Phosphorylation of p47^phox by p21-activated kinase (Pak). (A) Peptides derived from the C-terminus of human p47^phox were incubated with recombinant GST-Pak1 and [³²P]ATP, as described. The numbers under the bars indicate the location of the peptides in p47^phox (amino acids). Results are depicted as the mean ± SD of cpm measurement on 4 different fields per peptide. (B) In vitro kinase assays were performed as described using GST-Pak1 alone (lane 1), recombinant wild-type GST-p47^phox (lane 2), or recombinant GST-p47^phox quadruple mutant S303/304/320/328A (lane 3). (C) 2-D map of phosphopeptides derived from trypsin-digested GST-p47^phox (left) or GST-p47^phox S303/304/320/328A (right) after phosphorylation by GST-Pak1. The original (ori) application spot of the sample is indicated by an arrow.