KN-93 inhibition of G protein signaling is independent of the ability of Ca2+/calmodulin-dependent protein kinase II to phosphorylate phospholipase Cbeta3 on 537-Ser.

Stimulation of the phospholipase Cbeta (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 Galphai-coupled (formyl-Met-Leu-Phe, fMLP) or Galphaq-coupled [M1 muscarinic and oxytocin (OT)] receptors. The inhibitory effect of KN-93 was also observed when PLCbeta3 was stimulated directly by Galphaq or Gbetagamma in overexpression assays. CaMK II phosphorylated PLCbeta3 but not PLCbeta1 in vitro. Phosphorylation occurred exclusively on 537Ser in the X-Y linker region of PLCbeta3. 537Ser was also phosphorylated in the basal state in cells and phosphorylation was enhanced by ionomycin treatment. However, mutation of 537Ser to Glu had no effect on inhibition of Galphaq or Gbetagamma-stimulated PLCbeta3 activity by KN-93. KN-93 also inhibited Galphaq -stimulated PLCbeta1 activity, even though this enzyme is not a substrate for CaMK II. These data indicate that phosphorylation of PLCbeta3 by CaMK II is not directly involved in the inhibitory effect of KN-93 on phosphatidylinositide turnover.