How signaling through the T cell receptor (TCR) can lead to either differentiation (positive selection) or apoptosis (negative selection) is a major unanswered question in Immunology. Separate intracellular pathways of either selection process have been identified but how the TCR signals lead to these differential pathways is not clear. Positive selection involves the ras/raf yields MEK1 yields Erk1/2 pathway. Conversely, several proteins have been implicated solely in negative selection. These include the Nur77 transcription factor family members (Nur77, Nor-1), CD30, a member of the tumor necrosis factor receptor family and p38 and jnk MAP kinases. Nur77/Nor-1 transcription is induced following TCR stimulation. Using dominant negative and gain of function mutations, Nur77 family members were shown to be important in the process of TCR- mediated apoptosis. Some of the upstream regulators of Nur77 transcription were identified. These include the MEF2A/D transcription factors that regulate Nur77 promoter and Erk5, a newly identified MAP kinase, which function is poorly characterized. Erk5 interacts with MEF2D through its unique C-terminal domain. Interestingly, this region also contains a transcription activation activity that is regulated by its own kinase. Thus, Erk5 yields MEF2 yields Nur77/Nor-1 is a novel signal transduction pathway leading to apoptosis in T cells. In this proposal, several approaches are proposed to study this exciting and novel regulation in TCR signaling pathway.
In aim 1, the function of Nor-1 in the immune system will be studied by generating Nor-l-deficient mice. These mice will be bred to Nur77-/- mice to generate Nor-1/Nur77 double mutant mice. The role of Nor-1 in negative selection and other apoptotic pathways will be assessed.
In aim 2, Erk5 biochemical characterization will be continued. Its autophosphorylation sites will be identified and their function in transactivation will be determined using mutagenesis. The effect of Erk5 gain of function mutation and Erk5 dominant negative mutant on apoptosis and Nur77 activation will be examined. The molecular mechanism of differential requirement for Erk5 mediated MEF2 activation between T cells and non-T cells will be studied.
In aim 3 : The in vivo function of Erk5 in apoptosis will be assessed in transgenic mice harboring Erk5 constitutive activating mutant and Erk5 dominant negative mutant.
In aim 4, the function of Erk5 in the immune system will be studied by generating and characterizing Erk5-deficient mice.
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