Joseph Raji E, Min Lie, Xu Ruo, Musselman Eli D, Andreotti Amy H
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Biochemistry. 2007 May 8;46(18):5595-603. doi: 10.1021/bi700127c. Epub 2007 Apr 17.
During T cell signaling, Itk selectively phosphorylates a tyrosine within its own SH3 domain and a tyrosine within PLCgamma1. We find that the remote SH2 domain in each of these substrates is required to achieve efficient tyrosine phosphorylation by Itk and extend this observation to two other Tec family kinases, Btk and Tec. Additionally, we detect a stable interaction between the substrate SH2 domains and the kinase domain of Itk and find that addition of specific, exogenous SH2 domains to the in vitro kinase assay competes directly with substrate phosphorylation. On the basis of these results, we show that the kinetic parameters of a generic peptide substrate of Itk are significantly improved via fusion of the peptide substrate to the SH2 domain of PLCgamma1. This work is the first characterization of a substrate docking mechanism for the Tec kinases and provides evidence of a novel, phosphotyrosine-independent regulatory role for the ubiquitous SH2 domain.
在T细胞信号传导过程中，Itk选择性地磷酸化其自身SH3结构域内的一个酪氨酸以及PLCγ1内的一个酪氨酸。我们发现，这些底物中的每个远程SH2结构域都是Itk实现高效酪氨酸磷酸化所必需的，并将这一观察结果扩展到另外两个Tec家族激酶Btk和Tec。此外，我们检测到底物SH2结构域与Itk的激酶结构域之间存在稳定的相互作用，并发现向体外激酶测定中添加特定的外源SH2结构域会直接与底物磷酸化竞争。基于这些结果，我们表明，通过将肽底物与PLCγ1的SH2结构域融合，Itk的通用肽底物的动力学参数得到了显著改善。这项工作首次对Tec激酶的底物对接机制进行了表征，并为普遍存在的SH2结构域的一种新的、不依赖磷酸酪氨酸的调节作用提供了证据。
