Gelsolin Modulates Phospholipase C Activity In Vivo through Phospholipid Binding

Gelsolin and CapG are actin regulatory proteins that remodel the cytoskeleton in response to phosphatidylinositol 4,5-bisphosphate (PIP(2)) and Ca(2+) during agonist stimulation. A physiologically relevant rise in Ca(2+) increases their affinity for PIP(2) and can promote significant interactions with PIP(2) in activated cells. This may impact divergent PIP(2)- dependent signaling processes at the level of substrate availability. We found that CapG overexpression enhances PDGF-stimulated phospholipase C(γ) (PLC(γ)) activity (Sun, H.-q., K. Kwiatkowska, D.C. Wooten, and H.L. Yin. 1995. J. Cell Biol. 129:147–156). In this paper, we examined the ability of gelsolin and CapG to compete with another PLC for PIP(2) in live cells, in semiintact cells, and in vitro. We found that CapG and gelsolin overexpression profoundly inhibited bradykinin-stimulated PLC(β). Inhibition occurred at or after the G protein activation step because overexpression also reduced the response to direct G protein activation with NaF. Bradykinin responsiveness was restored after cytosolic proteins, including gelsolin, leaked out of the overexpressing cells. Conversely, exogenous gelsolin added to permeabilized cells inhibited response in a dose-dependent manner. The washout and addback experiments clearly establish that excess gelsolin is the primary cause of PLC inhibition in cells. In vitro experiments showed that gelsolin and CapG stimulated as well as inhibited PLC(β), and only gelsolin domains containing PIP(2)-binding sites were effective. Inhibition was mitigated by increasing PIP(2) concentration in a manner consistent with competition between gelsolin and PLC(β) for PIP(2). Gelsolin and CapG also had biphasic effects on tyrosine kinase– phosphorylated PLC(γ), although they inhibited PLC(γ) less than PLC(β). Our findings indicate that as PIP(2) level and availability change during signaling, cross talk between PIP(2)-regulated proteins provides a selective mechanism for positive as well as negative regulation of the signal transduction cascade.