Investigating the function and stability of CD45-associated protein, a protein implicated in T cell activation

Recently, a novel lymphocyte-specific transmembrane protein, CD45 associated protein or CD45ap, was identified due to its constitutive association with CD45, a transmembrane protein tyrosine phosphatase essential to the activation of T cells. Although little is known about the role of CD45ap in T cell activation, its potential importance in the T cell response has been demonstrated by the fact that the expression of CD45ap is lymphocyte specific rather than being present in all cells that express CD45 and that CD45ap-null mice have been shown to have reduced T cell proliferation upon stimulation. It was also shown that CD45ap could associate with kinases essential to the T cell response such as Lck and ZAP-70, stimulating suggestions that CD45ap may function as an adaptor molecule. Expression of CD45ap as a bacterial fusion protein yielded a highly degraded and unstable protein suggesting that the mechanism responsible for CD45ap degradation in eukaryotic cells may be conserved in E. coli. Subsequent deletion analysis demonstrated that a 40 amino acid residue deletion from the C-terminus of CD45ap could stabilize its expression. However, the same 40 amino acid residue deletion from CD45ap did not alter the half life of the molecule in eukaryotic L fibroblast cells. The stable recombinant form of CD45ap was shown in vitro to outcompete CD45 for binding to Lck. This correlates with other in vitro findings that demonstrated CD45ap significantly decreased the rate at which CD45 could dephosphorylate the F505 Lck mutant (believed to be constitutively active) but only slightly decreased the rate at which CD45 could dephosphorylate the F394 Lck mutant (believed to be constitutively inactive). This suggests that CD45ap can serve as an effective modulator of the CD45- Lck interaction as well as a promoter of T cell activation by its ability to favour the CD45-mediated activation rather than deactivation of Lck. In further support of CD45ap serving as a promoter of T cell activation, BW 5147 T cells overexpressing CD45ap demonstrated prolonged and increased phosphorylation of various T cell proteins upon CD3 stimulation. In addition, the WW domain of CD45ap was also shown to interact with a 50 kDa tyrosine phosphorylated protein providing another possible route through which CD45ap may promote T cell activation. Narrowing the potential roles that CD45ap may play in T cell signaling, CD45ap was found not to affect that rate of CD45 transport to the cell surface or the turnover rate of CD45 in L cells. However, CD45ap expression in L cells resulted in increased total but not cell surface levels of CD45, implicating CD45ap in the maintenance of subcellular pools of CD45.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat