The distinct roles of Ras and Rac in PI3-kinase dependent protrusion during EGF-stimulated cell migration

Phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol, generating secondary messengers that direct a variety of cellular responses. In EGF-stimulated carcinoma cells, PI3K activity is required for the formation of actin-rich protrusions, such as lamellipodia. There are two actin polymerization transients in EGF-stimulated MTLn3 cells, with peaks of barbed end formation at the leading edge at 1 and 3 min. PI3K is only required for the late peak of barbed end formation at 3 min.; Given the differential requirement of PI3K activity in the two-actin polymerization transients, it is important to define the spatial and the temporal regulation of PI3K activation and PIP3 production during EGF-stimulated protrusion. We measured the kinetics of EGF-stimulated PIP3 production at the leading edge of the lamellipod using a saponin-permeabilization/fixation method and a monoclonal anti-PIP3 antibody. Anti-PIP3 immunostaining exhibited a rapid and persistent response, as the activity reached maximal level at 1 min and remained elevated at 3 min. However, membrane PIP3 turnover is extremely fast, as Ly294002 treatment abolished leading edge PIP3 within 10sec. This means that the sustained elevation of leading edge PIP3 in EGF-stimulated cells requires persistent PI3K activation.; Both Rac and Ras have been implicated in growth factor-induced membrane ruffling and have been shown to activate Class IA PI3K. However, the coordination of Rac and Ras activation with PI3K dependent lamellipod extension has not been characterized during EGF-stimulated protrusion. Inhibition of Rac1 activity by siRNA-mediated knockdown or pharmacological inhibition had no effect on EGF-stimulated lamellipod extension or PIP3 production at the leading edge of carcinoma cells. In contrast, microinjection of inhibitory anti-Ras antibodies or pharmacological inhibition of Ras abolished EGF-stimulated protrusion and PIP3 production at the leading edge. Moreover, the activation kinetics of Ras, but not Rac, was coincident with those of leading edge PIP 3 production. Despite the fact that lamellipod extension was independent of Rac, Rac activity was required for cell motility, as Rac1 siRNA treatment inhibited cell migration. We conclude that Ras, but not Rac, plays a dominant role in regulating PI3K activation and PIP3-dependent lamellipod extension in EGF-stimulated cells.