KN-93 inhibition of G protein signaling is independent of the ability of Ca2+/calmodulin-dependent protein kinase II to phosphorylate phospholipase Cβ3 on 537-Ser

Stimulation of the phospholipase C beta (PLC) signaling pathway results in intracellular Ca2+ release and subsequent activation of calmodulin (CaM) and CaM kinase II (CaMK II). KN-93, an inhibitor of CaMK II, reduced the stimulation of phosphatidylinositide (PI) turnover by G alpha (i)-coupled (formyl-Met-Leu-Phe, fMLP) or G alpha (q)-coupled [M1 muscarinic and oxytocin (OT)] receptors. The inhibitory effect of KN-93 was also observed when PLC beta (3) was stimulated directly by G alpha (q) or G beta gamma in overexpression assays. CaMK II phosphorylated PLC beta (3) but not PLC beta (1) in vitro. Phosphorylation occurred exclusively on (537)Ser in the X-Y linker region of PLC beta (3). (537)Ser was also phosphorylated in the basal state in cells and phosphorylation was enhanced by ionomycin treatment. However, mutation of (537)Ser to Glu had no effect on inhibition of G alpha (q) or G beta gamma -stimulated PLC beta (3) activity by KN-93. KN-93 also inhibited G alpha (q) -stimulated PLC beta (1) activity, even though this enzyme is not a substrate for CaMK II. These data indicate that phosphorylation of PLC beta (3) by CaMK II is not directly involved in the inhibitory effect of KN-93 on phosphatidylinositide turnover. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.