Demonstration of a direct interaction between p56(lck) and the cytoplasmic domain of CD45 in vitro

p56(lck) is a potential in vivo substrate for the tyrosine-specific phosphatase, CD45. In this study, recombinant purified p56(lck) 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 of. 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 p56(lck) (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 p56(lck), 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 p56(lck) to CD45, and when added together worked synergistically to compete for p56(lck) 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. p56(lck) is a potential in vivo substrate for the tyrosine-specific phosphatase, CD45. In this study, recombinant purified p56(lck) 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 p56(lck) (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 p56(lck), 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 p56(lck) to CD45, and when added together worked synergistically to compete for p56(lck) 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,