Characterization of P-Rex1 for its role in fMet-Leu-Phe-induced superoxide production in reconstituted COSphox cells

P-Rex1 (phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1) is a Rac-specific guanine nucleotide exchange factor activated by Gβγ subunits and by PtdIns(3,4,5)P3. Recent studies indicate that P-Rex1 plays an important role in signaling downstream of neutrophil chemoattractant receptors. Here we report that heterologous expression of P-Rex1, but not Vav1, reconstitutes formyl peptide receptor 1 (FPR1)-mediated NADPH oxidase activation in the transgenic COSphox cells expressing gp91phox, p22phox, p67phox and p47phox. A successful reconstitution requires the expression of a full-length P-Rex1 with intact DH and PH domains, and is accompanied by P-Rex1 membrane localization as well as Rac1 activation. P-Rex1-dependent superoxide generation in the reconstituted COSphox cells was further enhanced by expression of the novel PKC isoform PKCδ and by overexpression of Akt. Heterologous expression of P-Rex1 in COSphox cells potentiated fMet-Leu-Phe-induced Akt phosphorylation, whereas expression of a constitutively active form of Akt enhanced Rac1 activation. In contrast, a dominant negative Akt mutant reduced the fMet-Leu-Phe stimulated superoxide generation as well as Rac1 activation. These results demonstrate that in COSphox cells, P-Rex1 is a critical component for FPR1-mediated signaling leading to NADPH oxidase activation, and there is a crosstalk between the P-Rex1-Rac pathway and Akt in superoxide generation.