Structural Studies of a T cell Specific Tyrosine Kinase

描述（由申请人提供）：我们在免疫蛋白激酶白细胞介素-2酪氨酸激酶（Itk）中发现了一种新的脯氨酸驱动的构象转换。脯氨酸顺式/反式异构化的Itk Src同源2（SH 2）结构域内介导的一致性特异性识别Itk信号的合作伙伴和一个重要的监管与无处不在的肽基脯氨酰异构酶亲环素A（CypA）的相互作用。该提案的重点是明确定义脯氨酸异构化在Itk调节装置中的作用。我们在结构和功能层面与Itk和CypA合作的丰富经验使我们处于剖析Itk监管机制的理想位置。脯氨酸异构化是最容易通过NMR光谱研究的内在构象交换事件。因此，我们的方法是锚定在结构研究，提供必要的原子水平的信息，以充分检查这些细胞内信号蛋白在T细胞中的功能。我们的结构见解已经在小鼠模型系统中进行了测试，提供了进一步的证据表明Itk调控与CypA靶向的单个脯氨酸残基有关。此外，Itk内的脯氨酸异构化事件可能代表另一种形式的翻译后“修饰”，类似于蛋白质磷酸化。像磷酸化的氨基酸侧链，关键脯氨酸残基可以控制配体结合，并作为识别位点的监管伙伴，如肽基脯氨酰异构酶。我们对Itk和CypA的详细分析将为识别控制免疫细胞信号传导的蛋白质中的其他脯氨酸开关奠定基础。

DESCRIPTION (provided by applicant): We have discovered a novel proline-driven conformational switch in the immunological protein kinase Interleukin-2 tyrosine kinase (Itk). Proline cis/trans isomerization within the Itk Src homology 2 (SH2) domain mediates conformer-specific recognition of Itk signaling partners and an important regulatory interaction with the ubiquitous peptidyl-prolyl isomerase cyclophilin A (CypA). This proposal is focused on clearly defining the role of proline isomerization in the Itk regulatory apparatus. Our extensive experience in working with Itk and CypA both at the structural and functional level places us in an ideal position to dissect the Itk regulatory mechanism. Proline isomerization is an intrinsic conformational exchange event that is most easily studied by NMR spectroscopy. Thus, our approach is anchored in structural studies that provide the indispensable atomic-level information required to fully examine the functions of these intracellular signaling proteins in T cells. Our structural insights have already been tested in mouse model systems providing further evidence that Itk regulation is linked to a single proline residue that is the target of CypA. Moreover, the proline isomerization event within Itk may represent another form of posttranslational 'modification' akin to protein phosphorylation. Like phosphorylated amino acid side chains, critical proline residues could control ligand binding and serve as recognition sites for regulatory partners such as the peptidyl-prolyl isomerases. Our detailed analysis of Itk and CypA will lay the groundwork for identification of other proline switches within the proteins that control immune cell signaling.