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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：
9115636
负责人：
ANNE R BRESNICK
金额：
$ 40.49万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
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/9115636
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 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 KRAS2 gene Knock-in Mouse Light Lipids Malignant Neoplasms Malignant neoplasm of pancreas Malignant neoplasm of prostate 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 knockin animal lipid metabolism mouse model mutant neoplastic cell new therapeutic target 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（PI 3 Ks）调节广泛的细胞功能，包括生长和存活、代谢、脂质和蛋白质合成以及运动。在I类酶中，只有PI 3 K β受受体酪氨酸激酶（RTK）和G蛋白偶联受体（GPCR）的调节，后者通过PI 3 K β催化亚基（p110 β）与G蛋白偶联受体（GPCR）的直接结合来调节。亚单位。我们最近通过鉴定和突变G β的p110 β结合位点，提供了关于GPCR调控PI 3 K β的第一个机制信息。令人惊讶的是，PI 3 K β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，都需要它与G β的相互作用。亚单位。此外，当我们在乳腺肿瘤细胞中用GPCR解偶联突变体取代内源性p110 β时，细胞显示出体外3D侵袭减少，原位模型中肿瘤生长减少，体内转移测定中转移减少。值得注意的是，破坏GPCR对PI 3 K β的输入比消除PI 3 K β脂质激酶活性更能降低肿瘤生长。这些数据表明，GCPR与PI 3 K β的偶联在肿瘤的发展和转移中起着关键作用。这种偶联可能为癌症化疗提供一个重要的新的药物靶点。除了G？，p110 β还结合小的GT3 Rab 5。我们鉴定并突变了p110 β中的Rab 5结合位点。表达Rab 5-非偶联p110 β的细胞在巨胞饮作用中表现出明显的缺陷，巨胞饮作用是一种不依赖网格蛋白的内吞过程，肿瘤细胞利用这种过程获得支持快速生长的营养物质。这些数据表明Rab 5-PI 3 K相互作用可以为某些肿瘤提供新的药物靶点。最后，已发表的研究表明，PTEN肿瘤抑制因子在细胞中与p110形成复合物，而体外数据显示，PTEN优先结合p85调节亚基的二聚体。这些数据似乎是不相容的，因为p85被认为是不二聚化时，结合到p110催化亚基。我们已经开发了操纵p85多聚体状态的新工具，我们可以用它来定义p85和p110如何与PTEN相互作用。具体目标1检查GPCR信号PI 3 K β在前列腺癌和子宫内膜癌小鼠模型，使用条件和整个动物敲入突变p110 β。目的2提出Rab 5-PI 3 K β相互作用对巨胞饮调节的机制研究，并研究Rab 5-p110 β结合在树突状细胞功能和K-Ras驱动的胰腺癌模型中的作用。最后，目标3使用分析性超离心，其测量与形状无关的低聚物的分子量，以定义PTEN和PI 3 Ks之间的相互作用。总之，我们已经确定了新的突变体，制造了新的生物学工具，并设计了新的实验策略，以更好地定义PI 3 K在细胞生物学和人类疾病中的作用。