Phosphorylation of serine 1105 by protein kinase A inhibits phospholipase Cbeta3 stimulation by Galphaq.
The mechanism by which protein kinase A (PKA) inhibits Galphaq -stimulated phospholipase C activity of the beta subclass (PLCbeta ) is unknown. We present evidence that phosphorylation of PLCbeta3 by PKA results in inhibition of Galphaq -stimulated PLCbeta3 activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLCbeta3 revealed a single phosphoserine as the putative PKA site, and peptide mapping yielded one phosphopeptide. The residue was identified as Ser1105 by direct sequencing of reverse-phase high pressure liquid chromatography-isolated phosphopeptide and by site-directed mutagenesis. Overexpression of Galphaq with PLCbeta3 or PLCbeta (Ser1105--> Ala) mutant in COSM6 cells resulted in a 5-fold increase in [3H]phosphatidylinositol 1,4,5-trisphosphate formation compared with expression of Galphaq, PLCbeta3, or PLCbeta3 (Ser1105 --> Ala mutant alone. Whereas Galpha1-stimulated PLCbeta3, activity was inhibited by 58-71% by overexpression of PKA catalytic subunit, Galphaq-stimulated PLCbeta3 (Ser1105 --> Ala) mutant activity was not affected. Furthermore, phosphatidylinositide turnover stimulated by presumably Galpha1-coupled M1 muscarinic and oxytocin receptors was completely inhibited by pretreating cells with 8-[4-chlorophenythio]-cAMP in RBL-2H3 cells expressing only PLCbeta3. These data establish that direct phosphorylation by PKA of Ser1105 in the putative G-box of PLCbeta3 inhibits Galphaq-stimulated PLCbeta3 activity. This can at least partially explain the inhibitory effect of PKA on Galphaq-stimulated phosphatidylinositide turnover observed in a variety of cells and tissues.
Yue
C
C
Department of Biochemistry and Molecular Biology, University of Texas Houston Medical School, Houston, Texas 77225, USA.
Dodge
K L
KL
Weber
G
G
Sanborn
B M
BM
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D054799:Phospholipase C beta; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism*; D013552:Swine; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000235:genetics
D007527:Isoenzymes; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins