Molecular mechanism of the inhibition of phospholipase C β3 by protein kinase C

Activation of protein kinase C (PKC) can result from stimulation of the receptor-G protein-phospholipase C (PLC beta) pathway. In turn, phosphorylation of PLC beta by PKC may play a role in the regulation of receptor-mediated phosphatidylinositide (PI) turnover and intracellular Ca2+ release. Activation of endogenous PRC by phorbol 12-myristate 13 acetate inhibited both G alpha(q)-coupled (oxytocin and M1 muscarinic) and G alpha(i)-coupled (formyl-Met-Leu-Phe) receptor-stimulated PI turnover by 50-100% in PHM1, HeLa, COSM6, and RBL-2H3 cells expressing PLC beta(3). Activation of conventional PKCs with thymeleatoxin similarly inhibited oxytocin or formyl-Met-Leu-Phe receptor-stimulated PI turnover. The PKC inhibitory effect was also observed when PLC beta(3) was stimulated directly by G alpha(q) or G beta gamma in overexpression assays. PKC phosphorylated PLC beta(3) at the same predominant site in vivo and in vitro. Peptide sequencing of in vitro phosphorylated recombinant PLC beta(3) and site-directed mutagenesis identified Ser(1105) as the predominant phosphorylation site. Ser(1105) is also phosphorylated by protein kinase A (PKA; Yue, C., Dodge, K. L., Weber, G., and Sanborn, B. M. (1998) J. Biol. Chem. 273, 18023-18027). Similar to PKA, the inhibition by PKC of G alpha(q)-stimulated PLC beta(3) activity was completely abolished by mutation of Ser(1105) to Ala. In contrast, mutation of Ser(1105) or Ser(26), another putative phosphorylation target, to Ala had no effect on inhibition of G beta gamma-stimulated PLC beta(3) activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit G alpha(q)-stimulated PLC beta(3) as a result of phosphorylation of Ser(1105). Moreover, PKC and PKA both inhibit G beta gamma-stimulated activity by mechanisms that do not involve Ser(1105).