Non-canonical signal pathway for Crk in breast cancer

描述(由申请人提供)：含有Src同源2(SH2)和Src同源3(SH3)结构域的蛋白Crk是一类接头蛋白的原型成员，该接头蛋白在酪氨酸激酶下游的信号传递中发挥重要作用。通过促进SH2和SH3结构域介导的蛋白质复合体的组装，过去20年积累的证据阐明了Crk信号转导的典型途径，即SH2结构域与酪氨酸磷酸化蛋白结合，而N-末端SH3结构域(SH3N)与激活rac1、Rap1和RAS的鸟氨酸-核苷酸交换因子结合。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，并且siRNA介导的Crk的敲除逆转了这些癌症的恶性和转移特征。这些观察结果使人们迫切需要了解Crk促进恶性转化的机制，希望能利用新的信息来开发治疗方法，特别是对易发生侵袭和转移的肿瘤。在这一应用中，我们通过鉴定位于羧基末端SH3(SH3C)结构域的两个以前未被鉴定的酪氨酸磷酸化位点，确定了一种新的CRK信号传导模式。SH3C结构域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环中，而Tyr239位于连接子和SH3C的交界处，包含一个与SH3C对SH3N的负调控和自动钳制有关的Crk区域。需要检验的中心假设是，除了作为接头蛋白的传统作用外，Crk还通过在SH3C的Tyr239和Tyr251上被磷酸化而在信号转导中发挥非常规、非规范的作用，磷酸化将定义具有SH2或PTB结构域的蛋白质的新结合部位，从而参与新的磷酸化依赖的信号通路。由于Tyr251和Tyr239都显示在EGF刺激下酪氨酸磷酸化水平上升，并且与WT Crk相比，Y251F Crk突变体促进细胞迁移和侵袭EGF的能力减弱，我们假设机制研究将立即与升高EGFR的侵袭性三阴性乳腺癌细胞相关。这些研究可能对制定治疗策略以描述和靶向表达Crk的肿瘤具有重要意义，也为在广泛的人类癌症中探索这种生物学提供了理论基础。

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.