title: Membrane ruffling requires coordination between type Ialpha phosphatidylinositol phosphate kinase and Rac signaling.
abstract: 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.
authors.affiliation: Molecular and Cellular Pharmacology Program, Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
authors.forename: Renee L
authors.initials: RL
authors.lastname: Doughman
authors.forename: Ari J
authors.initials: AJ
authors.lastname: Firestone
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Wojtasiak
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Bunce
authors.forename: Richard A
authors.initials: RA
authors.lastname: Anderson
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure*; D002867:Chromones / Q000494:pharmacology; D003001:Cloning, Molecular; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D006801:Humans; D018613:Microscopy, Confocal; D015778:Minor Histocompatibility Antigens; D009025:Morpholines / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D020744:rac GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002867:Chromones; D017931:DNA Primers; D004791:Enzyme Inhibitors; D015778:Minor Histocompatibility Antigens; D009025:Morpholines; D010982:Platelet-Derived Growth Factor; D011994:Recombinant Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase; D020744:rac GTP-Binding Proteins