Regulation of K-Ras by a Farnesyl-electrostatic Switch

法尼基静电开关对 K-Ras 的调节

• 批准号: 8107100
• 负责人: MARK Reid PHILIPS
• 金额: $ 31.65万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8107100
• 关键词: 2-cyclopentyl-5-(5-isoquinolylsulfonyl)-6-nitro-1H-benzo(D)imidazole; Affinity; Agonist; Alleles; Amino Acids; Antineoplastic Agents; Apoptosis; Autophagocytosis; Binding; Biochemistry; C-terminal; Calcium; Calcium Channel; Calpain; Cancer Biology; Cancer Etiology; Cancer Model; Cell Death; Cell Death Signaling Process; Cell Line; Cell membrane; Cell physiology; Cells; Cellular biology; Characteristics; Charge; Collaborations; Colorectal Neoplasms; Complementarity Determining Regions; Complex; Cultured Cells; Electrophysiology (science); Electrostatics; Endoplasmic Reticulum; Energy Transfer; Exons; Face; Fibroblasts; Gene Mutation; Genes; Golgi Apparatus; Grant; Growth; HCT116 Cells; Homeostasis; Human; In Vitro; Inositol; Intracellular Membranes; Knock-in Mouse; Lipids; Lung; Lymphocyte; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Measurement; Mediating; Membrane; Mitochondria; Modeling; Modification; Mus; Mutate; Mutation; Oncogenes; Oncogenic; Outer Mitochondrial Membrane; Pathway interactions; Phospholipids; Phosphorylation; Phosphorylation Site; Positioning Attribute; Post-Translational Protein Processing; Predisposition; Principal Investigator; Process; Protein Isoforms; Protein Kinase C; RAS genes; Recombinants; Regulation; Rodent; Role; Serine; Signal Transduction; Stretching; System; Testing; Toxic effect; Tumor Cell Line; Tumorigenicity; Work; Xenograft Model; bryostatin; cancer therapy; carcinogenesis; farnesylation; homologous recombination; in vivo; inorganic phosphate; mammalian genome; mutant; neoplastic cell; prevent; programs; protein protein interaction; ras Proteins; receptor; research study; response; tripolyphosphate; tumor

DESCRIPTION (provided by applicant): Of the three ras genes, kras is most frequently mutated in human cancer. Ras proteins are highly homologous but differ extensively in their C-terminal hypervariable regions that direct post-translational modifications (e.g. farnesylation) and membrane targeting. We discovered a post-translational modification unique to K-Ras: protein kinase C (PKC) mediated phosphorylation. We found that phosphorylation of K-Ras at serine 181 partially neutralized the adjacent polybasic stretch of amino acids and thereby activated a farnesyl-electrostatic switch that resulted in release of K-Ras from the plasma membrane and association with intracellular membranes, including the endoplasmic reticulum (ER), Golgi apparatus and the outer mitochondrial membrane. Most intriguing, K-Ras translocation to internal membranes was associated with cell death. Bryostatin 1, a potent PKC agonist, showed anti-tumor activity that was dependent on K-Ras serine 181. In the first cycle of this grant we proposed to expand upon these discoveries with three Aims: 1) Regulation of the farnesyl-electrostatic switch, 2) Mechanisms of phospho-K-Ras mediated apoptosis and 3) The Role of C-terminal phosphorylation of K-Ras in mouse tumor models. Much progress has been made on each aim. Most exciting are our discoveries that K-Ras signals for cell death from the cytoplasmic face of the ER, that Bcl-XL is required for phospho-K-Ras mediated cell death, and that phospho-K-Ras forms a trimolecular complex with Bcl-XL and the IP3 receptor (IP3R) and regulates the calcium channel activity. We have also been successful in constructing a double knock-in mouse that harbors a conditional oncogenic K-Ras allele that lacks the phosphorylation site at amino acid 181 (LSL-K-Ras12D181A). In this competing renewal application we propose to continue our studies with three aims: Aim 1: Regulation of the IP3 Receptor (IP3R) by phospho-K-Ras. We will characterize both structurally (protein-protein interactions) and functionally (electrophysiology) the molecular interactions between phospho-K-Ras, Bcl-XL and IP3R. We will ascertain if phospho-K-Ras alters mitochondrial calcium homeostasis. We will determine if IP3R, calpain and autophagy are required for phospho-K-Ras mediated cell death. Aim 2: Analysis of K-Ras Phosphorylation at Serine 181 in vivo. We will use our newly created LSL-K-Ras12D181A mice in two Cre- driven tumor models to test the hypothesis that phosphorylation at serine 181 negatively regulates K-Ras oncogenicity and we will use the same models to show that the efficacy of bryostatin 1 depends on phosphorylation of serine 181. Aim 3: Analysis of K-Ras Phosphorylation at Serine 181 in Human Tumor Cells. We will correlate susceptibility of human tumor cells lines with K-Ras mutation status and generate isogenic lines of human tumor cells with and without a phosphorylation site at position 181. We anticipate that our mechanistic studies of the cell biology of phospho-K-Ras along with our in vivo and human tumor cell analyses will reveal unique features of this important oncogene that can be exploited in developing anti-cancer drugs. PUBLIC HEALTH RELEVANCE: Oncogenes are genes that cause cancer. K-Ras is the most important human oncogene. We discovered that K-Ras can be modified by the addition of a phosphate group and that this modification inhibits its cancer-promoting activity. We propose to study the cell biology and physiology of K-Ras phosphorylation to better understand how to exploit this process to develop anti-cancer drugs.

