Abstract
The CD45 protein tyrosine phosphatase (PTPase) has been shown to regulate the activity of Lck and Fyn protein tyrosine kinases in T cells. However, it is not clear that these constitute the only CD45 substrates. Moreover, the manner by which PTPase activity and substrate recruitment are regulated, is poorly understood. Previous in vitro studies suggest that the first cytoplasmic PTPase domain (D1) of CD45 is the active PTPase, which may be regulated by an enzymatically inactive second PTPase domain (D2). However, the function of CD45 D2 in vivo is unknown. In this study, reconstitution of CD45(-) T cells with specific CD45 PTPase mutants allowed demonstration of a critical role for D2 in TCR-mediated interleukin (IL)-2 production. Specifically, replacement of CD45 D2 with that of the LAR PTPase to form a CD45/LAR:D2 chimera, abrogates CD45-dependent IL-2 production. This effect cannot be accounted for by loss of PTPase activity per se. The expression of D1 substrate-trapping mutants reveals an in vivo interaction between CD45 and TCR-zeta that is dependent on CD45 D2. Thus, cells expressing CD45 lacking D2 exhibit abnormal TCR-mediated signaling characterized by hyperphosphorylation of zeta and deficient ZAP-70 phosphorylation. These data suggest an essential role for CD45 D2 in TCR-regulated IL-2 production through substrate recruitment of the zeta chain.
Highlights
Analysis demonstrated that CD45 can down-regulate Fyn and Lck activity by dephosphorylating their positive regulatory autophosphorylation sites [9, 10]
To establish a regulatory role for domain 2 (D2) among transmembrane protein tyrosine phosphatase (PTPase) and identify in vivo substrates critical to our understanding of CD45 function, we address the physiologic role of CD45 D2 and substrate recruitment of in TCR-mediated signal transduction
Similar results were obtained using various anti-CD3 concentrations. These results suggest that CD45 D2 plays an important regulatory role in CD45 function and this is not explained by loss of D2 PTPase activity per se
Summary
Analysis demonstrated that CD45 can down-regulate Fyn and Lck activity by dephosphorylating their positive regulatory autophosphorylation sites [9, 10]. CD45Ϫ J-AS Jurkat cells were stably transfected to express either wild-type (wt) CD45, or PTPase mutations in the context of the smallest (CD45RO or CD45(0)) extracellular domain. Expression of chimeric proteins containing active CD45 D1 (in the presence of wt or inactive D2) in CD45Ϫ cells was necessary and sufficient to reconstitute TCR-mediated tyrosine phosphorylation of cellular proteins and dephosphorylate Lck at its carboxyl-terminal regulatory site [34, 36].
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