Role of Ser³⁶ phosphorylation of p66^Shc in endothelial activation.

<p><i>A</i>. Basal and TNFα-stimulated phosphorylation of p66^Shc on Ser³⁶ in HUVEC overexpressing the p66^Shc Ala³⁶ mutant (HUVEC/p66^ShcAla³⁶). Cells were stimulated with 50 ng/ml TNFα for 0.5 h or left untreated. When indicated, cells were pre-incubated with 30 mM SP600125 or DMSO for 2 h prior to TNFα stimulation. Each pair of representative immunoblots shows p66^Shc phosphorylation on Ser³⁶ (top left) and Shc protein content (bottom left) in HUVEC/wt, HUVEC/p66^Shc, HUVEC/p66^ShcAla³⁶, and HUVEC/mock, respectively. The ratio of phosphorylated to total p66^Shc protein in the four cell lines is also shown (<i>right</i>; HUVEC/wt, filled bars; HUVEC/p66^Shc, open bars; HUVEC/p66^ShcAla³⁶, grey bars; HUVEC/mock, light grey bars). *<i>P</i><0.05, TNFα-stimulated HUVEC/p66^Shc vs. HUVEC/p66^ShcAla³⁶; ^#<i>P</i><0.05, SP600125-treated HUVEC/p66^Shc vs. untreated HUVEC/p66^Shc. <i>B</i>. Leukocyte transmigration test. HL-60 cells were added to HUVEC/w.t. and HUVEC treated with Ad/p66^Shc, Ad/p66^ShcAla³⁶, or Ad/mock for 48 h, and left to migrate for 18 h at 37°C. Studies were carried out under basal conditions or following stimulation with 50 ng/ml TNFα for 4 h. Migrated cells were stained and measured by OD at 450 nm. Data represents the mean of triplicates from two independent experiments, and are normalized to basal HUVEC/wt. *<i>P</i><0.05 vs. no TNFα; ^§P<0.05 vs. HUVEC/wt; ^#<i>P</i><0.05 vs. HUVEC/p66^ShcAla³⁶. <i>C</i>. Effects of Ser/Ala³⁶ mutation on E-Selectin mRNA levels in HUVEC. HUVEC/wt (filled bars), HUVEC/p66^Shc (open bars), HUVEC/<i>Ala</i>³⁶ (grey bars), and HUVEC/mock (light grey bars) were treated with 50 ng/ml TNFα for 1 h or left untreated. E-Selectin gene expression was evaluated using qRT-PCR. *<i>P</i><0.05, TNFα-stimulated cells vs. unstimulated cells; ^#<i>P</i><0.05, HUVEC/p66^Shc vs. controls. <i>D</i>. Cartoon illustrating the signaling pathway mediating the stimulatory effect of TNFα on E-Selectin gene expression in HUVEC. MKKs, MKK-7 and MKK-4; IKK, IκBα kinase; ROS, reactive oxygen species.