描述（由申请人提供）：在三个RAS基因中，KRAS在人类癌症中最常见。 RAS蛋白具有高度同源性，但在其C末端高变量区域有很大差异，该区域直接直接翻译后修饰（例如Farneylation）和膜靶向。我们发现了K-RAS独有的翻译后修饰：蛋白激酶C（PKC）介导的磷酸化。 We found that phosphorylation of K-Ras at serine 181 partially neutralized the adjacent polybasic stretch of amino acids and thereby activated a farnesyl-electrostatic switch that resulted in release of K-Ras from the plasma membrane and association with intracellular membranes, including the endoplasmic reticulum (ER), Golgi apparatus and the outer mitochondrial membrane.最吸引人的K-Ras转移到内膜与细胞死亡有关。 Bryostatin 1是一种有效的PKC激动剂，表现出抗肿瘤活性，取决于K-Ras丝氨酸181。在我们提议的第一个赠款中，我们提议扩展以三个目的扩展这些发现：1）调节Farnesyl-Electrostatic Switch，2）2）phose-k-ras介导的Apoptise和3端的ptental tentry tentry tentry tenter的作用。型号。每个目标都取得了很多进展。最令人兴奋的是，我们发现的是，K-RAS的ER细胞质脸部的细胞死亡信号，磷酸化-K-RAS介导的细胞死亡是必需的，并且磷酸-K-RAS与Bcl-XL和IP3受体（IP3R）形成三型分子复合物，并调节钙通道活性。我们还成功地构建了一只双敲门小鼠，该小鼠具有条件性致癌K-Ras等位基因，该等位基因缺乏氨基酸181（LSL-K-RAS12D181A）的磷酸化位点。在这种竞争性更新应用中，我们建议以三个目的继续我们的研究：AIM 1：Phosho-K-Ras对IP3受体（IP3R）的调节。我们将在结构上（蛋白质 - 蛋白质相互作用）和功能（电生理学）表征磷酸化磷酸化，BCL-XL和IP3R之间的分子相互作用。我们将确定磷酸-K-RAS是否改变线粒体钙稳态。我们将确定IP3R，钙蛋白酶和自噬是否需要磷酸k-Ras介导的细胞死亡。 AIM 2：在体内丝氨酸181上K-RAS磷酸化的分析。我们将使用新创建的LSL-K-RAS12D181A小鼠在两个型肿瘤模型中测试以下假设：丝氨酸181处的磷酸化负调节K-RAS致癌性，我们将使用相同的模型来表明Broyostatin 1对Broyostatin 1的疗效依赖于Serine 181 Serine 181 k-ras k.-aim k ras sy in sim k-ras。肿瘤细胞。我们将与K-RAS突变状态的人类肿瘤细胞的敏感性相关联，并在181位的具有和没有磷酸化位点的人类肿瘤细胞中生成等源性线。我们预计，我们对磷酸化的机械研究的机械性研究以及我们的体内和人类肿瘤细胞分析将揭示出这种重要养殖者的独特特征。 公共卫生相关性：癌基因是引起癌症的基因。 K-Ras是最重要的人类癌基因。我们发现可以通过添加磷酸盐组来修改K-RAS，并且这种修饰抑制其癌症促进活性。我们建议研究K-RAS磷酸化的细胞生物学和生理学，以更好地了解如何利用这一过程来开发抗癌药物。