GELSOLIN BINDING TO PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE IS MODULATED BY CALCIUM AND PH

The actin cytoskeleton of nonmuscle cells undergoes extensive remodeli ng during agonist stimulation, Lamellipodial extension is initiated by uncapping of actin nuclei at the cortical cytoplasm to allow filament elongation, Many actin filament capping proteins are regulated by pho sphatidylinositol 4,5-bisphosphate (PIP2), which is hydrolyzed by phos pholipase C. It is hypothesized that PIP2 dissociates capping proteins from filament ends to promote actin assembly, However, since actin po lymerization often occurs at a time when PIP2 concentration is decreas ed rather than increased, capping protein interactions with PIP2 may n ot be regulated solely by the bulk PIP2 concentration. We present evid ence that PIP2 binding to the gelsolin family of capping proteins is e nhanced by Ca2+. Binding was examined by equilibrium and nonequilibriu m gel filtration and by monitoring intrinsic tryptophan fluorescence, Gelsolin and CapG affinity for PIP2 were increased 8- and 4-fold, resp ectively, by mu M Ca2+, and the Ca2+ requirement was reduced by loweri ng the pH from 7.5 to 7.0. Studies with the NH2- and COOH-terminal hal ves of gelsolin showed that PIP2 binding occurred primarily at the NH2 -terminal half, and Ca2+ exposed its PIP2 binding sites through a chan ge in the COOH-terminal half, Mild acidification promotes PIP2 binding by directly affecting the NH2-terminal sites, Our findings can explai n increased PIP2-induced uncapping even as the PIP2 concentration drop s during cell activation. The change in gelsolin family PIP2 binding a ffinity during cell activation can impact divergent PIP2-dependent pro cesses by altering PIP2 availability, Cross-talk between these protein s provides a multilayered mechanism for positive and negative modulati on of signal transduction from the plasma membrane to the cytoskeleton .