Molecular Mechanism of the Inhibition of Phospholipase C β3 by Protein Kinase C

Abstract

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 Gαq-coupled (oxytocin and M1 muscarinic) and Gαi-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 Gαq 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 Gαq-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 PLCβ 3 activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit Gαq-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.

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