Abstract
The actin cytoskeleton of nonmuscle cells undergoes extensive remodeling 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 phosphatidylinositol 4,5-bisphosphate (PIP2), which is hydrolyzed by phospholipase C. It is hypothesized that PIP2 dissociates capping proteins from filament ends to promote actin assembly. However, since actin polymerization often occurs at a time when PIP2 concentration is decreased rather than increased, capping protein interactions with PIP2 may not be regulated solely by the bulk PIP2 concentration. We present evidence that PIP2 binding to the gelsolin family of capping proteins is enhanced by Ca2+. Binding was examined by equilibrium and nonequilibrium gel filtration and by monitoring intrinsic tryptophan fluorescence. Gelsolin and CapG affinity for PIP2 were increased 8- and 4-fold, respectively, by microM Ca2+, and the Ca2+ requirement was reduced by lowering the pH from 7.5 to 7.0. Studies with the NH2- and COOH-terminal halves of gelsolin showed that PIP2 binding occurred primarily at the NH2-terminal half, and Ca2+ exposed its PIP2 binding sites through a change in the COOH-terminal half. Mild acidification promotes PIP2 binding by directly affecting the NH2-terminal sites. Our findings can explain increased PIP2-induced uncapping even as the PIP2 concentration drops during cell activation. The change in gelsolin family PIP2 binding affinity during cell activation can impact divergent PIP2-dependent processes by altering PIP2 availability. Cross-talk between these proteins provides a multilayered mechanism for positive and negative modulation of signal transduction from the plasma membrane to the cytoskeleton.
Highlights
Phosphoinositides are important in signal transduction, both as precursors to signaling molecules and as physical anchors and regulators of proteins [1, 2]
CapG Binding to PIP2—Small zone gel filtration analyses showed that CapG bound to PIP2 micelles in a dose-dependent manner
Actin polymerization in response to agonist activation is frequently associated with a rise in cytosolic Ca21, changes in PIP2 content, and intracellular pH
Summary
Expression and Purification of Recombinant CapG, Gelsolin, and Gelsolin Domains—Gelsolin has six semihomologous domains (S1– 6), which can be further divided into two functional halves [32]. Small Zone Gel Filtration—The assay was similar to that described previously for studying lipid binding to most actin regulatory proteins [33, 35, 38] This is because small proteins bound to PIP2 micelles or mixed vesicles migrate faster than the unbound proteins. Proteins were incubated with lipid for 30 min at room temperature, and 100 ml of the mixture was chromatographed at 4 °C through a Superdex 75 HR 10/30 column (Pharmacia Biotech Inc.), equilibrated with pH 7.0 or 7.5 buffers containing 25 mM Hepes, 100 mM KCl, 0.5 mM b-mercaptoethanol, 0.4 mM EGTA with or without CaCl2. 100 ml of the equilibration buffer containing CapG was incubated with PIP2 micelles for 30 min and loaded onto the column. Values shown are mean 6 S.E., determined at pH 7.5
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