FUNCTIONAL DOMAIN IN AN ARGININE-RICH CARBOXYL-TERMINAL REGION OF P47PHOX

Activation of the neutrophil respiratory burst oxidase involves phosph orylation-dependent translocation of the cytosolic proteins p47phox an d p67phox to the plasma membrane, a process in intact cells that is in hibited by staurosporine. We now report that in a cell-free oxidase sy stem, staurosporine and protein kinase C pseudosubstrate PKC(19-36) bo th inhibited p47phox phosphorylation but had no effect on superoxide g eneration. In contrast, p47phox phosphorylation, translocation, and su peroxide generation were inhibited by a peptide, p47phox(323-332) (AYR RNSVRFL), based on a putative serine phosphorylation domain. This effe ct was specific for the 323-332 peptide, as it was not observed with t wo peptides based on other p47phox phosphorylation domains. All three peptides served as substrates for phosphorylation, but the extent of p eptide phosphorylation did not correlate with inhibition of oxidase fu nction. p47phox(325-330), which represents the serine phosphorylation motif of p47phox(323-332), did not inhibit translocation or superoxide production despite its ability to block phosphorylation of p47phox. T hese data indicate the presence of functionally important sites within the p47phox(323-332) peptide. Although serine 328 is in a consensus p hosphorylation motif, the lack of correlation in the cell-free system between p47phox phosphorylation and either protein translocation or su peroxide formation suggests that a phosphorylation-independent functio n resides in the 323-332 segment of p47phox.