Felberg Jackie, Lefebvre Dennis C, Lam Mindy, Wang Yanni, Ng David H W, Birkenhead Darlene, Cross Jennifer L, Johnson Pauline
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
J Biol Chem. 2004 Jan 30;279(5):3455-62. doi: 10.1074/jbc.M309537200. Epub 2003 Nov 17.
CD45 is a transmembrane, two-domain protein-tyrosine phosphatase expressed exclusively in nucleated hematopoietic cells. The Src family kinase, Lck, is a major CD45 substrate in T cells and CD45 dephosphorylation of Lck is important for both T cell development and activation. However, how the substrate specificity of phosphatases such as CD45 is achieved is not well understood. Analysis of the interaction between the cytoplasmic domain of CD45 and its substrate, Lck, revealed that the active, membrane-proximal phosphatase domain of CD45 (CD45-D1) bound to the phosphorylated Lck kinase domain, the SH2 domain, and the unique N-terminal region of Lck. The second, inactive phosphatase domain (CD45-D2) bound only to the kinase domain of Lck. CD45-D2 was unable to bind phosphotyrosine, and its interaction with the kinase domain of Lck was independent of tyrosine phosphorylation. The binding of CD45-D2 was localized to subdomain X (SD10) of Lck. CD45-D2 bound similarly to Src family kinases but bound Csk to a lesser extent and did not bind significantly to the less related kinase, Erk1. CD45 dephosphorylated Lck and Src at similar rates but dephosphorylated Csk and Erk1 at lower rates. Replacement of Erk1 SD10 with that of Lck resulted in the binding of CD45-D2 and the conversion of Erk1 to a more efficient CD45 substrate. This demonstrates a role for CD45-D2 in binding substrate and identifies the SD10 region in Lck as a novel site involved in substrate recognition.
CD45是一种跨膜的双结构域蛋白酪氨酸磷酸酶，仅在有核造血细胞中表达。Src家族激酶Lck是T细胞中主要的CD45底物，Lck的CD45去磷酸化对于T细胞发育和激活都很重要。然而，像CD45这样的磷酸酶的底物特异性是如何实现的，目前还不太清楚。对CD45胞质结构域与其底物Lck之间相互作用的分析表明，CD45的活性、靠近膜的磷酸酶结构域（CD45-D1）与磷酸化的Lck激酶结构域、SH2结构域以及Lck独特的N端区域结合。第二个无活性的磷酸酶结构域（CD45-D2）仅与Lck的激酶结构域结合。CD45-D2无法结合磷酸酪氨酸，其与Lck激酶结构域的相互作用不依赖于酪氨酸磷酸化。CD45-D2的结合定位于Lck的亚结构域X（SD10）。CD45-D2与Src家族激酶的结合方式类似，但与Csk的结合程度较低，与关系较远的激酶Erk1没有明显结合。CD45以相似的速率使Lck和Src去磷酸化，但使Csk和Erk1去磷酸化的速率较低。用Lck的SD10替换Erk1的SD10会导致CD45-D2的结合，并使Erk1转变为更有效的CD45底物。这证明了CD45-D2在结合底物方面的作用，并确定Lck中的SD10区域是参与底物识别的新位点。
