Non-canonical signal pathway for Crk in breast cancer

乳腺癌中 Crk 的非经典信号通路

• 批准号: 8402232
• 负责人: RAYMOND B BIRGE
• 金额: $ 32.37万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-12 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8402232
• 关键词: Adaptor Signaling Protein; Address; Adenocarcinoma; Affinity; Apoptotic; Behavior; Binding; Binding Sites; Biological Assay; Biological Process; Biology; Breast; Breast Cancer Cell; Cancer Patient; Cancer Prognosis; Cancer cell line; Cell physiology; Cell-Cell Adhesion; Cells; Chickens; Clinical Research; Data; Detection; Development; EGF gene; Embryo; Epidermal Growth Factor Receptor; Eukaryotic Cell; Fatty acid glycerol esters; Fibroblasts; Generations; Glioblastoma; Guanine Nucleotide Exchange Factors; Human; Immigration; Intervention; Lung; Malignant - descriptor; Malignant Neoplasms; Mediating; Modeling; N-terminal; Names; Neoplasm Metastasis; Oncogene Proteins; Ovarian; PTB Domain; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Positive Lymph Node; Predisposition; Prognostic Marker; Property; Protein Binding; Protein Tyrosine Kinase; Proteins; Receptor Protein-Tyrosine Kinases; Recruitment Activity; Regulation; Research; Role; SH3 Domains; Screening procedure; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Small Interfering RNA; Stomach; Stomach Carcinoma; Structure; Testing; Therapeutic; Translating; Tyrosine; Tyrosine Phosphorylation; Tyrosine Phosphorylation Site; base; cancer cell; cell behavior; cell growth; cell motility; cell transformation; cis trans isomerization; clinically significant; in vivo; innovation; malignant breast neoplasm; malignant phenotype; member; mouse model; mutant; novel; overexpression; polyclonal antibody; protein complex; protein expression; protein function; protein protein interaction; proto-oncogene protein c-crk; sarcoma; src Homology Region 2 Domain; stoichiometry; synovial sarcoma; therapeutic development; therapeutic target; triple-negative invasive breast carcinoma; tumor

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.

描述（由申请人提供）：包含 Src 同源 2 (SH2) 和 Src 同源 3 (SH3) 结构域的蛋白 Crk 是一类接头蛋白的典型成员，在酪氨酸激酶下游信号传导中发挥重要作用。通过促进 SH2 和 SH3 结构域介导的蛋白质复合物的组装，过去二十年积累的证据阐明了 Crk 信号传导的经典途径，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被列举，因为 Crk 在几种不同的癌症中经常过度表达，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 敲低可逆转这些癌症的恶性和转移特征。这些观察结果使得了解 Crk 促进恶性转化的机制变得更加紧迫，希望能够利用新信息来开发治疗方法，特别是针对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 SH3 (SH3C) 结构域（一种没有明确生物学功能的非典型 SH3 结构域）内的两个先前未表征的酪氨酸磷酸化位点，确定了 Crk 的新信号传导范式。 Tyr251 位于 Crk SH3C 的高度保守 RT 环中，而 Tyr239 位于连接子和 SH3C 的边界处，并包含与 SH3C 对 SH3N 的负调节和自动钳位有关的 Crk 区域。要测试的中心假设是，除了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、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性信号传导途径。由于 Tyr251 和 Tyr239 在 EGF 刺激后酪氨酸磷酸化均升高，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，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分析和靶向表达 Crk 的肿瘤的治疗策略具有重要意义，同时也为在广泛的人类癌症中探索这种生物学特性提供了理论依据。 公共健康相关性：含有 Src Homology-2 (SH2) 和 Src Homology-3 (SH3) 的蛋白质是真核细胞中最通用的蛋白质之一，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 致癌基因产品的新功能。近年来，越来越多的证据表明，Crk 蛋白在人类肿瘤中过度表达，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。