权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.

前体mRNA转录物的选择性剪接是产生多肽多样性的广泛手段从一个基因。超过60%的人类基因是通过选择性剪接表达的，然而，剪接调节的机制知之甚少。这个实验室发现胰岛素调节蛋白激酶C-<$II（PKC <$II）在其靶组织中的选择性剪接：肌肉，脂肪，肝脏和细胞，功能性胰岛素受体，如主动脉平滑肌细胞、胚胎成纤维细胞和HeLa细胞。我们鉴定了富含丝氨酸/精氨酸（SR）家族剪接蛋白的成员，前体mRNA中的增强子调节外显子包含，作为响应于胰岛素我们首先研究了SRp 40，一种剪接增强子，并将Akt鉴定为一种调节其表达的激酶。通过精氨酸/丝氨酸（RS）结构域中残基的磷酸化起作用。我们假设Akt 作为剪接调节的分子开关，在几个步骤中也调节其他SR蛋白激酶例如Clk，一个四种双功能LAMMER激酶家族。激酶如Clk 1（也称为Clk/Sty）和Clk 2磷酸化SR蛋白并改变它们在剪接体中的相互作用。揭开Akt 调节Clk将为胰岛素作用增加另一个调节水平。这项研究的长期目标是确定胰岛素如何通过激活各种激酶调节核剪接位点选择，剪接因子目前的目的是研究（1）Clk 1和Clk 2磷酸化在PKC？选择性剪接，（2）确定SRp 55在PKC剪接中的功能，（3）鉴定剪接体参与胰岛素激活剪接体的复合物和核内剪接因子的耗竭提取物，使用体外剪接测定来确定参与胰岛素调节剪接的顺式元件。的发现胰岛素调节蛋白激酶C Ⅱ的剪接，蛋白激酶C Ⅱ是一种参与多种胰岛素反应的激酶，水平，表明还有其他靶基因的这一途径，也必须剪接在一个类似的方式。独特的系统将揭示激酶调节的本质，重点是PKC Ⅱ，剪接和糖尿病。项目叙述鉴于糖尿病及其并发症所遇到的问题的严重性，胰岛素作用对医疗保健有巨大的影响，因为它是与疾病相关的第六大原因。死在美国。需要定义导致糖尿病发病的因素并发现新的潜力治疗目标是优先考虑的。胰岛素刺激其靶点后前体mRNA的加工组织是在糖尿病状态下改变的知之甚少的区域。了解胰岛素受体信号通路，目的是确定胰岛素作用如何反映在细胞核中，胰岛素反应组织将使我们能够确定通过胰岛素信号的特异性，受体调节代谢功能，导致胰岛素抵抗。该提案旨在研究胰岛素信号级联中的新激酶Clk/Sty及其核底物SR 蛋白质，它们修饰RNA加工以改变基因表达。胰岛素中的SR蛋白发生改变阻力