title: Activation of nuclear factor-kappaB via T cell receptor requires a Raf kinase and Ca2+ influx. Functional synergy between Raf and calcineurin.
abstract: Signals transduced via the TCR activate the transcription factor nuclear factor-kappaB (NF-kappaB), which, in turn, is critical to the transcriptional induction of many genes important for the proliferation and expression of a differentiated phenotype. Treatment of T cells with the protein kinase C activator PMA in combination with Ca2+ ionophores mimics this process, and the two agents are often substituted for TCR stimulation, bypassing the TCR. Here we identify intracellular signaling components involved in activation of NF-kappaB following TCR stimulation. TCR signaling was triggered by treating Jurkat T cells with PHA or anti-CD3 Abs, and NF-kappaB activation was monitored by electrophoretic mobility shift assays and/or by kappaB-dependent reporter assays. Contrary to the idea that protein kinase C is involved in TCR-mediated activation of NF-kappaB, high doses of staurosporine did not interfere with activation of NF-kappaB by PHA, while the same dose of staurosporine completely blocked activation by PMA. PHA-induced kappaB-dependent reporter activity was, however, effectively blocked by a dominant negative form of Raf-1, suggesting a critical role for a Raf kinase. The TCR-mediated activation of NF-kappaB was also dependent on a Ca2+ influx, because the Ca2+ channel blocker, SK&F 96365, as well as other agents that prevented the Ca2+ influx, inhibited NF-kappaB activation. Cotransfection of a constitutively active form of calcineurin largely substituted for the Ca2+ requirement and reversed the blockade by SK&F 96365. Consistent with these observations, coexpression of constitutively active forms of Raf-1 and calcineurin synergistically induced kappaB-dependent reporter activity, suggesting a physiologically relevant functional interaction between the kinase and the phosphatase.
authors.affiliation: Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: T
authors.initials: T
authors.lastname: Kanno
authors.forename: U
authors.initials: U
authors.lastname: Siebenlist
mesh_terms: D019703:Calcineurin; D002118:Calcium / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology; D002148:Calmodulin-Binding Proteins / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000502:physiology*; D014407:Tumor Cells, Cultured
chemical_list: D002148:Calmodulin-Binding Proteins; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D011948:Receptors, Antigen, T-Cell; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002118:Calcium