Insulin Signaling Pathways Regulating PKCBeta Splicing

调节 PKCβ 剪接的胰岛素信号通路

基本信息

批准号：
7778227
负责人：
DENISE Ratzlaff COOPER
金额：
$ 42.11万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-08-15 至 2012-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7778227
关键词：
1-Phosphatidylinositol 3-Kinase 3&apos Splice Site Abbreviations Actins Address Alternative Splicing Antisense Oligonucleotides Area Arginine Binding Binding Proteins Binding Sites Biological Assay C-terminal Cell Extracts Cell Nucleus Cell physiology Cells Cessation of life Code Complementary DNA Complex DNA Polymerase II DNA Sequence Rearrangement Data Diabetes Mellitus Disease Documentation Elements Embryo Endocrine system Enhancers Esters Event Evolution Excision Exercise Exons F-Actin Family Fatty acid glycerol esters Fibroblasts Fibronectins Gene Expression Gene Targeting Genes Genetic Transcription Glucose Glucosephosphate Dehydrogenase Goals Healthcare Hela Cells Hormonal Hormones Human Human Genome IGF1 gene In Vitro Insulin Insulin Receptor Insulin Resistance Insulin Signaling Pathway Introns Ligands Liver Mass Spectrum Analysis Mediating Messenger RNA Metabolic Modeling Molecular Monitor Muscle Muscle Cells Mutate Nature Nuclear Nuclear Extract Pathway interactions Phosphatidylinositols Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Phosphotransferases Play Prevalence Principal Investigator Process Protein Binding Protein Dephosphorylation Protein Isoforms Protein Kinase Protein Kinase C Protein Splicing Protein-Serine-Threonine Kinases Proteins Proto-Oncogene Proteins c-akt Pyrimidine Pyrimidines RNA RNA Polymerase II RNA Processing RNA Recognition Motif RNA Splicing Reaction Receptor Protein-Tyrosine Kinases Receptor Signaling Regulation Repression Research Resistance Role SH2B gene STY kinase Serine Serum Signal Pathway Signal Transduction Signal Transduction Pathway Signaling Protein Site Site-Directed Mutagenesis Skeletal Muscle Small Interfering RNA Smooth Muscle Myocytes Sodium Dodecyl Sulfate-PAGE Solid Specificity Spliceosomes Starvation Stress Surface System TNF gene Techniques Text Tissues Transcript Translations U1 Small Nuclear Ribonucleoprotein Untranslated Regions Variant Western Blotting cell type cis acting element design diabetic embryonic stem cell glucose uptake human ARMET protein insulin sensitivity insulin signaling mRNA Precursor member myotonic dystrophy protein kinase numb protein overexpression polypeptide protein function protein tyrosine phosphatase 1B public health relevance receptor response src-Family Kinases therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Alternative splicing of pre-mRNA transcripts is a widespread means for producing polypeptide diversity from a single gene. Over 60% of human genes are expressed through alternative splicing, however, mechanisms of splicing regulation are poorly understood. This lab discovered that insulin regulates the alternative splicing of protein kinase C-(II (PKC(II) in its target tissues: muscle, fat, liver and in cells with functional insulin receptor such as aortic smooth muscle cells, embryonic fibroblasts, and HeLa cells. We identified members of the Serine/Arginine-rich (SR) family splicing proteins that bind to splicing enhancers in the pre-mRNA to regulate exon inclusion as the factors phosphorylated in response to insulin. We first studied SRp40, a splicing enhancer and identified Akt as a kinase that regulated its function via phosphorylation of residues in the arginine/serine (RS) domain. We hypothesize that Akt acts as a molecular switch in splicing regulation at several steps by also regulating other SR protein kinases such as Clk, a family of four dual function LAMMER kinases. Kinases such as Clk1 (also called Clk/Sty) and Clk2 phosphorylate SR proteins and alter their interactions in the spliceosome. Unraveling how Akt regulates Clk will add another level of regulation to insulin action. The long-term goal of the research is to determine how insulin regulates nuclear splice site selection via the activation of various kinases and splicing factors. The current aims will investigate (1) the roles of Clk1 and Clk2 phosphorylation in PKC( alternative splicing, (2) determine how SRp55 functions in PKC( splicing, and (3) identify spliceosome complexes involved in the insulin activated spliceosome and depletion of splicing factors from nuclear extracts using in vitro splicing assays to define cis-elements involved in insulin regulated splicing. The discovery that insulin regulates splicing of PKC(II, a kinase involved in insulin responses at multiple levels, indicates that there are also other target genes of this pathway that must also be spliced in a similar manner. The unique system will reveal the nature of kinase regulation, focusing on PKC(II in splicing and diabetes. PUBLIC HEALTH RELEVANCE Given the magnitude of the problems encountered with diabetes and its complications, understanding insulin action has an immense impact on healthcare since it is the sixth leading cause of disease-related death in the US. The need to define the factors contributing to diabetes onset and identify new potential therapeutic targets is a priority. The processing of pre-mRNA following insulin stimulation of its target tissues is a poorly understood area that is altered in the diabetic state. Understanding the insulin receptor signaling pathways with the goal of defining how insulin action is reflected in the nucleus of insulin responsive tissues will allow us to determine the specificity of signaling through the insulin receptor to regulate metabolic functions causing resistance to insulin action. This proposal is designed to investigate a new kinase in the insulin signaling cascade, Clk/Sty, and its nuclear substrates, SR proteins, which modify RNA processing to alter gene expression. SR proteins are altered in insulin resistance.

