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：
8012334
负责人：
DENISE Ratzlaff COOPER
金额：
$ 10万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-01 至 2010-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8012334
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 receptor response src-Family Kinases therapeutic target

项目摘要

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. Project Narrative 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.

前MRNA转录本的替代剪接是产生多肽多样性的一种宽度手段来自一个基因。但是，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。该实验室发现胰岛素调节蛋白激酶C-¿II（pKc¿ii）的替代剪接在其目标时机：肌肉，脂肪，肝脏和带有带有的细胞功能性胰岛素受体，例如主动脉平滑肌细胞，胚胎成纤维细胞和HELA细胞。我们确定了丝氨酸/精氨酸（SR）家族剪接蛋白的成员前MRNA中的增强剂调节外显子包容，因为这些因素响应于胰岛素。我们首先研究了SRP40，这是一种剪接增强子，并将AKT鉴定为调节其的激酶通过在精氨酸/丝氨酸（RS）结构域中残留物磷酸化的功能。我们假设AKT行动作为在几个步骤中剪接调节中的分子开关，还通过控制其他SR蛋白激酶例如CLK，一个四个双重功能板酶的家族。激酶，例如CLK1（也称为Clk/sty）和CLK2磷酸化SR蛋白并改变其在剪接体中的相互作用。揭开AKT的方式调节CLK将为胰岛素作用增加另一个级别的调节。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。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¿剪接中的功能，（3）识别剪接体胰岛素激活的剪接体和核因子的耗竭涉及的复合物使用体外剪接测定的提取物来定义参与胰岛素调节剪接的顺式元素。这胰岛素调节PKC¿II的胰岛素的发现，这是一种参与多个胰岛素反应的激酶级别，表明该途径的其他靶基因也必须在A中剪接类似的方式。独特的系统将揭示激酶调节的性质，重点介绍剪接和糖尿病。项目叙述鉴于糖尿病及其并发症遇到的问题的大小，请理解胰岛素作用对医疗保健有影响，因为它是与疾病有关的第六个主要原因在美国死亡。定义导致糖尿病发作并确定新潜力的因素的需求治疗靶标是优先事项。胰岛素刺激其靶标后，前MRNA的处理组织是一个知识渊博的区域，在糖尿病状态下发生了改变。了解胰岛素接收器信号通路的目的是定义如何反映在胰岛素响应时机将使我们能够通过胰岛素确定信号传导的特异性受体调节代谢功能，导致对胰岛素作用的抗性。该建议的设计研究胰岛素信号级联A及其核底物SR中的新激酶蛋白质，将RNA加工修饰以改变基因表达。 SR蛋白在胰岛素中改变反抗。