Demonstration of a direct interaction between p56lck and the cytoplasmic domain of CD45 in vitro.
p56lck is a potential in vivo substrate for the tyrosine-specific phosphatase, CD45. In this study, recombinant purified p56lck was found to specifically associate with recombinant CD45 cytoplasmic domain protein, but not to the cytoplasmic domain of another related tyrosine phosphatase, receptor protein-tyrosine phosphatase alpha. Under equilibrium binding conditions, the binding was saturable and occurred at a 1:1 molar stoichiometry. A fusion protein containing only the amino-terminal region of p56lck (residues 34-150) also bound to recombinant CD45, and further analysis of this region indicated that glutathione S-transferase fusion proteins of the unique amino-terminal region and the SH2 domain, but not the SH3 domain of p56lck, bound to recombinant CD45. The SH2 domain protein bound with a higher affinity than the amino-terminal region, but both were able to compete for the binding of p56lck to CD45, and when added together worked synergistically to compete for p56lck binding. The SH2 domain interaction with CD45 was specific as glutathione S-transferase-SH2 fusion proteins from p85 alpha subunit of phosphatidylinositol 3-kinase and SHC did not bind to CD45. In addition, this interaction occurred in the absence of any detectable tyrosine phosphorylation on CD45, suggesting a nonconventional SH2 domain interaction.
Ng
D H
DH
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
Watts
J D
JD
Aebersold
R
R
Johnson
P
P
D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D017493:Leukocyte Common Antigens / Q000302:isolation & purification / Q000378:metabolism*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D009419:Nerve Tissue Proteins / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D019061:src-Family Kinases / Q000302:isolation & purification / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases; D017493:Leukocyte Common Antigens; C516850:PTPRG protein, human; D017027:Protein Tyrosine Phosphatases; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5