Demonstration that the leukocyte common antigen CD45 is a protein tyrosine phosphatase.
It has been proposed on the basis of amino acid sequence homology that the leukocyte common antigen CD45 represents a family of catalytically active, receptor-linked protein tyrosine phosphatases [Charbonneau, H., Tonks, N. K., Walsh, K. A., & Fischer, E. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7182-7186]. The present study confirms that CD45 possesses intrinsic protein tyrosine phosphatase (PTPase) activity. First, a mouse monoclonal antibody to CD45 (mAb 9.4) specifically eliminated, by precipitation, PTPase activity from a high Mr fraction containing CD45, prepared by gel filtration (Sephacryl S200) of a Triton X-100 extract of human spleen. Second, PTPase activity was demonstrated in a highly purified preparation of CD45 that was eluted with a high pH buffer from an affinity column, constructed from the same antibody. Third, on sucrose density gradient centrifugation, PTPase activity was only found in those fractions that contained CD45 as determined by Western analysis. When CD45 was caused to aggregate, first by reacting it with mAb 9.4 and then adding a secondary, cross-linking anti-mouse mAb, the PTPase activity shifted to the same higher Mr fractions that contained CD45. No shift in CD45 or PTPase was observed following addition of a control IgG2a. On this basis, it is concluded that CD45 is a protein tyrosine phosphatase.
Tonks
N K
NK
Department of Biochemistry, University of Washington, Seattle 98195.
Charbonneau
H
H
Diltz
C D
CD
Fischer
E H
EH
Walsh
K A
KA
D000943:Antigens, Differentiation / Q000302:isolation & purification*; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology; D002499:Centrifugation, Density Gradient / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006649:Histocompatibility Antigens / Q000302:isolation & purification*; D006801:Humans; D017493:Leukocyte Common Antigens; D008562:Membrane Glycoproteins / Q000302:isolation & purification*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification*; D017027:Protein Tyrosine Phosphatases; D013154:Spleen / Q000201:enzymology* / Q000276:immunology
D000943:Antigens, Differentiation; D006649:Histocompatibility Antigens; D008562:Membrane Glycoproteins; D010749:Phosphoprotein Phosphatases; D017493:Leukocyte Common Antigens; D017027:Protein Tyrosine Phosphatases