Activation of PKB, ERK1/2, Ras, and Rap1.

(A) Increase of cAMP inhibits TCR/CD3 plus CD28–mediated but not phorbol ester–mediated activation of PKB and ERK1/2. Purified primary human T cells were cultured either with media, with anti-CD3 plus anti-CD28 mAbs, or with phorbol ester plus anti-CD28 mAb followed by cross-linking with rabbit antimouse immunoglobulin for the indicated time intervals. Where indicated, cells were pretreated with forskolin and IBMX. At the indicated time intervals cell lysates were prepared and activation of PKB and ERK1/2 was examined by SDS-PAGE and Western blot with phospho-specific antibodies. Immunoblots were stripped and reprobed with ERK1/2-specific antibody, which recognizes total nonphosphorylated ERK1/2 to confirm equal loading and ERK1/2 protein expression. (B) Increase of cAMP inhibits TCR/CD3 plus CD28–mediated but not phorbol ester–mediated activation of Ras but inhibits both TCR/CD3 plus CD28– and phorbol ester–mediated activation of Rap1. In the same cell lysates activation of Ras and Rap1 was examined by pull-down assays using glutathione beads coated with Raf1RBD-GST for Ras activation and RalGDSRBD-GST for Rap1 activation. Incubation of cells with forskolin and IBMX alone did not result in activation of either Ras or Rap1 (data not shown). Results are representative of 3 experiments.