The T-cell antigen receptor utilizes Lck, Raf-1, and MEK-1 for activating mitogen-activated protein kinase. Evidence for the existence of a second protein kinase C-dependent pathway in an Lck-negative Jurkat cell mutant.
T-cell antigen receptor (TCR) ligation of an Lck-deficient Jurkat mutant, J.CaM1, with anti-CD3 or anti-TCR beta monoclonal antibodies failed to induce tyrosine phosphorylation and activation of p42MAPK. The same stimuli activated mitogen-activated protein (MAP) kinase in J.CaM1 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 kinase (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, and p42MAPK in J.CaM1 cells during treatment with the phorbol ester phorbol 12-myristate 13-acetate, which activates protein kinase C (PKC). This demonstrates the presence of a PKC-dependent pathway which functions 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 Shc proteins or their association with Grb2 which forms a complex with the guanine nucleotide exchange factor hSOS. However, the same stimuli induced tyrosine phosphorylation of another putative guanine nucleotide exchange factor, p95Vav, in Jurkat but not J.CaM1 cells. Moreover, Lck was reversibly co-immunoprecipitated with p95Vav, and the stoichiometry of binding increased in anti-CD3-treated Jurkat cells. Phorbol 12-myristate 13-acetate did not induce tyrosine phosphorylation of p95Vav. These data show that the TCR activates MAP kinase by way of a signaling cascade, which depends upon Lck, and may be mediated by downstream events involving PKC or p95Vav which act on Raf-1 and MEK-1.
Gupta
S
S
Department of Medicine, UCLA School of Medicine 90024.
Weiss
A
A
Kumar
G
G
Wang
S
S
Nel
A
A
D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D048369:MAP Kinase Kinase 1; D020930:MAP Kinase Kinase Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D019000:Phosphotyrosine; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D011518:Proto-Oncogene Proteins; D011948:Receptors, Antigen, T-Cell; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D020930:MAP Kinase Kinase Kinases; C087095:insulin-stimulated MEK kinase; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D013755:Tetradecanoylphorbol Acetate