TCR-Ras-Erk signaling and agonist-induced IL-2 secretion in Jurkat T cells overexpressing RasGRP.

(A) Jurkat cells transformed with either the empty vector pBMGNeo (Neo) or the same vector containing full-length human RasGRP (GRP) were induced with 5.0 μmol/L CdCl₂ for 24 hours, as indicated, then examined for RasGRP expression by immunoblotting using the H176 antibody. (B) Cells expressing BMGNeo or RasGRP were induced with CdCl₂ for 24 hours then stimulated with either 50 ng/mL PMA for 10 minutes or 0.156 μg/mL OKT3 for various periods. Cells were then harvested, lysed, and assayed for Ras-GTP, phospho-Erk and total Erk. (C) Cells from the same cultures as in (A) were stimulated with various concentrations of OKT3 for 15 minutes at 37°C and then directly lysed in SDS sample buffer. The lysates were then probed for phospho-Erk. The bands representing phospho-Erk were quantified by densitometry and plotted versus the concentration of OKT3. Samples were also probed with an anti-Erk antibody to verify that similar amounts of total Erk protein were present (data not shown). (D) Cells were induced with CdCl₂ as indicated for 24 hours then stimulated for 48 hours with the calcium ionophore A23187 (A, 0.5 μmol/L) and either PMA (24 nmol/L) or bryostatin-1 (Bryo, 10 nmol/L). IL-2 in the medium was then measured. Values are the average of quadruplicate samples, with the standard error of the mean indicated.