Fig 1: Gi and c-Abl Mediate VEGFR2 Phosphorylation at Y951 by VEGF(A–D) HPAECs treated without or with pertussis toxin (PTX; 50 µM) were stimulated with VEGF for indicated time points. Phosphorylation of VEGFR2 and c-Abl1 was determined using phosphospecific antibodies. Immunoblot with c-Abl1 and VEGFR2 expression was used as loading control. (A) shows a representative immunoblot, and (B)–(D) show densitometry. One-way ANOVA, p < 0.05 (B). One-way ANOVA, p < 0.001 (C and D). Paired t test, two-tailed, ***p < 0.001 relative to unstimulated HPAECs (B–D).(E and F) Lysate from S1pr1+/+ or S1pr1-/- TAECs were immunoprecipitated with anti-VEGFR2 antibody, and immunocomplexes were probed with c-Abl1 or VEGFR2 antibodies. A representative immunoblot is shown in (E), and (F) shows fold change in VEGFR2-c-Abl interaction normalized against total VEGFR2. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1+ tumor ECs.(G and H) Lysates from indicated TAECs were immunoblotted with either phospho-Src, phospho-c-Abl1, c-Src, or c-Abl1 antibodies. A representative immunoblot is shown in (G), and (H) shows densitometry. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1+ tumor ECs.(I) Indicated TAECs seeded on polycarbonate membrane were treated with or without imatinib (40 µM for 1 h), and EC migration was determined as described in Figure 1C. One-way ANOVA, p < 0.05. Paired t test, two-tailed, **p < 0.01 relative to untreated S1PR1+ tumor ECs; NS relative to imatinib-treated S1PR1+ or S1PR1- TAECs. Data representative of mean ± SEM from multiple experiments.(J–M) HPAECs transfected with c-Abl1 siRNA for 72 h were stimulated with VEGF for indicated time points. VEGFR2 phosphorylation and Rac1 activity were determined as described in Figure 5A. Immunoblotting with c-Abl antibody was used to determine c-Abl1 depletion. Total Rac1, total VEGFR2, and actin were used as loading control. A representative immunoblot is shown in (J), and (K)–(M) show densitometries. One-way ANOVA, p < 0.01 (K–M). Paired t test, two-tailed, **p < 0.001 relative to un-stimulated siScr or c-Abl-siRNA-transfected cells; ##p < 0.01 relative to siScr HPAECs following 10 and 120 min VEGF stimulation (K–M). All western blots show data from multiple experiments expressed as mean ± SEM.(N) Model showing influence of S1P-S1PR1 signaling on VEGF-VEGFR2-mediated angiogenesis that augments EC migration and tumor vascularization and growth. Cancer cells generate S1P and VEGF. S1P ligates S1PR1 in S1PR1+ ECs. Additionally, activation of SPHK induce S1PR1 activity in a paracrine manner. S1PR1, in turn, stimulates Gi, which promotes c-Abl1 activity by VEGF. Activated c-Abl1 phosphorylates VEGFR2 on Y951, reducing VEGFR2 binding with unknown endocytic protein, causing VEGFR2 to remain on the EC surface and prolonging Rac1 activity in a Tiam1-dependent manner that increases EC migration and efficient tumor vascularization. In ECs lacking S1PR1, VEGF leads to VEGFR2 phosphorylation at Y1175, followed by receptor internalization. Internalized receptor induces ERK activity but transiently activates Rac1, leading to reduced EC migration and impairing tumor vascularization and growth.