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：
8920160
负责人：
ANNE R BRESNICK
金额：
$ 3.37万
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 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8920160
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 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 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（由适用提供）：I类磷酸肌醇3-激酶（PI3KS）调节广泛的细胞功能，包括生长和生存，代谢，脂质和蛋白质合成以及运动性。在I类酶中，只有受体酪氨酸激酶（RTKS）和G蛋白偶联受体（GPCR）对PI3K进行调节，而后者是通过PI3K催化亚基（P110英寸）与G'与G'的直接结合？亚基。最近，我们通过识别和突变G'的P110英寸结合位点提供了有关PI3K''GPCR调节的第一个机械信息。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-null肿瘤细胞生长的能力，都需要与G'相互作用吗？亚基。此外，当我们用乳腺肿瘤细胞中的GPCR脉冲突变体代替内源性P110英寸时，该细胞在体外模型中显示出降低的体外3D浸润，肿瘤的生长降低，并在体内转移分析中增加了转移。值得注意的是，与消除PI3K脂质激酶活性相比，扰乱GPCR输入对PI3K的生长降低了。这些数据表明，与PI3K的GCPR耦合在肿瘤发育和转移中起关键作用。这种耦合可以为癌症化学疗法提供重要的新药物。除G≧外，P110≧还结合了小的GTPase Rab5。我们在p110≧中鉴定并突变了Rab5结合位点。表达RAB5脉冲的P110≧的细胞在大型细胞增多症中表现出明显的缺陷，这是一种由肿瘤细胞用于获得支持快速生长的养分的非网状蛋白非依赖性内吞过程。这些数据表明RAB5-PI3K≧相互作用可以为某些肿瘤提供新的药物靶标。最后，已发表的研究表明，PTEN肿瘤抑制剂在细胞中与p110°形成复合物，而体外数据表明，PTEN优选地结合了p85调节亚基的二聚体。这些数据似乎是不兼容的，因为当绑定到P110催化亚基时，p85不被二聚。我们开发了用于操纵p85的多药状态的新型工具，我们可以用来定义p85和p110与PTEN相互作用。特定的AIM 1检查使用突变体P110的条件和全动物敲击，在前列腺和子宫内膜癌的小鼠模型中对PI3K的GPCR信号传导。 AIM 2关于RAB5-PI3K在相互作用中调节大型细胞细胞增多症的建议机械研究，并检查RAB5-P110的作用。树突状细胞功能和胰腺癌的K-RAS驱动模型中的结合。最后，AIM 3使用分析性超速离心，该分析超离心与形状独立于形状的分子量来定义PTEN和PI3KS之间的相互作用。综上所述，我们已经确定了新型突变体，制造了新型的生物学工具并设计了新的实验策略，以更好地定义PI3K在细胞生物学和人类疾病中的作用。