Phosphorylation of eNOS on Thr497 is required for inhibition of NO production by Tie2. (A) The effect of Tie2 on NO production stimulated by VEGFR-2 was measured in transfected COS-7 cells. Cells were cotransfected with expression vectors coding for eNOS in combination with VEGFR-2 and WT Tie2 (left panels) or in separate sets of experiments the kinase-dead K855A-Tie2 constructs (right panel). Levels of NO released in the medium after transfection were measured, as in Figure 1B. Expression levels of the transfected proteins were monitored by Western blotting (bottom panels). The inactivity of K855A-Tie2, in contrast to WT-Tie2, was determined by anti-phosphotyrosine Western blotting of Tie2 IPs (inset, left panel). NO released from COS-7 cells transfected with expression vectors coding for T497A-eNOS single mutant (B) or T497A/S1179D-eNOS double mutant (C), in combination with VEGFR-2, Tie2, or both, was measured by chemiluminescence, as described above. Equal transfection levels were monitored by Western blotting using anti–VEGFR-2, anti-Tie2, and anti-eNOS antibodies. Data are the average ± SEM of at least 4 experiments; *P < .05.
