Regulating IRS-proteins by Ser/Thr Phosphorylation

通过 Ser/Thr 磷酸化调节 IRS 蛋白

• 批准号: 7414864
• 负责人: Morris F. White
• 金额: $ 32.16万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7414864
• 关键词: Acute; Alleles; Antibody Formation; Build-it; Cells; Chronic; Complex; Data; Diabetes Mellitus; Docking; Funding; Genes; Genetic Polymorphism; Goals; Homologous Gene; Human; IRS1 gene; IRS2 gene; Inflammation; Insulin; Insulin Resistance; JUN kinase; Libraries; Life; Link; Literature; Mediating; Metabolic; Metabolic Diseases; Metabolic stress; Metabolism; Molecular; Mus; N-terminal; Non-Insulin-Dependent Diabetes Mellitus; Nutrient; Pancreas; Phospho-Specific Antibodies; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Process; Production; Proteins; Publications; Regulation; Risk; Rodent; Role; Serine/Threonine Phosphorylation; Signal Pathway; Signal Transduction; Signaling Protein; Site; Sorting - Cell Movement; Source; Stream; Tissues; Tyrosine Phosphorylation; United States National Institutes of Health; Validation; cell growth; cytokine; human IRS2 protein; insulin receptor substrate 1 protein; insulin receptor tyrosine kinase; insulin sensitivity; insulin signaling; link protein; mutant; prototype; response

DESCRIPTION (provided by applicant): This application entitled "Regulating IRS-proteins by Ser/Thr Phosphorylation" is the amened competing renewal ofDK38712-16. IRS1 is a prototype docking protein that links the activated insulin receptor tyrosine kinase to down-stream signaling pathways. Tyrosine phosphorylation of IRS 1, and its homolog IRS2, activates the PI3K and ras/MAPK cascades, which promote cellular growth, survival and metabolism. Nutrient excess, acute and chronic inflammation, or proinflammatory cytokines regulate IRS-protein signaling through multisite Ser/Thr-phosphorylation. During the previous funding period, we revealed the inhibitory role of Ser307 phosphorylation in rodent IRS1 (Ser312 in human IRS1), and showed the utility of polyclonal phosphospecific antibodies to study these processes in murine and human cells and tissues. Phosphorylation of Ser307 is mediated, at least in part, by the association of the activated NH2-terminal JUN kinase (JNK) with IRS1, linking insulin signaling to the inhibitory effects of metabolic stress and proinflammatory cytokines. IRS1and IRS2 each contain at least 40 Ser/Thr-phosphorylation sites (probably more), but how they interact to regulate insulin action in various tissues under acute and chronic metabolic stress is important to resolve. Here, we propose 5 Specific Aims to reveal how Ser/Thr-phosphorylation of Irs1 and Irs2 modulates insulin sensitivity and contirbutes to life threatening metabolic diseases that can progress to diabetes. 1. Prepare and validate a comprehensive mAb library against observed S/T-phosphorylation sites in Irs1 and Irs2 (apS/TmAbIrs1 and apS/TmAbIrs2 libraries). Support is requested in year 1 to generate the library against Irs1 (apS/TmAbIrs1); the apS/TmAbIrs2 library already underway and supported by other sources. 2. Utilize the pS/TmAblrs1 and pS/TmAbIrs2 libraries to decode the S/T-phosphorylation that occurs on Irs1 and Irs2 during metabolic stress. 3. Investigate the regulatory function of Ser307 in Irs1 using mice in which the Irs1 alleles are replaced with mutant A307Irs1 alleles. 4. Determine whether Ser302 in Irsl plays a positive or negative regulatory role upon Irsl signaling in murine tissues. 5. Establish the role of the Jnk-directed "kinase interaction motif (KIM) in Irsl. This proposal is important because dysregulated insulin signaling contributes to life threatening metabolic disease that progresses to type 2 diabetes when pancreatic b-cells fail to secrete sufficient insulin quickly enough to compensate for insulin resistance. Ser/Thr-phosphorylation of Irs 1 and Irs2 is common cause of insulin resistance. The production and validation of the apS/TmAbIrs1 and apS/TmAbIrs2 libraries to detect and quantify the phosphorylation sites provides a rational experimental platform to decoding the complex regulatory mechanism that controls the insulin response throughout life.

描述（由申请人提供）：本申请题为“通过Ser/Thr磷酸化调节IRS-蛋白质”，是DK38712-16的经修改的竞争性更新。 IRS1 是一种原型对接蛋白，可将激活的胰岛素受体酪氨酸激酶与下游信号通路连接起来。 IRS 1 及其同源物 IRS2 的酪氨酸磷酸化可激活 PI3K 和 ras/MAPK 级联，从而促进细胞生长、存活和代谢。营养过剩、急性和慢性炎症或促炎细胞因子通过多位点丝氨酸/苏氨酸磷酸化调节 IRS 蛋白信号传导。在之前的资助期间，我们揭示了啮齿动物 IRS1 中 Ser307 磷酸化（人类 IRS1 中 Ser312）的抑制作用，并展示了多克隆磷酸化特异性抗体在研究小鼠和人类细胞和组织中的这些过程中的实用性。 Ser307 的磷酸化至少部分是由活化的 NH2 末端 JUN 激酶 (JNK) 与 IRS1 的结合介导的，将胰岛素信号传导与代谢应激和促炎细胞因子的抑制作用联系起来。 IRS1 和 IRS2 各自至少包含 40 个 Ser/Thr 磷酸化位点（可能更多），但在急性和慢性代谢应激下它们如何相互作用以调节各种组织中的胰岛素作用是重要的解决方案。在这里，我们提出了 5 个具体目标，以揭示 Irs1 和 Irs2 的 Ser/Thr 磷酸化如何调节胰岛素敏感性并导致危及生命的代谢性疾病（可进展为糖尿病）。 1. 针对 Irs1 和 Irs2 中观察到的 S/T 磷酸化位点制备并验证综合 mAb 文库（apS/TmAbIrs1 和 apS/TmAbIrs2 文库）。在第一年请求支持生成针对 Irs1 (apS/TmAbIrs1) 的库； apS/TmAbIrs2 库已经在进行中并得到其他来源的支持。 2. 利用 pS/TmAblrs1 和 pS/TmAbIrs2 文库解码代谢应激期间 Irs1 和 Irs2 上发生的 S/T 磷酸化。 3. 使用 Irs1 等位基因被突变 A307Irs1 等位基因取代的小鼠研究 Ser307 在 Irs1 中的调节功能。 4. 确定Irsl中的Ser302对小鼠组织中的Irsl信号传导是否发挥正向或负向调节作用。 5. 确定 Jnk 导向的“激酶相互作用基序 (KIM) 在 Irsl 中的作用。该提议很重要，因为当胰腺 B 细胞无法分泌足够的胰岛素时，胰岛素信号传导失调会导致危及生命的代谢性疾病，进而发展为 2 型糖尿病足够快地补偿 Irs 1 和 Irs2 的 Ser/Thr 磷酸化是胰岛素抵抗的常见原因 apS/TmAbIrs1 和用于检测和定量磷酸化位点的 apS/TmAbIrs2 文库提供了一个合理的实验平台，用于解码控制整个生命周期胰岛素反应的复杂调节机制。