Signal Transduction by Tyrosine Phosphorylation

通过酪氨酸磷酸化进行信号转导

• 批准号: 8460088
• 负责人: TONY R. HUNTER
• 金额: $ 63.12万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8460088
• 关键词: Agar; Anchorage-Independent Growth; Apoptosis; Apoptotic; Autophagocytosis; Biological Assay; Cell Cycle Progression; Chemicals; Colon; Colon Carcinoma; Colorectal Cancer; Drug Targeting; Family; Family member; Feedback; Glioblastoma; Goals; Imaging Techniques; In Vitro; Individual; Intention; Investigation; Isoenzymes; Life; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Mutation; Normal Cell; Nude Mice; Oncogene Proteins; Oncogenic; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Play; Point Mutation; Process; Protein Kinase; Protein Kinase C; Role; Signal Pathway; Signal Transduction; Somatic Mutation; System; Testing; Tumor Suppressor Proteins; Tyrosine Phosphorylation; addiction; atypical protein kinase C; cancer cell; cancer genome; cancer therapy; cellular imaging; genome sequencing; in vivo; loss of function mutation; mouse model; mutant; novel; public health relevance; ras Proteins; receptor; therapeutic target; tumor; tumorigenesis; tumorigenic

DESCRIPTION (provided by applicant): To identify protein kinases with functional somatic mutations that contribute to the process of tumorigenesis, protein kinases newly implicated in cancer through the presence of putative driver mutations identified in recent cancer genome sequencing efforts will be characterized. Initially, the focus will be on four such kinases, DAPK3, SGK085 and MLK4, which score highly in the devised ranking system as being the most likely to play a role in cancer, and protein kinase C family members. In vitro and in vivo analysis of kinase activity will be carried out in order to determine whether individual mutations in DAPK3, SGK085 and MLK4 increase or decrease activity, attempt to identify substrates for the mutant and WT kinases and define in what signaling pathway(s) they act, and determine the effects of expressing mutant and WT kinases in normal and cancer cells, assaying for changes in proliferation, cell cycle progression, apoptosis and autophagy, morphological transformation, anchorage independent growth in soft agar, and tumorigenesis in nude mice. This will define whether each mutant kinase has gain- or loss-of-function mutations, whether it acts as an oncoprotein or opposes the effects of its normal tumor suppressor kinase counterpart, and whether the kinase might serve as a new drug target for cancer therapy. The protein kinase C (PKC) family of kinases has been extensively studied in cancer, through their role as receptors for tumor promoting chemicals, but few cancer mutations have been identified, and there is a debate regarding whether activation or inactivation of PKC family of kinases is important for cancer. The discovery of nonsynonymous point mutations in many PKCs, mainly in colorectal cancer (CRC) and glioblastoma multiforme (GBM), provides an opportunity to determine if PKCs are activated or inactivated in cancer and determine how these mutations contribute to tumorigenesis. Mutations observed in the PKC family of isozymes in cancer utilizing live-cell imaging techniques will be characterized. Additionally, all the PKC mutations we are studying in CRC occur in the context of activating K-Ras mutations. K-Ras is a substrate for PKC, and phosphorylation of K-Ras by PKC alters its subcellular targeting and causes K-Ras to promote apoptosis. The intention is to determine if loss of function mutations in PKC can promote the survival of colon cancer cells harboring K-Ras mutations, by suppressing this K-Ras-induced apoptotic feedback loop, thereby fine-tuning oncogenic K-Ras addiction. An investigation of the mutations in the atypical PKC, aPKC6, in GBM will be carried out to determine their effect on the activity of aPKC6 using similar approaches and their transforming potential will be tested in a new mouse model for GBM.

描述（由申请方提供）：为了鉴定具有有助于肿瘤发生过程的功能性体细胞突变的蛋白激酶，将表征通过在最近的癌症基因组测序工作中鉴定的推定驱动突变的存在而新涉及癌症的蛋白激酶。最初，重点将放在四种这样的激酶，DAPK 3，SGK 085和MLK 4，它们在设计的排名系统中得分很高，因为它们最有可能在癌症中发挥作用，以及蛋白激酶C家族成员。将进行激酶活性的体外和体内分析，以确定DAPK 3、SGK 085和MLK 4中的单个突变是否增加或降低活性，尝试鉴定突变体和WT激酶的底物并确定它们在什么信号传导途径中起作用，并确定在正常和癌细胞中表达突变体和WT激酶的作用，测定增殖的变化，细胞周期进程、凋亡和自噬、形态转化、软琼脂中的锚定非依赖性生长和裸鼠中的肿瘤发生。这将确定每个突变激酶是否具有获得或丧失功能的突变，它是否作为癌蛋白或对抗其正常肿瘤抑制激酶对应物的作用，以及激酶是否可能作为癌症治疗的新药靶点。 蛋白激酶C（PKC）家族的激酶通过其作为肿瘤促进化学物质的受体的作用在癌症中被广泛研究，但很少有癌症突变被鉴定，并且关于PKC家族的激酶的激活或失活对癌症是否重要存在争论。在许多PKC中发现非同义点突变，主要是在结直肠癌（CRC）和多形性胶质母细胞瘤（GBM）中，这为确定PKC在癌症中是否被激活或失活以及确定这些突变如何促进肿瘤发生提供了机会。利用活细胞成像技术在癌症中的PKC家族同工酶中观察到的突变将被表征。此外，我们在CRC中研究的所有PKC突变都发生在激活K-Ras突变的背景下。K-Ras是PKC的底物，PKC对K-Ras的磷酸化改变了其亚细胞靶向并导致K-Ras促进凋亡。目的是确定PKC功能缺失突变是否可以通过抑制这种K-Ras诱导的凋亡反馈环，从而微调致癌K-Ras成瘾，促进携带K-Ras突变的结肠癌细胞的存活。将使用类似的方法对GBM中的非典型PKC aPKC 6的突变进行研究，以确定其对aPKC 6活性的影响，并将在新的GBM小鼠模型中测试其转化潜力。