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Phosphorylation of Serine 1105 by Protein Kinase A Inhibits Phospholipase Cβ3 Stimulation by Gαq

1998; Elsevier BV; Volume: 273; Issue: 29 Linguagem: Inglês

10.1074/jbc.273.29.18023

1083-351X

Caiping Yue, Kimberly L. Dodge, Günther Weber, Barbara M. Sanborn,

Blood disorders and treatments

The mechanism by which protein kinase A (PKA) inhibits Gα q -stimulated phospholipase C activity of the β subclass (PLCβ) is unknown. We present evidence that phosphorylation of PLCβ 3 by PKA results in inhibition of Gα q -stimulated PLCβ 3 activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLCβ 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 Gα q with PLCβ 3 or PLCβ 3 (Ser 1105 → Ala) mutant in COSM6 cells resulted in a 5-fold increase in [ 3 H]phosphatidylinositol 1,4,5-trisphosphate formation compared with expression of Gα q , PLCβ 3 , or PLCβ 3 (Ser 1105 → Ala) mutant alone. Whereas Gα q -stimulated PLCβ 3 activity was inhibited by 58–71% by overexpression of PKA catalytic subunit, Gα q -stimulated PLCβ 3 (Ser 1105 → Ala) mutant activity was not affected. Furthermore, phosphatidylinositide turnover stimulated by presumably Gα q -coupled M1 muscarinic and oxytocin receptors was completely inhibited by pretreating cells with 8-[4-chlorophenythio]-cAMP in RBL-2H3 cells expressing only PLCβ 3 . These data establish that direct phosphorylation by PKA of Ser 1105 in the putative G-box of PLCβ 3 inhibits Gα q -stimulated PLCβ 3 activity. This can at least partially explain the inhibitory effect of PKA on Gα q -stimulated phosphatidylinositide turnover observed in a variety of cells and tissues.