Regulation of the Class IA PI 3-kinase PIK3CB

IA 类 PI 3 激酶 PIK3CB 的调节

基本信息

批准号：
8791287
负责人：
ANNE R BRESNICK
金额：
$ 40.49万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-05 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8791287
关键词：
1-Phosphatidylinositol 3-Kinase 1-Phosphatidylinositol 4-Kinase Animals Antigen Presentation Binding Binding Sites Biological Biological Assay Breast Cancer Cell Breeding Catalytic Domain Cell Proliferation Cell Survival Cell physiology Cells Cellular biology Chemicals Chemotherapy-Oncologic Procedure Clathrin Complex Coupling Data Defect Dendritic Cells Development Dimerization Drug Targeting EGF gene Endometrial Carcinoma Endometrial Hyperplasia Enzymes Equilibrium Fibroblasts G Protein-Coupled Receptor Signaling G protein coupled receptor kinase G-Protein-Coupled Receptors Growth Homodimerization Human In Vitro Knock-in Mouse Light Lipids Malignant Neoplasms Malignant neoplasm of pancreas Malignant neoplasm of prostate Mammary Neoplasms Measures Mediating Membrane Methods Modeling Molecular Weight Monomeric GTP-Binding Proteins Mus Mutate Mutation Neoplasm Metastasis Normal Cell Nutrient PIK3CB gene PTEN gene Pharmaceutical Preparations Phosphatidylinositols Phosphotransferases Physiological Play Process Prostate Protein Biosynthesis Publishing Receptor Protein-Tyrosine Kinases Recruitment Activity Regulation Role Shapes Signal Transduction Site System Testing Tumor Suppressor Proteins analytical ultracentrifugation base cancer cell cell motility design dimer human disease in vivo inhibitor/antagonist lipid metabolism mouse model mutant neoplastic cell novel prevent public health relevance rapid growth tool tumor tumor growth tyrosine receptor uptake

项目摘要

DESCRIPTION (provided by applicant): The Class I Phosphoinositide 3-kinases (PI3Ks) regulate a wide range of cellular functions, including growth and survival, metabolism, lipid and protein synthesis, and motility. Of the Class I enzymes, only PI3K¿ is regulated both by receptor tyrosine kinases (RTKs) and G-protein coupled receptors (GPCRs), the latter through direct binding of the PI3K¿ catalytic subunit (p110¿) to G¿? subunits. We recently provided the first mechanistic information about GPCR regulation of PI3K¿, by identifying and mutating the p110¿ binding site for G¿?. Surprisingly, many of the cellular activities of PI3K¿, including its abilityto transform fibroblasts and to support the growth of PTEN-null tumor cells, require its interactions with G¿? subunits. Moreover, when we replace endogenous p110¿ with a GPCR-uncoupled mutant in breast tumor cells, the cells show decreased in vitro 3D invasion, decreased tumor growth in an orthotopic model, and decreased metastasis in an in vivo metastasis assay. Notably, disrupting GPCR inputs to PI3K¿ caused a greater decrease in tumor growth than eliminating PI3K¿ lipid kinase activity. These data suggest that GCPR coupling to PI3K¿ plays critical roles in tumor development and metastasis. This coupling could provide an important new drug target for cancer chemotherapy. In addition to G¿?, p110¿ also binds the small GTPase Rab5. We identified and mutated the Rab5 binding site in p110¿. Cells expressing Rab5-uncoupled p110¿ show pronounced defects in macropinocytosis, a clathrin-independent endocytic process used by tumor cells to obtain nutrients that support rapid growth. These data suggest that Rab5-PI3K¿ interactions could provide a novel drug target for some tumors. Finally, published studies have shown that the PTEN tumor suppressor forms a complex with p110¿ in cells, whereas in vitro data shows that PTEN preferentially binds dimers of the p85 regulatory subunit. These data appear to be incompatible, as p85 is not thought to dimerize when bound to p110 catalytic subunits. We have developed novel tools for manipulating the multimeric state of p85, which we can use to define how p85 and p110¿ interact with PTEN. Specific Aim 1 examines GPCR signaling to PI3K¿ in mouse models of prostate and endometrial cancer, using conditional and whole animal knock-ins of mutant p110¿. Aim 2 proposes mechanistic studies on the regulation of macropinocytosis by Rab5-PI3K¿ interactions, and examines the role of Rab5-p110¿ binding in dendritic cell function and in a K-Ras-driven model of pancreatic cancer. Finally, Aim 3 uses analytical ultracentrifugation, which measures the molecular weight of oligomers independently of shape, to define interactions between PTEN and PI3Ks. Taken together, we have identified novel mutants, made novel biological tools, and devised new experimental strategies, to better define the role of PI3K¿ in cell biology and human disease.

描述（由申请人提供）：I 类磷酸肌醇 3-激酶 (PI3K) 调节多种细胞功能，包括生长和存活、代谢、脂质和蛋白质合成以及运动性。受受体酪氨酸激酶 (RTK) 和 G 蛋白偶联受体 (GPCR) 调节，后者通过直接结合 PI3K¿催化亚基 (p110¿) 至 G¿ ? 我们最近提供了有关 PI3K 的 GPCR 调控的第一个机制信息。，通过识别和突变 p110¿ G 的结合位点令人惊讶的是，PI3K 的许多细胞活性，包括其转化成纤维细胞和支持 PTEN 缺失肿瘤细胞生长的能力，需要与 G¿ 相互作用此外，当我们替换内源性p110¿在乳腺肿瘤细胞中使用 GPCR 解偶联突变体，细胞显示出体外 3D 侵袭减少、原位模型中肿瘤生长减少以及体内转移测定中转移减少，值得注意的是，破坏了 PI3K 的 GPCR 输入。比消除 PI3K 更能减少肿瘤生长？这些数据表明 GCPR 与 PI3K 偶联。除了 G¿ 之外，这种偶联还可以为癌症化疗提供重要的新药物靶点。？，p110¿还结合小 GTP 酶 Rab5。我们鉴定并突变了 p110 中的 Rab5 结合位点。表达 Rab5 未偶联 p110 的细胞¿显示出巨胞饮作用的明显缺陷，巨胞饮作用是肿瘤细胞用来获取支持快速生长的营养物质的独立于网格蛋白的内吞过程。这些数据表明 Rab5-PI3K¿最后，已发表的研究表明，PTEN 肿瘤抑制因子与 p110 形成复合物。在细胞中，而体外数据显示 PTEN 优先结合 p85 调节亚基的二聚体，这些数据似乎不相容，因为 p85 在与 p110 催化亚基结合时不会形成二聚体。 p85，我们可以用它来定义 p85 和 p110¿与 PTEN 相互作用。具体目标 1 检查 PI3K 的 GPCR 信号传导。在前列腺癌和子宫内膜癌的小鼠模型中，使用突变体 p110 的条件性和整体动物敲入目标 2 提出通过 Rab5-PI3K 调节巨胞饮作用的机制研究相互作用，并检查 Rab5-p110 的作用¿最后，Aim 3 使用分析超速离心，测量寡聚体的分子量（与形状无关），以定义 PTEN 和 PI3K 之间的相互作用。鉴定了新的突变体，制造了新的生物工具，并设计了新的实验策略，以更好地定义 PI3K 的作用。在细胞生物学和人类疾病方面。