Regulatory mechanisms of the Crk Adapter protein

描述(由申请人提供)：CRK家族适配器通过将活性酶招募到信号网络中，调节由各种外部和内部刺激引起的蛋白质复合体的及时形成，并有助于信号转导事件的特异性。尽管接头蛋白没有内在的催化或转录活性，但它们的异常行为可能导致致癌转化。我们的长期目标是：(I)提供Crk介导的信号现象的结构基础；(Ii)确定Crk和Abl之间相互作用的分子特征，从而导致Abl的反式激活；(Iii)了解细胞信号调节现象的物理化学基础；以及(Iv)设计能够控制导致癌症的异常信号转导事件的候选药物。这项研究背后的有力假设是，Crk的结合活性受到一种新的自我抑制机制的调节。值得注意的是，亲环素A(CypA)有效地催化了亲环素A(CypA)催化的内源异构化反应，从而进一步控制了自身抑制作用。我们还发现，由c-Abl磷酸化的酪氨酸残基(Y222)组成的P221独立地进行顺式/反式异构化，这也是由CypA催化的。我们的目标是： 1.描述接头-SH3CCrk片段顺式和反式构象的结构和动力学特征，以了解P238位的顺式/反式异构化如何调节接头与SH3C域之间的相互作用。 2.确定分子内SH3N-SH3C相互作用的分子基础，这是自抑制机制的基础。 3.表征全长Crk(c-Crk11)的结构和动力学性质，以了解不同的调控机制如何整合到Crk的生理细胞形式中。 4.体外和体内检测自身抑制和反式/顺式异构化对Crk生物学功能的影响。 在细胞生理功能过程中，Crk蛋白参与了许多信号事件的调节，但它也可能导致肿瘤的发生。在这项提案中，我们将研究调节Crk蛋白活性的机制，即该蛋白是如何“开启”和“关闭”的。我们希望最终将所获得的知识转化为新的策略，合理设计能够抑制癌细胞增殖的候选药物。

DESCRIPTION (provided by applicant): Crk-family adaptors mediate the timely formation of protein complexes elicited by a variety of external and intrinsic stimuli and contribute to the specificity of signal transduction events by recruiting active enzymes into signaling networks. Despite the fact that adaptor proteins have no intrinsic catalytic or transcriptional activity, their aberrant behavior may lead to oncogenic transformation. Our long-term objectives are to (i) provide the structural basis of Crk-mediated signaling phenomena; (ii) determine the molecular features of the interaction between Crk and Abl, which results in Abl trans-activation; (iii) understand the physicochemical basis of regulatory phenomena in cell signaling; and (iv) design drug candidates that could control aberrant signal transduction events leading to cancer. The strong hypothesis behind the proposed research is that the binding activity of Crk is regulated by a novel autoinhibitory mechanism. Notably, autoinhibition is further controlled by an intrinsic switch afforded by prolyl cis-trans isomerization whose kinetics is effectively catalyzed by cyclophilin A (CypA). We have additionally identified that P221, which lies by the tyrosine residue (Y222) that becomes phosphorylated by c-Abl, undergoes independently cis/trans isomerization that is also catalyzed by CypA. We aim to: 1. Delineate the structural and dynamic features of both the cis and trans conformers of the linker-SH3C Crk fragment, to understand how cis/trans isomerization at P238 regulates the interaction between the linker and the SH3C domain. 2. Determine the molecular basis of the intramolecular SH3N-SH3C interaction, which forms the basis of an autoinhibitory mechanism. 3. Characterize the structural and dynamic properties of full-length Crk (c-Crkll), to understand how distinct regulatory mechanisms integrate in the context of the physiological cellular form of Crk. 4. Determine the effect of the autoinhibition and proline cis/trans isomerization on the biological function of Crk, both in vitro and in vivo. Crk proteins are involved in mediating many signaling events during the physiological cellular function, but it may also result in tumorigenesis. In this proposal, we will investigate the mechanisms that regulate the activity of the Crk protein, that is, how the protein is switched "on" and "off." We hope to ultimately translate the gained knowledge into new strategies for the rational design of drug candidates that could inhibit proliferation of cancerous cells.