Non-canonical signal pathway for Crk in breast cancer

描述（由申请人提供）：Src Homology 2 （SH2）和Src Homology 3 （SH3）结构域蛋白Crk是一类接头蛋白的典型成员，在酪氨酸激酶的下游信号传导中起重要作用。通过促进由SH2和SH3结构域介导的蛋白复合物的组装，过去二十年积累的证据已经阐明了Crk信号的典型途径，其中SH2结构域结合酪氨酸磷酸化蛋白，n端SH3结构域（SH3N）结合激活Rac1、Rap1和Ras的鸟嘌呤核苷酸交换因子。近年来，Crk在人类癌症中的临床意义已经被列举出来，因为Crk在几种不同的癌症中经常过表达，包括乳腺癌、卵巢癌、胃癌、肺癌、胶质母细胞瘤和肉瘤，sirna介导的Crk敲低可以逆转这些癌症的恶性和转移特征。这些观察结果导致了了解Crk促进恶性转化的机制的新的紧迫性，希望新的信息可以被利用来开发治疗方法，特别是对于具有侵袭和转移倾向的肿瘤。在这项应用中，我们通过鉴定位于SH3羧基末端（SH3C）结构域内的两个以前未被表征的酪氨酸磷酸化位点，确定了Crk的新信号范式，这是一个没有明确生物学功能的非典型SH3结构域。Tyr251位于Crk SH3C的高度保守的rt环，而Tyr239位于连接子和SH3C的边界，包含一个涉及SH3C对SH3N负调控和自动夹紧的Crk区域。要验证的中心假设是，除了作为接头蛋白的传统作用外，Crk在信号传导中具有非正统、非规范的作用，因为它在SH3C中的Tyr239和Tyr251上被磷酸化，磷酸化将为具有SH2或PTB结构域的蛋白质定义新的结合位点，从而参与新的磷酸化依赖信号通路。由于Tyr251和Tyr239在EGF刺激下酪氨酸磷酸化水平升高，而与WT Crk相比，Y251F Crk突变体促进细胞向EGF迁移和侵袭的能力减弱，我们假设机制研究将与EGFR升高的侵袭性三阴性乳腺癌细胞立即相关。这些研究可能对研究和靶向表达crk的肿瘤的治疗策略具有重要意义，但也为在广泛的人类癌症中探索这一生物学提供了理论依据。

DESCRIPTION (provided by applicant): The Src Homology 2 (SH2) and Src Homology 3 (SH3) domain-containing protein Crk is the prototypical member of a class of adaptor proteins that play essential roles in signaling downstream of tyrosine kinases. By promoting the assembly of protein complexes mediated by the SH2 and SH3 domains, evidence accumulated over the past two decades has elucidated a canonical pathway for Crk signaling whereby the SH2 domain binds tyrosine phosphorylated proteins and the N-terminal SH3 domain (SH3N) binds guanine-nucleotide exchange factors that activate Rac1, Rap1, and Ras. The clinical significance of Crk in human cancer has been enumerated in recent years, as Crk is frequently over-expressed in several different cancers, including breast, ovarian, gastric, lung, glioblastoma, and sarcomas and siRNA-mediated knockdown of Crk reverses the malignant and metastatic features of these cancers. These observations have led to a new urgency to understand the mechanisms by which Crk promotes malignant transformation in the hope that new information can be exploited to develop therapeutics, particularly for tumors with a predisposition towards invasion and metastasis. In this application we have identified a new signaling paradigm for Crk by the identification of two previously uncharacterized tyrosine phosphorylation sites located within the carboxyl-terminal SH3 (SH3C) domain, an atypical SH3 domain that has no clear biological function. Tyr251 is located in the highly conserved RT-loop of the Crk SH3C while Tyr239 is located at the boundary of the linker and SH3C and comprises a region of Crk implicated in the negative regulation and auto-clamping of the SH3C to the SH3N. The central hypothesis to be tested is that in addition to its conventional role as an adaptor protein, Crk has an unorthodox, non-canonical role in signaling by virtue of being phosphorylated on Tyr239 and Tyr251 in the SH3C, phosphorylation which will define new binding sites for proteins with SH2 or PTB domains, and hence engage novel phosphorylation dependent signaling pathways. Since both Tyr251 and Tyr239 show elevation in tyrosine phosphorylation upon EGF stimulation, and the Y251F Crk mutant has a diminished ability to promote cell migration and invasion towards EGF compared to WT Crk, we hypothesize that mechanistic studies will be immediately relevant to invasive triple negative breast cancer cells that have elevated EGFR. These studies may have important implications in the development of therapeutic strategies to profile and target Crk-expressing tumors, but also provide rationale for exploring this biology in a wide spectrum of human cancers.