1179A eNOS and HA-Akt overnight. 36 h after transfection, we placed cells into dialysed serum-replete, phosphate-free Dulbecco's minimum essential medium supplemented with 80 Ci per ml of 32 P orthophosphoric acid for 3 h. Some cells were pretreated with wortmannin (500 nM) in the phosphate-free medium for 1 h and during the labelling. The lysates were collected and the eNOS was solubilized and partially purified by ADP Sepharose-affinity chromatography as previously described. 32 P incorporation into eNOS was visualized after SDS-PAGE (7.5%) by autoradiography, and the amount of eNOS protein was verified by western blotting for eNOS. For in vitro phosphorylation studies, we incubated recombinant eNOS purified from Escherichia Coli with wild-type or kinase-inactive Akt immunoprecipitated from transfected COS cells. eNOS was incubated with 32 P β₯-ATP (2 l, specific activity 3000Ci per mmol), ATP (50 M) and DTT (1 mM), in a buffer containing HEPES (20 mM, pH ΒΌ 7:4), MnCl 2 (10 mM), MgCl 2 (10 mM) and immunoprecipitated Akt for 20 min at room temperature. In experiments examining the in vitro phosphorylation of wild-type and mutant eNOS we incubated recombinant Akt (1 g) purified from baculovirus-infected SF9 cells with wild-type or S1179A eNOS (2.4 g, purified from E. coli) using essentially the same conditions as above. Proteins were resolved by SDS-PAGE and 32 P incorporation and the amount of protein was determined by Coomassie staining as above. In studies identifying the labelled eNOS peptide, we incubated immunoprecipitated Akt with recombinant eNOS as above. The sample was run on SDS-PAGE and the eNOS band digested in gel, and the resultant tryptic fragments were purified by RP-HPLC. Peptide mass and 32 P incorporation were monitored and the prominent labelled peak was further analysed by mass spectrometry. In other experiments, peptides corresponding to the potential Akt phosphorylation site were synthesized, purified by HPLC and verified by mass spectrometry (W. M.