THE T-CELL ANTIGEN RECEPTOR UTILIZES LCK, RAF-1, AND MEK-1 FOR ACTIVATING MITOGEN-ACTIVATED PROTEIN-KINASE - EVIDENCE FOR THE EXISTENCE OF A 2ND PROTEIN-KINASE C-DEPENDENT PATHWAY IN AN LCK-NEGATIVE JURKAT CELL MUTANT

T cell antigen receptor (TCR) ligation of an Lck-deficient Jurkat muta nt, J.CaM1, with anti-CD3 or anti-TCR beta monoclonal antibodies faile d to induce tyrosine phosphorylation and activation of p42(MAPK). The same stimuli activated mitogen-activated protein (MAP) kinase in J.CaM 1 cells transfected with Lck, demonstrating that Lck plays a critical role in MAP kinase activation. Utilizing immunocomplex kinase assays, we demonstrated that TCR/CD3 ligation activated a MAP kinase kinase ki nase (Raf-1) as well as a MAP kinase kinase (MEK-1) in Jurkat but not in J.CaM1 cells. It was possible, however, to activate Raf-1, MEK-1, a nd p42(MAPK) in J.CaM1 cells during treatment with the phorbol ester p horbol 12-myristate 13-acetate, which activates protein kinase C (PKC) . This demonstrates the presence of a PKC-dependent pathway which func tions independently from Lck in MAP kinase activation. Stimulation of Jurkat cells with either anti-TCR beta or anti-CD3 monoclonal antibody failed to induce substantial tyrosine phosphorylation of She proteins or their association with Grb2 which forms a complex with the guanine nucleotide exchange factor hSOS. However, the same stimuli induced ty rosine phosphorylation of another putative guanine nucleotide exchange factor, p95(Vav), in Jurkat but not J.CaM1 cells. Moreover, Lck was r eversibly co-immunoprecipitated with p95(Vav), and the stoichiometry o f binding increased in anti-CD3-treated Jurkat cells. Phorbol 12-myris tate 13-acetate did not induce tyrosine phosphorylation of p95(Vav). T hese data show that the TCR activates MAP kinase by way of a signaling cascade, which depends upon Lck, and may be mediated by downstream ev ents involving PKC or p95(Vav) which act on Raf-1 and MEK-1.