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 (PLCβ) pathway. In turn, phosphorylation of PLCβ by PKC may play a role in the regulation of receptor-mediated phosphatidylinositide (PI) turnover and intracellular Ca²⁺ release. Activation of endogenous PKC by phorbol 12-myristate 13-acetate inhibited both Gα(q)-coupled (oxytocin and M1 muscarinic) and Gα(i)-coupled (formyl-Met-Leu-Phe) receptor-stimulated PI turnover by 50-100% in PHM1, HeLa, COSM6, and RBL-2H3 cells expressing PLCβ₃. 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 PLCβ₃ was stimulated directly by Gα(q) or Gβγ in overexpression assays. PKC phosphorylated PLCβ₃ at the same predominant site in vivo and in vitro. Peptide sequencing of in vitro phosphorylated recombinant PLCβ₃ and site-directed mutagenesis identified Ser¹¹⁰⁵ as the predominant phosphorylation site. Ser¹¹⁰⁵ 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 Gα(q)-stimulated PLCβ₃ activity was completely abolished by mutation of Ser¹¹⁰⁵ to Ala. In contrast, mutation of Ser¹¹⁰⁵ or Ser²⁶, another putative phosphorylation target, to Ala had no effect on inhibition of Gβγ-stimulated PLCβ₃ activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit Gα(q)-stimulated PLCβ₃ as a result of phosphorylation of Ser¹¹⁰⁵. Moreover, PKC and PKA both inhibit Gβγ-stimulated activity by mechanisms that do not involve Ser¹¹⁰⁵.