Cross-linking of CD150 on CD4 T cells results in activation of the serine/threonine kinase Akt/PKB.

(A) Triggering of CD150 on activated CD4 T cells does not induce detectable phosphorylation of MAPK ERK1/2. Negatively selected CD4 T cells from C57Bl/6 mice were preactivated with anti-CD3 plate-bound plus IL-2 for 48 hours then rested in IL-2 (50 U/mL) for 24 hours to induce CD150 surface expression before CD150 cross-linking. Anti-CD3 (145.2C11, 10 μg/mL) as a positive control, IgG as a negative control, or 9D1 (10 μg/mL) antibodies were cross-linked with isotype-specific secondary antibodies for 10 minutes. Cell lysates were subjected to SDS-PAGE and Western blotted for phospho-ERK (top panel) or total ERK1/ERK2 (bottom panel). (B) CD150 cross-linking on activated CD4 T cells induces rapid phosphorylation of the serine/threonine kinase Akt on Ser473. Cells were preactivated as described in panel A before treatment with IgG for 10 minutes (negative control), 2C11 for 10 minutes (positive control), or 9D1 (all antibodies used at 10 μg/mL) for the indicated time points. Top panel is Western blot with anti–phospho-serine-473 Akt. Bottom panel is Western blot total Akt.