The major phosphorylation site of the NADPH oxidase component p67phox is Thr233

Phosphorylation of p67^phox was shown to increase two- to three-fold upon stimulation by PMA, N-formylmethionyl-leucylphenylalanine or serum-opsonized zymosan. Phosphopeptide mapping showed one major tryptic peptide for p67^phox immunoprecipitated from resting or stimulated cells. In vitro phosphorylation of p67^phox by isolated cytosol or mitogen-activated protein kinase also generated the same phosphopeptide. Results of cyanogen bromide digestion and HPLC–MS suggested that Thr²³³ was the phosphorylated residue. Mutagenesis of Thr²³³ to alanine resulted in loss of phosphorylation in vitro. In the present work, Thr²³³ has been identified as the major phosphorylation site of p67^phox, which is situated in a proline-rich domain.