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

Abstract

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.

The Adenomatous Polyposis Coli-associated Guanine Nucleotide Exchange Factor Asef Is Involved in Angiogenesis

“…Our findings that Asef is required for bFGF-and VEGF-mediated angiogenesis raise the possibility that Asef and APC function downstream of growth factors acting through receptor tyrosine kinases. In line with this notion, we have recently found that Asef functions downstream of EGF and hepatocyte growth factor in epithelial cells (19,28). In this study, we showed that bFGF and VEGF induce the accumulation and colocalization of Asef and APC in membrane ruffles and lamellipodia and increase the amounts of the Asef-APC complex.…”

Rac1 and Cdc42 Differentially Modulate Cigarette Smoke–Induced Airway Cell Migration through p120-Catenin–Dependent and –Independent Pathways

“…48 Seemingly, the p120ctn/Cdc42/Rac1 pathway could be activated by constitutively active EGFR mutant in the absence of smoke exposure, eventually causing lung adenocarcinoma in patients who have never smoked. In addition to EGFR, 42,43 Rac1 and its downstream effects on cell motility could be activated by platelet-derived growth factor receptor 49 and insulin receptor, 50 as previously reported. Thus, the EGFR/p120ctn-dependent and EGFR/ p120ctn-independent pathways may be operative in smokers, former smokers, and nonsmokers.…”

“…43 These data highlight the potential significance of Rac1 in advanced disease as tumor-suppressing p120ctn is lost from cells. Activation of Rac1 via an EGFR/p120ctn-independent pathway is feasible because its activation is complex with multiple signaling inputs that include EGFR-independent receptor tyrosine kinases, integrins, and cadherins.…”

“…The American Journal of Pathology -ajp.amjpathol.orgcells, 42,43 which was probably achieved by phosphorylation and activation of Asef, a Rac1-Cdc42 guanine nucleotide exchange factor functioning downstream of EGFR. 43 These data highlight the potential significance of Rac1 in advanced disease as tumor-suppressing p120ctn is lost from cells.…”

Structural basis for the recognition of Asef by adenomatous polyposis coli

“…At the center of this new interface, Asef-Tyr175 hydrogen bonds to Asef-Asp209, Asef-Glu212, and also makes van der Waals contacts with Asef-Trp231 and Asef-Trp242 from the SH3 domain. Interestingly, AsefTyr175 has been reported to be a phosphorylation site by Src family tyrosine kinases, and could be involved in the regulation of the activity of Asef [31]. In addition to Asef-Tyr175, Asef-His173, Asef-His174, and Asef- His177 from the ABR region are also involved in this new interface with the SH3 domain ( Figure 6B).…”