. Treatment of labelled alpha-G(i)-2 from basal and PMA-treated cells produced a pattern of pe ptides which was identical with those found when the tryptic phosphope ptide was treated with V8 protease. We tentatively suggest that, on al pha-G(i)-2, Ser(144) is phosphorylated through the action of protein k inase C and Ser(207) is phosphorylated upon elevation of the intracell ular concentrations of cyclic AMP.