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：
7466756
负责人：
DENISE Ratzlaff COOPER
金额：
$ 28.63万
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/7466756
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 Classification Code Complementary DNA Complex Condition DNA Polymerase II DNA Sequence Rearrangement Data Diabetes Mellitus Disease Documentation Elements 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 Heterogeneous Nuclear RNA Hormonal Hormones Human Human Genome IGF1 gene In Vitro Insulin Insulin Receptor Insulin Resistance Insulin Signaling Pathway Insulin-Like Growth Factor I Introns KLK3 gene Ligands Liver Mass Spectrum Analysis Mediating Messenger RNA Metabolic Modeling Molecular Monitor Muscle Muscle Cells Mutate Myoblasts Nature Nuclear Nuclear Extract Numbers Oligonucleotides Pathway interactions Phosphatidylinositols Phosphoinositide-3-Kinase, Catalytic, Gamma Polypeptide Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Phosphotransferases Play Prevalence Principal Investigator Process Protein Binding Protein Dephosphorylation Protein Isoforms Protein Kinase Protein Kinase C Protein Overexpression Protein Splicing Protein-Serine-Threonine Kinases Proteins Proto-Oncogene Proteins c-akt Public Health Pyrimidine Pyrimidines RAC-Alpha Serine/Threonine Kinase 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 human SH2B protein insulin sensitivity insulin signaling mRNA Precursor member myotonic dystrophy protein kinase numb protein polypeptide protein function protein tyrosine phosphatase 1B 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MRNA转录本的替代剪接是从单个基因产生多肽多样性的普遍手段。超过60％的人类基因是通过替代剪接表达的，但是，剪接调节的机制知之甚少。 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前MRNA调节外显子纳入胰岛素的磷酸化，我们首先研究了SRP40，这是一种剪接增强子，并确定AKT是一种激酶，通过在精液/丝氨酸（RS）域中通过SPITIC进行了SPICER的STRICTARTIAL STICALS SCHOTIEL SCORTULTAR SCTICTS SCOTCONT SCHOTIONT AKT磷酸化。蛋白质激酶，例如四个双重功能板激酶的家族，例如CLK1（也称为CLK/Sty）和CLK2磷酸化SR蛋白，并改变其在剪接中的相互作用。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的选择。当前的目的将研究（1）CLK1和CLK2磷酸化在PKC中的作用（替代剪接，（2）确定SRP55在PKC（拼接和（3）中涉及胰岛素激活的夹层和插入型核酸内液体涉及的夹杂物的剪接体复合物中的SRP55功能（剪接，（3））剪接。胰岛素调节PKC的剪接（ii，一种在多个级别涉及胰岛素反应的激酶，表明该途径的其他目标基因也必须以类似的方式剪接。独特的系统将揭示与PKC相关的动力酶调节的性质并发症，理解胰岛素作用对医疗保健产生了巨大影响，因为它是美国与疾病相关的第六个主要原因。胰岛素刺激其靶组织后，前MRNA的处理是一个知识渊博的区域，在糖尿病状态下发生了改变。理解胰岛素受体信号通路的目的是确定如何在胰岛素反应组织的核中反映胰岛素作用，这将使我们能够通过胰岛素受体来确定信号传导的特异性以调节代谢功能，从而导致抗胰岛素作用。该建议旨在研究胰岛素信号级联，clk/sty及其核底物SR蛋白中的新型激酶，该蛋白会修改RNA加工以改变基因表达。 SR蛋白在胰岛素抵抗中改变。