Membrane Ruffling Requires Coordination between Type Iα Phosphatidylinositol Phosphate Kinase and Rac Signaling

• DOI: 10.1074/jbc.m211397200
• Publication Date: 2003-06-13T17:43:06Z
• Authors: Renee L. Doughman; Ari J. Firestone; Michelle L. Wojta...; Matthew W. Bunce; Richard A. Anderson
• Keywords: Platelet-Derived Growth Factor; 0301 basic medicine; Microscopy, Confocal; Base Sequence; Morpholines; Cell Membrane; Fibroblasts; Recombinant Proteins; Cell Line; rac GTP-Binding Proteins; Minor Histocompatibility Antigens; Phosphotransferases (Alcohol Group Acceptor); 03 medical and health sciences; Amino Acid Substitution; Chromones; Mutagenesis, Site-Directed; Humans; Cloning, Molecular; Enzyme Inhibitors; DNA Primers; Signal Transduction

Membrane ruffle formation requires remodeling of cortical actin filaments, a process dependent upon the small G-protein Rac. Growth factors stimulate actin remodeling and membrane ruffling by integration of signaling pathways that regulate actin-binding proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the activity of many actin-binding proteins and is produced by the type I phosphatidylinositol phosphate kinases (PIPKIs). Here we show in MG-63 cells that only the PIPKIalpha isoform is localized to platelet-derived growth factor (PDGF)-induced membrane ruffles. Further, expression of kinase dead PIPKIalpha, which acts as a dominant negative mutant, blocked membrane ruffling, suggesting that PIPKIalpha and PIP2 participate in ruffling. To explore this, PIPKIalpha was overexpressed in serum-starved cells and stimulated with PDGF. In serum-starved cells, PIPKIalpha expression did not stimulate actin remodeling, but when these cells were stimulated with PDGF, actin rapidly reorganized into foci but not membrane ruffles. PIPKIalpha-mediated formation of actin foci was independent of both Rac1 and phosphatidylinositol 3-kinase activities. Significantly, coexpression of dominant active Rac1 with PIPKIalpha in PDGF-stimulated cells resulted in membrane ruffling. The PDGF- and Rac1-stimulated ruffling was inhibited by expression of kinase-dead PIPKIalpha. Combined, these data support a model where the localized production of PIP2 by PIPKIalpha is necessary for actin remodeling, whereas formation of membrane ruffles required Rac signaling.