Phosphorylation of serine 1105 by protein kinase A inhibits phospholipase Cβ3 stimulation by Gαq

The mechanism by which protein kinase A (PKA) inhibits G alpha(q)-stimulated phospholipase C activity of the beta subclass (PLC beta) is unknown. We present evidence that phosphorylation of PLC beta(3) by PKA results in inhibition of G alpha(q)-stimulated PLC beta(3) activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLC beta(3) revealed a single phosphoserine as the putative PKA site, and peptide mapping yielded one phosphopeptide. The residue was identified as Ser(1105) by direct sequencing of reverse-phase high pressure liquid chromatography-isolated phosphopeptide and by site-directed mutagenesis. Overexpression of G alpha(q) with PLC beta(3) or PLCP, (Serll05 --> Ala) mutant in COSM6 cells resulted in a 5-fold increase in [H-3]phosphatidylinositol 1,4,5trisphosphate formation compared with expression of G alpha(q), PLC beta(3), or PLC beta(3), (Ser(1105) --> Ala) mutant alone. Whereas G alpha(q)-stimulated PLC beta(3) activity was inhibited by 58-71% by overexpression of PKA catalytic subunit, G alpha(q)-stimulated PLC beta(3) (Ser(1105) --> Ala) mutant activity was not affected. Furthermore, phosphatidylinositide turnover stimulated by presumably G alpha(q)-coupled M1 muscarinic and oxytocin receptors was completely inhibited by pretreating cells with 8-[4-chlorophenythio]cAMP in RBL-2H3 cells expressing only PLCP beta(3). These data establish that direct phosphorylation by PKA of Ser(1105) in the putative G-box of PLC beta(3) inhibits G alpha(q)-stimulated PLC beta(3) activity. This can at least partially explain the inhibitory effect of PKA on G alpha(q)-stimulated phosphatidylinositide turnover observed in a variety of cells and tissues.