The distinct roles of Ras and Rac in PI 3-kinase-dependent protrusion during EGF-stimulated cell migration

Abstract

Cell migration involves the localized extension of actin-rich protrusions, a process that requires Class I phosphoinositide 3-kinases (PI 3-kinases). Both Rac and Ras have been shown to regulate actin polymerization and activate PI 3-kinase. However, the coordination of Rac, Ras and PI 3-kinase activation during epidermal growth factor (EGF)-stimulated protrusion has not been analyzed. We examined PI 3-kinase-dependent protrusion in MTLn3 rat adenocarcinoma cells. EGF-stimulated phosphatidyl-inositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] levels showed a rapid and persistent response, as PI 3-kinase activity remained elevated up to 3 minutes. The activation kinetics of Ras, but not Rac, coincided with those of leading-edge PtdIns(3,4,5)P3 production. Small interfering RNA (siRNA) knockdown of K-Ras but not Rac1 abolished PtdIns(3,4,5)P3 production at the leading edge and inhibited EGF-stimulated protrusion. However, Rac1 knockdown did inhibit cell migration, because of the inhibition of focal adhesion formation in Rac1 siRNA-treated cells. Our data show that in EGF-stimulated MTLn3 carcinoma cells, Ras is required for both PtdIns(3,4,5)P3 production and lamellipod extension, whereas Rac1 is required for formation of adhesive structures. These data suggest an unappreciated role for Ras during protrusion, and a crucial role for Rac in the stabilization of protrusions required for cell motility.

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

“…7F). Thus, in MTLn3 cells, where we find increased RhoA activity and decreased Rac activity, protrusion is Cdc42-dependent and Rac-independent [24,26]; Rac activity and Rac-dependent protrusion is restored upon inhibition of ROCK (Fig. 6).…”

“…7E). MTC cells treated with Rac1 siRNA were less well spread than cells treated with control siRNA, and in contrast to MTLn3 cells [26], showed a marked decrease in EGF-stimulated protrusion (Fig. 7F).…”

“…We have previously shown that in control MTLn3 cells, EGF-stimulated protrusion is unaffected by knockdown of Rac1 [43], but is inhibited by knockdown of Cdc42 [24]. Given the dramatic increase in Rac1 activation caused by inhibition of ROCK, we tested whether the functional roles of Rac and Cdcd42 were altered in Y27632-treated cells.…”

“…Rac1 siRNA [26] and Cdc42 siRNA [27] were previously described. The Rac1 siRNA oligo was: 5′-AAAGAGAUCGGUGCUGUCAAA-3′.…”

“…3A, panel 4) but decreased motility (Table 2). We therefore tested whether Rho inhibition might enhance protrusion through affects on Rac1 (Rac 2 and Rac 3 expression is barely detectable in MTLn3 cells [26]). MTLn3 cells microinjected with C3T (Fig.…”