Phosphatidylinositol 3-kinase is a target for protein tyrosine nitration.
A major mechanism of injury associated with the production of nitric oxide (NO*) in vivo is due to its diffusion-limited reaction with superoxide to form peroxynitrite, which in turn may cause nitration of protein tyrosine residues. To assess the physiological role of tyrosine nitration, it is crucial to identify the proteins that become nitrated. Therefore, we treated lysates from RAW 264.7 cells with 1 mM peroxynitrite and immunoprecipitated tyrosine nitrated proteins. This treatment resulted in the nitration of several proteins, with molecular weights ranging from 60-250 kD. One of these proteins was immunologically identified as the p85 regulatory subunit of the phosphatidylinositol 3-kinase, a key enzyme involved in the signal transduction cascade initiated by many agonists including growth factors. Treatment of RAW 264.7 macrophages with the NO* donor spermine NONOate also induced a nitration of the p85 subunit, demonstrating that this covalent modification also occurs in intact cells. Immunoprecipitation of the p110 catalytic subunit of the phosphatidylinositol 3-kinase co-immunoprecipitated p85 in control lysates. However, p85 could not be detected in the same immunoprecipitates when the lysates had been preincubated with 1 mM peroxynitrite, indicating that the nitration of the p85 subunit may abrogate its interaction with the p110 subunit.
Hellberg
C B
CB
Molecular Cardiovascular Research Center, Case Western Reserve, University School of Medicine and University Hospitals of Cleveland, Cleveland, Ohio, 44106-4958, USA.
Boggs
S E
SE
Lapetina
E G
EG
D000818:Animals; D002460:Cell Line; D051379:Mice; D008970:Molecular Weight; D009566:Nitrates / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009589:Nitrogen Oxides; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D013096:Spermine / Q000031:analogs & derivatives / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D009566:Nitrates; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; D011506:Proteins; C091861:spermine nitric oxide complex; C039642:peroxynitric acid; D013096:Spermine; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases