Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF

Rac1 has a crucial role in epidermal growth factor (EGF)-induced membrane ruffling, lamellipodial protrusion, and cell migration. Several guanine nucleotide exchange factors (GEFs) including Sos1, Sos2, Tiam1 and Vav2 have been shown to transduce the growth signal from the EGF receptor to Rac1. To clarify the role of each GEF, we time-lapse imaged the EGF-induced activity change of Rac1 in A431 cells transfected with siRNA targeting each Rac1 GEF. Because knockdown of these GEFs suppressed EGF-induced Rac1 activation only partially, we looked for another Rac1 GEF downstream of the EGF receptor and found that Asef, a Rac1-Cdc42 GEF bound to the tumor suppressor APC, also contributed to EGF-induced Rac1 activation. Intriguingly, EGF stimulation induced phosphorylation of Tyr94 within the APC-binding region of Asef in a manner dependent on Src-family tyrosine kinases. The suppression of EGF-induced Rac1 activation in siRNA-treated cells was restored by wild-type Asef, but not by the Tyr94Phe mutant of Asef. This observation strongly argues for the positive role of Tyr94 phosphorylation in EGF-induced Asef activation following the activation of Rac1.