INTERACTION OF THE GTP-BINDING PROTEIN G(I2) WITH A PROTEIN-KINASE A-LIKE KINASE IN MOUSE FIBROBLASTS

We have previously shown that the GTP-binding protein, G(i)ds of mouse Balb/c3T3 cells is linked to a serine kinase which phosphorylates the alpha-subunit of G(i) itself. In this report we show that G(i) is cou pled to a second protein kinase. This Kinase does not phosphorylate G( i) but phosphorylates another protein bound non-covalently to G(i). Ph osphorylation of the G(i)-linked protein induces its release from G(i) . Kinase activity is slightly enhanced by GTP gamma S, suggesting that this kinase may be physiologically regulated by G(i). In an attempt t o identify the kinase we have examined the effect of peptide substrate s and inhibitors on Kinase activity. We found that the protein kinase A inhibitory peptide, PKI 5-24, inhibited the Kinase activity but at c oncentrations above those usually required to block protein kinase A. The protein kinase A substrate peptide, kemptide, acted as a substrate of the kinase, and was an inhibitor of the phosphorylation of the G(i )-linked protein. However, a protein kinase A, catalytic subunit antib ody failed to react with any proteins linked to G(i). A protein kinase C inhibitory peptide had no effect on phosphorylation of the G(i)-lin ked protein. Thus, the identity of this kinase has not been resolved b ut it may form part of the signalling system of activated G(i) in fibr oblasts.