title: Demonstration of protein tyrosine phosphatase activity in the second of two homologous domains of CD45.
abstract: It has been reported that alteration of deletion of critical residues within one of the two homologous protein tyrosine phosphatase (PTPase)-like domains of CD45 completely abolishes all activity, suggesting that only the more N-terminal domain is catalytically active. However, we now demonstrate, by two independent techniques, that the second (C-terminal) domain is also a viable phosphatase. Limited proteolysis by endoproteinase Lys-C or trypsin increased the phosphatase activity toward reduced, carboxymethylated, and maleylated lysozyme approximately 8-fold. A 50-kDa fragment, isolated by ion exchange chromatography, was found to be responsible for this activity. N-terminal sequencing revealed that this fragment includes less than half of the first phosphatase domain and most, if not all, of the second. In a second experiment, 109 residues, including the presumed catalytic region, were removed from domain I by site-directed mutagenesis. Expression of this construct in a mammalian cell line resulted in increased PTPase activity over nontransfected control cells. Isolation of the recombinant CD45 by immunoprecipitation and immunoaffinity chromatography revealed that it had phosphatase activity. Both of these experimental approaches demonstrate that the second conserved PTPase domain of CD45 is a functioning PTPase, but that external regulation may be required to express its activity in the context of the native molecule.
authors: Department of Biochemistry, University of Washington, Seattle 98195.
authors: X
authors: X
authors: Tan
authors: D R
authors: DR
authors: Stover
authors: K A
authors: KA
authors: Walsh
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D017493:Leukocyte Common Antigens / Q000737:chemistry / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000201:enzymology
chemical_list: D017493:Leukocyte Common Antigens; D017027:Protein Tyrosine Phosphatases