Ng D H, Watts J D, Aebersold R, Johnson P
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
J Biol Chem. 1996 Jan 19;271(3):1295-300. doi: 10.1074/jbc.271.3.1295.
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.
p56lck是酪氨酸特异性磷酸酶CD45在体内的潜在底物。在本研究中，发现重组纯化的p56lck与重组CD45胞质结构域蛋白特异性结合，但不与另一种相关酪氨酸磷酸酶——受体蛋白酪氨酸磷酸酶α的胞质结构域结合。在平衡结合条件下，这种结合是可饱和的，且以1:1的摩尔化学计量比发生。仅包含p56lck氨基末端区域（第34 - 150位氨基酸残基）的融合蛋白也与重组CD45结合，对该区域的进一步分析表明，p56lck独特的氨基末端区域和SH2结构域的谷胱甘肽S - 转移酶融合蛋白能与重组CD45结合，而SH3结构域则不能。SH2结构域蛋白比氨基末端区域具有更高的亲和力，但二者都能竞争p56lck与CD45的结合，并且当一起添加时能协同竞争p56lck的结合。SH2结构域与CD45的相互作用具有特异性，因为来自磷脂酰肌醇3激酶p85α亚基和SHC的谷胱甘肽S - 转移酶 - SH2融合蛋白不与CD45结合。此外，这种相互作用在CD45上没有任何可检测到的酪氨酸磷酸化的情况下发生，提示存在一种非传统的SH2结构域相互作用。
