| Summary: | 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.
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