T cell activation is central to initiating an immune response. Two signals are required: an antigen-specific signal through the T cell receptor (TCR) and an antigen-independent costimulatory signal, primarily provided by CD28 in naive T cells. Recent work has focused on therapeutic inhibition of inappropriate T cell activation, as in autoimmune diseases, by blocking CD28 costimulation. We have identified a novel adaptor, ALX, which is related to the adaptor RIBP/TSAd/Lad. RIBP-deficient mice are grossly normal, although T cells are somewhat impaired in IL-2 production and proliferation. Older RIBP-deficient mice have increased rates of autoimmune disease stemming from an inability to down-regulate T cell responses through apoptosis. Perhaps the mild phenotype in the RIBP-deficient mice is due to compensation by a related molecule, which we propose is ALX. Interestingly, overexpression of ALX in Jurkat T cells inhibits TCR/CD28 activation of the IL-2 promoter. In particular, overexpression of ALX in Jurkat T cells inhibits TCR/CD28 activation of the RE/AP composite element, the primary site of CD28 transcriptional upregulation of the IL-2 promoter, while TCR-mediated AP-1 activation is unaffected by ALX overexpression. Therefore, ALX overexpression affects CD28 rather than TCR signaling. Recent data has shown that ALX binds to MEKK2, MEKK3 and MEK5, components of a map kinase pathway leading to activation of Erk5/BMK1. In this proposal, a series of interrelated aims, combining biochemical and genetic approaches, will test the hypothesis that ALX plays an important regulatory role in CD28 signaling leading to IL-2 upregulation during T cell activation through modulation of the MEKK/MEK5/Erk5 pathway.
Specific Aim #1 : Determining the role of ALX in modulating the MEKK/MEKS/Erk5 pathway. We have found that ALX associates with MEKK2, MEKK3 and MEKS. The role of ALX in regulating the MEKK/MEK5/Erk5 pathway during TCR/CD28 stimulation will be analyzed. In addition, the subcellular localization of these proteins during T cell activation will be examined.
Specific Aim #2 : To understand the role of ALX phosphorylation in modulating its function during T cell activation. We have shown that ALX becomes phosphorylated after TCR/CD28 stimulation, most likely on serine. Since ALX associates with the serine/threonine kinases MEKK2, MEKK3 and MEKS, the ability of these kinases to phosphorylate ALX on sites that axe phosphorylated in vivo upon activation of TCR/CD28 will be determined. Through mutation of these sites, the role that ALX phosphorylation plays in regulating RE/AP activation, as well as in the MEK5/Erk5 pathway, will be examined.
Specific Aim #3 : Generation and analysis of ALX-deficient mice. The analysis of CD28 function and T cell activation in ALX-deficient T cells will complement the biochemical approaches in Specific Aims 1 and 2 to define the role of ALX in CD28 signaling, IL-2 activation and the regulation of the MEK5/Erk5 pathway during T cell activation. These mice will be crossed to RIBP-deficient mice, to determine whether the additional deficiency of ALX exacerbates the phenotype.
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| Shapiro, Michael J; Nguyen, Chau T; Aghajanian, Haig et al. (2008) Negative regulation of TCR signaling by linker for activation of X cells via phosphotyrosine-dependent and -independent mechanisms. J Immunol 181:7055-61 |
| Perchonock, Claire E; Pajerowski, Anthony G; Nguyen, Chau et al. (2007) The related adaptors, adaptor in lymphocytes of unknown function X and Rlk/Itk-binding protein, have nonredundant functions in lymphocytes. J Immunol 179:1768-75 |
| Perchonock, Claire E; Fernando, Melissa C; Quinn 3rd, William J et al. (2006) Negative regulation of interleukin-2 and p38 mitogen-activated protein kinase during T-cell activation by the adaptor ALX. Mol Cell Biol 26:6005-15 |
