PHOSPHORYLATION OF THE RESPIRATORY BURST OXIDASE SUBUNIT P47(PHOX) ASDETERMINED BY 2-DIMENSIONAL PHOSPHOPEPTIDE MAPPING - PHOSPHORYLATION BY PROTEIN-KINASE-C, PROTEIN-KINASE-A, AND A MITOGEN-ACTIVATED PROTEIN-KINASE

The respiratory burst oxidase is responsible for superoxide (O-2(radic al anion)) production by phagocytes and B lymphocytes. This multicompo nent enzyme is dormant in resting cells but is activated on exposure o f the cells to an appropriate stimulus. Upon activation, several serin e residues on the cytosolic oxidase subunit p47(phox) become phosphory lated, Using two-dimensional tryptic phosphopeptide mapping, we studie d the phosphorylation of p47(phox) in P-32(i)-loaded Epstein-Barr viru s-transformed B lymphoblasts expressing wild type p47(phox) or any of several p47(phox) Ser --> Ala mutants. We were able to identify the la beled peptides from wild type p47(phox) as those containing Ser(303/30 4), Ser(315), Ser(320), Ser(328) and/or Ser(359/370), and Ser(345/348) ; no P-32-labeled Ser(310)-containing peptide was found. When purified p47(phox) was phosphorylated in vitro by various protein kinases, var ying phosphopeptide patterns were observed, Protein kinase C phosphory lated all the peptides except the one containing Ser(345/348); protein kinase A phosphorylated the peptide containing Ser(320) and one or bo th of the peptides containing Ser(328) and Ser(359/370); while mitogen activated protein kinase phophorylated only the peptide containing Se r(345/348). These findings suggest that these three kinases play disti nct roles in the activation of the respiratory burst oxidase, each of them catalyzing the phosphorylation of a different group of serines in p47(phox).