Membrane Ruffling Requires Coordination between Type Iα 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 (PIP 2 ) 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 PIPKI␣ isoform is localized to platelet-derived growth factor (PDGF)-induced membrane ruffles. Further, expression of kinase dead PIPKI␣, which acts as a dominant negative mutant, blocked membrane ruffling, suggesting that PIPKI␣ and PIP 2 participate in ruffling. To explore this, PIPKI␣ was overexpressed in serumstarved cells and stimulated with PDGF. In serumstarved cells, PIPKI␣ expression did not stimulate actin remodeling, but when these cells were stimulated with PDGF, actin rapidly reorganized into foci but not membrane ruffles. PIPKI␣-mediated formation of actin foci was independent of both Rac1 and phosphatidylinositol 3-kinase activities. Significantly, coexpression of dominant active Rac1 with PIPKI␣ in PDGF-stimulated cells resulted in membrane ruffling. The PDGF-and Rac1-stimulated ruffling was inhibited by expression of kinase-dead PIPKI␣. Combined, these data support a model where the localized production of PIP 2 by PIPKI␣ is necessary for actin remodeling, whereas formation of membrane ruffles required Rac signaling.

Activation of Type I Phosphatidylinositol 4-Phosphate 5-Kinase Isoforms by the Rho GTPases, RhoA, Rac1, and Cdc42

“…GTPases and PIP5K isoforms, may be decisive to yet coordinate the demand of PIP 2 for different cellular processes. Indeed, Rac1 was shown to associate with PIP5K-I␣ and PIP5K-I␤, but only PIP5K-I␣ seemed to induce actin filament uncapping downstream of Rac1 in permeabilized platelets (35) and to localize in Rac1-induced membrane ruffles (25).…”

“…The differential regulation and/or subcellular localization of the PIP5K isoforms obviously could represent a mechanism for cells to coordinate their PIP 2 production. Indeed, PIP5K-I␣ was found to specifically localize to membrane ruffles (25), PIP5K-I␥ targeted to focal adhesions (26) and nerve terminals (27), whereas the PIP 2 pool involved in endocytosis may primarily be produced by PIP5K-I␤ (28).…”

PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

“…[12][13][14][15] Increasing evidences indicate that PI5KIs, providing spatially and functionally distinct PIP2 pools, guarantee the regulation of specific cellular events: PI5KI␥, for instance, is required for focal adhesion and synaptic vesicle trafficking, 16,17 PI5KI␤ for constitutive receptor endocytosis, 18 whereas PI5KI␣ acts in the regulation of processes dependent on local changes of actin dynamics, such as membrane ruffling and phagocytosis. 19,20 In this report, we took advantage of the fusion protein consisting of GFP and the pleckstrin homology (PH) domain of PLC␦1, that is known to bind PIP2 with high affinity and specificity, [21][22][23] to monitor the dynamics and function of PIP2 during the cytotoxic event. We present evidences of localized changes in the concentration of PIP2 at the cytolytic synapse area where it is required for granule exocytosis.…”

PIP5KIγ is required for cardiovascular and neuronal development

“…All three isoforms can be stimulated by small GTPases (Rho, Rac, Cdc42, and ARF), as well as by phosphatidic acid. Although PIP5KI␣, PIP5KI␤, and PIP5KI␥ are all capable of synthesizing PIP 2 , these isoenzymes have significantly dissimilar primary structures and different expression levels in different tissues, and they appear to localize within different compartments within some cells (7)(8)(9)(10)(11)(12). For example, PIP5KI␣ localizes in membrane ruffles (8), PIP5KI␤ localizes near endosomes (9), and PIP5KI␥ is targeted to focal adhesions and nerve terminals (10)(11)(12).…”