Characterization of the C-terminal protein tyrosine phosphatase-like domain of CD45

• Main Author: Felberg, Jackie
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/12717

CD45 is a transmembrane two-domain protein tyrosine phosphatase (PTP) essential for thymocyte development and B cell maturation. Its tyrosine phosphatase activity is required for efficient signal transduction initiated by lymphocyte antigen receptors. As with most transmembrane two-domain phosphatases, the role of the second, C-terminal domain, D2, is unclear. To investigate the role of D2 and to determine if it plays a role in regulating CD45 activity, substrate specificity, or molecular interactions, recombinant proteins containing the first, N-terminal PTP domain, D1, without D2, and D2 without D1, were expressed and purified from E. coli and analyzed. It was found that a purified D1 recombinant protein dimerized, and was enzymatically active in the absence of PTP-D2, whereas no phosphatase activity was detected for D2 which was a monomer in solution. D2 enhanced activity of Dl in the monomeric full length CD45 cytoplasmic domain protein (D1/D2), and this was shown to be specific to a PTP domain, as the Lck SH2 domain could not substitute for D2. Specific binding of the recombinant 6His-Dl to a recombinant GST-D2 protein, and the association of N - and C- terminal tryptic fragments of 6His-Dl/D2 provided evidence for an intramolecular interaction occurring in the CD45 cytoplasmic domain. Co-purification of 6His-Dl and GST-D2 from E. coli indicated that this is a stable interaction resulting in increased stability of Dl. This interaction did not require the acidic region unique to D2. It was, however, affected by a destabilizing point mutation, Q1180G, in D2. No evidence was obtained for a role for D2 in determining substrate specificity using a panel of peptide substrates or between full length Src family kinase protein substrates. However in in vitro binding assays, 6His-D2 preferentially bound to its proposed physiological substrate, the Src family kinase p56lck, expressed as a GST fusion protein, compared to a related, non-Src family tyrosine kinase, GST-Csk. Using in vitro binding assays, it was determined that CD45-D2 bound to the kinase domain of Lck, and this interaction did not require the acidic region in D2. The interaction was independent of the tyrosine phosphorylation state of Lck, and 6His-D2 did not bind phosphotyrosine.