描述（由申请人提供）：前体mRNA转录物的选择性剪接是从单个基因产生多肽多样性的广泛手段。超过60%的人类基因是通过选择性剪接表达的，然而，人们对剪接调控机制知之甚少。该实验室发现，胰岛素在其靶组织：肌肉、脂肪、肝脏以及具有功能性胰岛素受体的细胞（如主动脉平滑肌细胞、胚胎成纤维细胞和 HeLa）中调节蛋白激酶 C-(II (PKC(II)) 的选择性剪接我们鉴定了富含丝氨酸/精氨酸 (SR) 家族剪接蛋白的成员，这些剪接蛋白与前体 mRNA 中的剪接增强子结合，作为响应胰岛素的磷酸化因子来调节外显子包含。首先研究了剪接增强子 SRp40，并确定 Akt 是一种激酶，通过精氨酸/丝氨酸 (RS) 结构域中残基的磷酸化来调节其功能，我们假设 Akt 在剪接调节中充当分子开关，同时还调节其他步骤。 SR 蛋白激酶，例如 Clk，是四个双功能 LAMMER 激酶家族，例如 Clk1（也称为 Clk/Sty）和 Clk2。磷酸化 SR 蛋白并改变它们在剪接体中的相互作用。解开 Akt 如何调节 Clk 将为胰岛素作用增加另一个水平的调节。该研究的长期目标是确定胰岛素如何通过激活各种激酶和剪接因子来调节核剪接位点选择。当前的目标将研究 (1) Clk1 和 Clk2 磷酸化在 PKC( 选择性剪接中的作用，(2) 确定 SRp55 在 PKC( 剪接中的功能)，以及 (3) 识别参与胰岛素激活剪接体和剪接消耗的剪接体复合物使用体外剪接测定从核提取物中提取因子来定义参与胰岛素调节剪接的顺式元件。胰岛素调节 PKC(II，一种在多个水平上参与胰岛素反应的激酶) 的剪接，表明该途径还存在其他靶基因也必须以类似的方式进行剪接。独特的系统将揭示激酶调节的本质，重点关注剪接和糖尿病中的 PKC(II)。公共健康相关性鉴于糖尿病及其并发症所遇到的问题的严重性，了解胰岛素作用对医疗保健具有巨大影响，因为它是导致疾病相关的第六大原因确定导致糖尿病发病的因素并确定新的潜在治疗靶点是当务之急。靶组织受到胰岛素刺激后，前体 mRNA 的加工过程是一个人们知之甚少的领域，但在糖尿病状态下，前体 mRNA 的加工过程会发生改变。了解胰岛素受体信号传导途径的目的是确定胰岛素作用如何反映在胰岛素反应组织的细胞核中，这将使我们能够确定通过胰岛素受体调节代谢功能导致胰岛素作用抵抗的信号传导的特异性。该提案旨在研究胰岛素信号级联中的一种新激酶 Clk/Sty 及其核底物 SR 蛋白，这些蛋白通过修改 RNA 加工来改变基因表达。 SR 蛋白在胰岛素抵抗中发生改变。