Singer Andrew L, Bunnell Stephen C, Obstfeld Amrom E, Jordan Martha S, Wu Jennifer N, Myung Peggy S, Samelson Lawrence E, Koretzky Gary A
Signal Transduction Program, Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2004 Apr 9;279(15):15481-90. doi: 10.1074/jbc.M313339200. Epub 2004 Jan 13.
The adaptor protein Src homology (SH)2 domain-containing and leukocyte-specific phosphoprotein of 76 kDa (SLP-76) is critical for signal transduction in multiple hematopoietic lineages. It links proximal and distal T cell receptor signaling events through its function as a molecular scaffold in the assembly of multimolecular signaling complexes. Here we studied the functional roles of sub-domains within the SLP-76 proline-rich region, specifically the Gads binding domain and the recently defined P1 domain. To gain a further understanding of the functions mediated by this region, we used three complementary approaches as follows: reconstitution of SLP-76-deficient cells with functional domain deletion mutants, blocking molecular associations through the expression of a dominant negative protein fragment, and directed localization of SLP-76 to assess the role of the domains in SLP-76 recruitment. We find the Gads binding domain and the P1 domain are both necessary for optimal SLP-76 function, and in the absence of these two regions, SLP-76 is functionally inert. Furthermore, we provide direct evidence that SLP-76 localization and, in turn, function are dependent upon association with Gads.
衔接蛋白含Src同源（SH）2结构域的76 kDa白细胞特异性磷蛋白（SLP-76）对多种造血谱系中的信号转导至关重要。它通过在多分子信号复合物组装中作为分子支架的功能，连接近端和远端T细胞受体信号事件。在此，我们研究了SLP-76富含脯氨酸区域内亚结构域的功能作用，特别是Gads结合结构域和最近定义的P1结构域。为了进一步了解该区域介导的功能，我们采用了以下三种互补方法：用功能结构域缺失突变体重建SLP-76缺陷细胞、通过表达显性负性蛋白片段阻断分子结合，以及对SLP-76进行定向定位以评估这些结构域在SLP-76募集中的作用。我们发现Gads结合结构域和P1结构域对于最佳的SLP-76功能都是必需的，并且在没有这两个区域的情况下，SLP-76在功能上是无活性的。此外，我们提供了直接证据表明SLP-76的定位以及进而其功能依赖于与Gads的结合。
