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Molecular analysis of the domains in CD45 that affect expression and function

University of British Columbia

The aim of this thesis was to further understand the molecular function of CD45, a transmembrane glycoprotein that has intrinsic tyrosine phosphatase activity. Expression of two isoforms of CD45, CD45RABC and CD45RO, and two mutated forms of CD45, one lacking protein tyrosine phosphatase (FTP) activity, and one lacking the cytoplasmic domain, were analyzed in a fibroblast cell line, L tk-. It was observed that only a proportion (10-30%) of CD45 expressed in all transfected cells was expressed on the surface. Deletion of the cytoplasmic tail of CD45 resulted in the expression of two CD45 proteins of 125 kDa and 160 kDa. From pulse-chase experiments, it was determined that the higher molecular weight form was derived from the lower molecular weight form. Mutational analysis of the of the cytoplasmic domain of CD45 indicated that a conserved glutamine in the second PTP domain of CD45, expressed in E. coli, resulted in the loss of CD45 PTP activity. This demonstrates that a mutation in domain II can affect FTP activity thought to reside in domain I. In order to determine the effect of mutations in CD45 on T cell signalling, it was first necessary to characterize a CD45-deficient T cell line. The BW5147 CD45-negative cell line was characterized with respect to p59fyn, a potential in vivo substrate for CD45. It was determined that the expression of CD45 resulted in the reduced tyrosine phosphorylation of p59fyn, yet CD45 had no appreciable effect on the in vitro kinase activity of p59fyn. However, a 120/130 kDa phosphoprotein was identified only in p59fyn immunoprecipitates from CD45-positive cells after an in vitro kinase assay and this occurred independent of T cell receptor mediated stimulation. These results implicate CD45 in regulating the associations of p59fyn in addition to regulating its tyrosine phosphorylation state.

Thesis/Dissertation

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2009-03-03

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http://hdl.handle.net/2429/5430

Microbiology and Immunology

University of British Columbia

UBCV

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