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Functional and enzymatic characterization of the receptor protein tyrosine phosphatase, CD45

University of British Columbia

CD45 is expressed exclusively on hematopoietic cells and is a major component of the lymphocyte cell surface. CD45 is required both in the development and in the antigen induced activation of lymphoid cells. Despite the demonstration of several in vitro substrates, only the src family kinases, p56lck and p59fyn have been clearly identified as potential in vivo subtrates. How the substrate specificity of CD45 is achieved in vivo is not known. CD45 isolated directly from cells and recombinant cytoplasmic CD45 did not discriminate between a variety of src-family phosphopeptides - indicating no substrate specificity at the peptide level. However, a direct interaction between p56lck and cytoplasmic CD45 was demonstrated in vitro. Several regions of p56lck could mediate this interaction, which was further modulated by the tyrosine phosphorylation state of p56lck and by the protein tyrosine phosphatase (PTP) activity of CD45. Overall, results demonstrated a complex interaction that provides insight into the enzyme/substrate relationship. To evaluate the substrate specificity and function of CD45 under cellular conditions, another receptor PTP, RPTPα was expressed in a CD45⁻, T cell receptor (TCR)+, BW5147 T lymphoma cell for comparative studies. Unlike CD45, high levels of RPTPα did not fully restore either proximal or distal TCR mediated signaling events. Furthermore, immunoprecipitated RPTPα was approximately one-seventh to one-tenth as active as CD45 when tested against artificial substrates. This difference in activity was also observed using recombinant enzymes. Therefore, CD45 is intrinsically a more efficient phosphatase, providing one reason why RPTPα is not able to substitute for CD45. This work reveals possible mechanisms that may account for CD45 substrate specificity under cellular environments. Ultimately, this information should assist in both the understanding of lymphoid cell biology and in the assessment of experimental and clinical targets towards modulation of lymphoid cell function.

Thesis/Dissertation

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2009-06-02

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http://hdl.handle.net/2429/8579

Microbiology and Immunology

University of British Columbia

UBCV

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