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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI054974-03
Application #
7076154
Study Section
Experimental Immunology Study Section (EI)
Program Officer
Mallia, Conrad M
Project Start
2004-07-01
Project End
2009-06-30
Budget Start
2006-07-01
Budget End
2007-06-30
Support Year
3
Fiscal Year
2006
Total Cost
$348,245
Indirect Cost
Name
University of Pennsylvania
Department
Pathology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Shapiro, Michael J; Spruce, Lynn; Sundsbak, Rhianna et al. (2010) Phosphorylation at serine 318 is not required for inhibition of T cell activation by ALX. Biochem Biophys Res Commun 396:994-8
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