Non-canonical signal pathway for Crk in breast cancer

描述（由申请人提供）：含Src同源2（SH 2）和Src同源3（SH 3）结构域的蛋白质Crk是一类衔接蛋白的原型成员，其在酪氨酸激酶下游的信号传导中起重要作用。通过促进由SH 2和SH 3结构域介导的蛋白质复合物的组装，过去二十年积累的证据已经阐明了Crk信号传导的经典途径，其中SH 2结构域结合酪氨酸磷酸化蛋白，N-末端SH 3结构域（SH 3 N）结合激活Rac 1、Rap 1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经常在几种不同的癌症中过表达，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敲低逆转了这些癌症的恶性和转移性特征。这些观察结果导致了一个新的紧迫性，以了解Crk促进恶性转化的机制，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。 在本申请中，我们通过鉴定位于羧基末端SH 3（SH 3C）结构域内的两个先前未表征的酪氨酸磷酸化位点，鉴定了Crk的新的信号传导范例，所述结构域是非典型的SH 3结构域，其没有明确的生物学功能。Tyr 251位于Crk SH 3C的高度保守的RT环中，而Tyr 239位于接头和SH 3C的边界处，并且包含参与SH 3C对SH 3 N的负调节和自动夹持的Crk区域。待测试的中心假设是，除了其作为衔接蛋白的常规作用之外，Crk在信号传导中具有非正统的、非典型的作用，这是由于在SH 3C中的Tyr 239和Tyr 251上被磷酸化，磷酸化将为具有SH 2或PTB结构域的蛋白质定义新的结合位点，并因此参与新的磷酸化依赖性信号传导途径。由于Tyr 251和Tyr 239在EGF刺激后显示酪氨酸磷酸化升高，并且与WT Crk相比，Y251 F Crk突变体促进细胞向EGF迁移和侵袭的能力降低，因此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直接相关。这些研究可能对开发治疗策略以描述和靶向表达Crk的肿瘤具有重要意义，但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。 公共卫生相关性：含有Src同源物-2（SH 2）和Src同源物-3（SH 3）的蛋白质是真核细胞中最通用的蛋白质之一，并且对于人类癌症中的信号传导至关重要。该提案中概述的研究定义了Crk癌基因产物的新功能。近年来，大量证据表明Crk蛋白在人类肿瘤中过表达，并且表达水平与癌细胞的侵袭性和恶性行为相关。Crk蛋白的这些特性使其成为潜在的癌症预后标志物和治疗靶点。

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. PUBLIC HEALTH RELEVANCE: Src Homology-2 (SH2) and Src Homology-3 (SH3) containing proteins are among the most versatile proteins in eukaryotic cells, and critically important for signaling in human cancers. The research outlined in this proposal defines a new function for the Crk oncogene product. In recent years, an emerging body of evidence suggests that Crk proteins are overexpressed in human tumors and the expression levels correlate with aggressive and malignant behavior of cancer cells. These properties of Crk proteins make them potential cancer prognosis markers and therapeutic targets.