Abstract
CD45 is a transmembrane protein tyrosine phosphatase playing an essential role during T-cell activation. This function relates to the ability of CD45 to regulate p56(lck), a cytoplasmic protein tyrosine kinase necessary for T-cell antigen receptor (TCR) signaling. Previous studies have demonstrated that CD45 is constitutively associated in T-lymphocytes with a transmembrane molecule termed CD45-AP (or lymphocyte phosphatase-associated phosphoprotein). Even though the exact role of this polypeptide is unclear, recent analyses of mice lacking CD45-AP have indicated that its expression is also required for optimal T-cell activation. Herein, we wished to understand better the function of CD45-AP. The results of our studies showed that in T-cells, CD45-AP is part of a multimolecular complex that includes not only CD45, but also TCR, the CD4 and CD8 coreceptors, and p56(lck). The association of CD45-AP with TCR, CD4, and CD8 seemed to occur via the shared ability of these molecules to bind CD45. However, binding of CD45-AP to p56(lck) could take place in the absence of other lymphoid-specific components, suggesting that it can be direct. Structure-function analyses demonstrated that such an interaction was mediated by an acidic segment in the cytoplasmic region of CD45-AP and by the kinase domain of p56(lck). Interestingly, the ability of CD45-AP to interact with Lck in the absence of other lymphoid-specific molecules was proportional to the degree of catalytic activation of p56(lck). Together, these findings suggest that CD45-AP is an adaptor molecule involved in orchestrating interactions among components of the antigen receptor signaling machinery. Moreover, they raise the possibility that one of the functions of CD45-AP is to recognize activated Lck molecules and bring them into the vicinity of CD45.
Highlights
G Supported in part by a studentship from Canderel. h Recipient of a fellowship from the Medical Research Council of Canada. i Holder of a Steve Fonyo studentship from the National Cancer Institute of Canada
As reported earlier [23, 24], large amounts of CD45-associated protein (CD45-AP) were found to coimmunoprecipitate with CD45.Unexpectedly, though, we observed that appreciable quantities of CD45-AP were present in immunoprecipitates of Lck, CD4, CD8, and T-cell antigen receptor (TCR)-associated CD3
We have examined the ability of CD45-AP to interact with various constituents of the antigen receptor signaling machinery in T-lymphocytes
Summary
The Src-related enzymes Lck and FynT initiate TCR-mediated signals by phosphorylating a signaling motif in the cytoplasmic domain of CD3 and termed ITAM (for immunoreceptor tyrosine-based activation motif) Following this phosphorylation, the Syk/Zap-70 family kinases are activated through binding of their tandem Src homology 2 (SH2) domains to doubly phosphorylated ITAMs. Together with Src family kinases, Zap-70 and Syk are responsible for subsequent tyrosine phosphorylation of several signal transduction molecules including phospholipase C-␥1, Cbl, Vav, Slp-76, and Lat. CD45 is a 180 –220-kDa transmembrane protein tyrosine phosphatase expressed on all nucleated hemopoietic cells [5, 6]. Previous studies have shown that CD45 is necessary for T-cell activation because of its ability to promote constitutive dephosphorylation of the inhibitory carboxyl-terminal tyrosine of p56lck, tyrosine 505 [7,8,9] This dephosphorylation is necessary for Lck to become activated and phosphorylate the TCR complex upon antigen stimulation. CD45-AP was reported to associate directly with Lck in transfected HeLa cells [25]; the affinity of this association was apparently low because it could be detected only through in vitro kinase reactions and not by Western blotting
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