Characterization of P-Rex1 for its role in fMet-Leu-Phe-induced superoxide production in reconstituted COS(phox) cells.
P-Rex1 (phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1) is a Rac-specific guanine nucleotide exchange factor activated by Gbetagamma subunits and by PtdIns((3,4,5))P(3). 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 COS(phox) cells expressing gp91(phox), p22(phox), p67(phox) and p47(phox). 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 COS(phox) cells was further enhanced by expression of the novel PKC isoform PKCdelta and by overexpression of Akt. Heterologous expression of P-Rex1 in COS(phox) 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 COS(phox) 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.
Nie
Baoming
B
Department of Pharmacology, University of Illinois, Chicago, 60612, United States.
Cheng
Ni
N
Dinauer
Mary C
MC
Ye
Richard D
RD
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000187:drug effects; D025461:Feedback, Physiological / Q000187:drug effects; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020662:Guanine Nucleotide Exchange Factors; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D051745:Protein Kinase C-delta; D051057:Proto-Oncogene Proteins c-akt; D013481:Superoxides; D020830:rac1 GTP-Binding Protein; C453280:PREX1 protein, human