Identification of Itk/Tsk Src homology 3 domain ligands
Identification of Itk/Tsk Src homology 3 domain ligands
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DOI：
10.1074/jbc.271.41.25646
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发表时间：
1996-10-11
影响因子：
4.8
通讯作者：
Berg, LJ
中科院分区：
文献类型：
--
作者：
Bunnell, SC;Henry, PA;Berg, LJ
酪氨酸激酶Itk/Tsk是Btk的T细胞特异性类似物，Btk是人类免疫缺陷x连锁无球蛋白血症和xid小鼠中存在缺陷的酪氨酸激酶。来自itk缺陷小鼠的T淋巴细胞对通过T细胞受体（TCR）传递的有丝分裂刺激难以耐受。为了深入了解Itk的生化作用，我们研究了Itk SH3结构域的结合特性。通过筛选偏置噬菌体展示文库，获得最佳Itk SH3结合基序；基于该基序的肽以高亲和力和选择性结合到Itk SH3结构域。对T细胞裂解物的初步研究表明，Itk SH3结构域与tcr刺激的jurkat细胞中的Cbl、Fyn和其他酪氨酸磷酸化蛋白结合。在洗涤剂强度增加的条件下，Sam 68、Wiskott-Aldrich综合征蛋白和hnRNP-K结合到Itk SH3结构域，但Cbl和Fyn没有结合。通过检测不同SH3结构域与Sam 68和WASP缺失变体相互作用的能力，我们证明了Src家族激酶的Itk-SH3结构域和SH3结构域与Sam 68和WASP中重叠但不同的富含脯氨酸的区域结合。
The tyrosine kinase Itk/Tsk is a T cell specific analog of Btk, the tyrosine kinase defective in the human immunodeficiency X-Linked agammaglobulinemia and in xid mice. T lymphocytes from Itk-deficient mice are refractory to mitogenic stimuli delivered through the T cell receptor (TCR). To gain insights into the biochemical role of Itk, the binding properties of the Itk SH3 domain were examined. An optimal Itk SH3 binding motif was derived by screening biased phage display libraries; peptides based on this motif bound with high affinity and selectivity to the Itk SH3 domain. Initial studies with T cell lysates indicated that the Itk SH3 domain bound Cbl, Fyn, and other tyrosine phosphoproteins from TCR-stimulated jurkat cells. Under conditions of increased detergent stringency Sam 68, Wiskott-Aldrich Syndrome protein, and hnRNP-K, but not Cbl and Fyn, were bound to the Itk SH3 domain. By examining the ability of different SH3 domains to interact with deletion variants of Sam 68 and WASP, we demonstrated that the Itk-SH3 domain and the SH3 domains of Src family kinases bind to overlapping but distinct sets of proline-rich regions in Sam 68 and WASP.