Bruyns E, Kirchgessner H, Meuer S, Schraven B
Institute of Immunology, Immunomodulation Laboratory, Ruprecht-Karls University of Heidelberg, Germany.
Int Immunol. 1998 Feb;10(2):185-94. doi: 10.1093/intimm/10.2.185.
In human and mouse lymphocytes the protein tyrosine phosphatase CD45, a key molecule involved in T cell activation, non-covalently associates with the tyrosine kinase p56(lck) and lymphocyte phosphatase-associated phosphoprotein (LPAP), a 32 kDa phosphoprotein of unknown function. In order to gain insight into the function of LPAP we have generated an LPAP-deficient Jurkat variant by means of antisense strategies. Analysis of the CD45-p56(lck) molecular complex in this cell line revealed that loss of LPAP does not alter the expression or the enzymatic activity of CD45 or p56(lck). In addition, the association between CD45 and p56(lck) is not affected in LPAP-deficient T cells. These data suggest that LPAP does not regulate the enzymatic activity of CD45 or p56(lck) and is not required for the association between these two proteins. In order to identify polypeptides that preferentially associate with LPAP we established a Jurkat variant expressing a chimeric receptor which was composed of the extracellular portion of the human HLA-A2.1 molecule and the full-length LPAP protein. Comparative two-dimensional analysis of CD45 and HLA-A2 immunoprecipitates obtained from these cells following metabolic labeling resulted in the identification of a 43 kDa protein that preferentially associates with LPAP under mild detergent conditions.
在人和小鼠淋巴细胞中，蛋白酪氨酸磷酸酶CD45是参与T细胞活化的关键分子，它与酪氨酸激酶p56(lck)和淋巴细胞磷酸酶相关磷蛋白(LPAP，一种功能未知的32 kDa磷蛋白)非共价结合。为了深入了解LPAP的功能，我们通过反义策略构建了LPAP缺陷的Jurkat变异体。对该细胞系中CD45-p56(lck)分子复合物的分析表明，LPAP的缺失不会改变CD45或p56(lck)的表达或酶活性。此外，在LPAP缺陷的T细胞中，CD45与p56(lck)之间的结合不受影响。这些数据表明，LPAP不调节CD45或p56(lck)的酶活性，且这两种蛋白之间的结合不需要LPAP。为了鉴定与LPAP优先结合的多肽，我们构建了一个表达嵌合受体的Jurkat变异体，该嵌合受体由人HLA-A2.1分子的细胞外部分和全长LPAP蛋白组成。对代谢标记后从这些细胞中获得的CD45和HLA-A2免疫沉淀复合物进行二维比较分析，结果鉴定出一种43 kDa的蛋白，在温和的去污剂条件下，它优先与LPAP结合。
