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(申请人提供)：I类磷脂酰肌醇3-激酶(PI3K)调节广泛的细胞功能，包括生长和存活、新陈代谢、脂肪和蛋白质合成以及运动。在I类酶中，只有PI3K�同时受受体酪氨酸激酶(RTK)和G蛋白偶联受体(GPCRs)的调节，后者通过PI3K�催化亚基(p110�)与G�？亚单位。最近，我们通过鉴定和突变PI3K�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PTEN缺失的肿瘤细胞的生长的能力，都需要它与G�？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�时，细胞在体外3D侵袭减少，在原位模型中肿瘤生长减少，在体内转移实验中减少转移。值得注意的是，与消除PI3K�脂蛋白激酶活性相比，干扰PI3K�的GPCR输入导致的肿瘤生长减少更大。这些数据表明GCPR与PI3K�偶联在肿瘤的发生和转移中起关键作用。这种偶联作用可能为癌症化疗提供一个重要的新药靶点。除G�？外，p110�还与小G蛋白酶Rab5结合。我们鉴定并突变了p110�中的Rab5结合位点。表达Rab5-非偶联p110�的细胞在巨噬细胞吞噬中显示出明显的缺陷，巨噬细胞吞噬是肿瘤细胞用来获得支持快速生长的营养物质的一种不依赖于笼蛋白的内吞过程。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，抑癌基因PTEN在细胞内与p110�形成复合体，而体外数据表明，PTEN优先与p85调节亚基的二聚体结合。这些数据似乎是不相容的，因为当P85与p110催化亚基结合时，不被认为是二聚的。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。特异性目标1在前列腺癌和子宫内膜癌的小鼠模型中，使用突变的p110�的条件性和全动物敲入，检查对PI3K�的gpcr信号。目的研究Rab5-PI3K�相互作用调节巨噬细胞吞噬功能的机制，探讨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使用分析超速离心法来定义PTEN和PI3K之间的相互作用。超速离心法测量与形状无关的低聚物的相对分子质量。综上所述，我们已经确定了新的突变体，制造了新的生物工具，并设计了新的实验策略，以更好地确定PI3K�在细胞生物学和人类疾病中的作用。