Regulation of the Class IA PI 3-kinase PIK3CB

IA 类 PI 3 激酶 PIK3CB 的调节

• 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
• 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

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类磷酸肌肽3-激酶（PI3Ks）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PI3K同时受到受体酪氨酸激酶（rtk）和G蛋白偶联受体（gpcr）的调节，后者通过PI3K催化亚基（p110）与G蛋白偶联受体（gpcr）的直接结合来调节。子单元。最近，我们通过鉴定和突变G′的p110′结合位点，首次提供了关于GPCR调控PI3K′的机制信息。令人惊讶的是，PI3K的许多细胞活性，包括转化成纤维细胞和支持无pten肿瘤细胞生长的能力，都需要它与G ？子单元。此外，当我们将乳腺肿瘤细胞中的内源性p110替换为gpcr不偶联突变体时，细胞在体外3D侵袭中表现出减少，在原位模型中表现出肿瘤生长减少，在体内转移试验中表现出转移减少。值得注意的是，破坏GPCR对PI3K的输入比消除PI3K脂质激酶活性更能降低肿瘤生长。这些数据表明GCPR与PI3K偶联在肿瘤的发展和转移中起关键作用。这种偶联可以为癌症化疗提供一个重要的新药物靶点。除了G ？p110 α也能结合小的GTPase Rab5。我们鉴定并突变了p110 α中的Rab5结合位点。表达rab5解偶联p110 α的细胞在巨噬细胞作用中表现出明显的缺陷，巨噬细胞作用是一种不依赖网格蛋白的内吞过程，用于肿瘤细胞获取支持快速生长的营养物质。这些数据表明Rab5-PI3K相互作用可能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被认为在与p110催化亚基结合时不会二聚化。我们已经开发了新的工具来操纵p85的多聚态，我们可以用它来定义p85和p110如何与PTEN相互作用。特异性目的1在前列腺癌和子宫内膜癌的小鼠模型中，使用条件敲入和全动物敲入突变体p110 α，检测GPCR信号传导到PI3K α。目的2提出通过Rab5-PI3K相互作用调控巨量红细胞增生的机制研究，并研究Rab5-p110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，Aim 3使用分析性超离心，测量寡聚物的分子量，独立于形状，来定义PTEN和pi3k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PI3K在细胞生物学和人类疾病中的作用。