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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：
7871748
负责人：
DENISE Ratzlaff COOPER
金额：
$ 12.98万
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/7871748
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 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.

mRNA 前体转录本的选择性剪接是产生多肽多样性的一种广泛手段来自单个基因。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可以调节蛋白激酶 C-II (PKCII) 在其靶组织中的选择性剪接：肌肉、脂肪、肝脏以及具有功能性胰岛素受体，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，这些剪接蛋白与剪接结合前体 mRNA 中的增强子可调节外显子包含，因为这些因子响应于磷酸化胰岛素。我们首先研究了 SRp40（一种剪接增强子），并确定 Akt 是调节其剪接增强子的激酶。通过精氨酸/丝氨酸 (RS) 结构域中残基的磷酸化发挥作用。我们假设 Akt 起作用作为剪接调节中的分子开关，还可以调节其他 SR 蛋白激酶例如 Clk，一个由四种双功能 LAMMER 激酶组成的家族。激酶，例如 Clk1（也称为 Clk/Sty） Clk2 磷酸化 SR 蛋白并改变它们在剪接体中的相互作用。揭开 Akt 的神秘面纱调节 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来调节核剪接位点选择拼接因素。当前的目标将研究 (1) Clk1 和 Clk2 磷酸化在 PKC 中的作用¿ 选择性剪接，(2) 确定 SRp55 在 PKC 剪接中如何发挥作用，以及 (3) 识别剪接体参与胰岛素激活剪接体和核内剪接因子消耗的复合物使用体外剪接测定提取物来定义参与胰岛素调节剪接的顺式元件。这发现胰岛素调节 PKC II 的剪接，PKC II 是一种参与多种胰岛素反应的激酶水平，表明该途径还有其他靶基因也必须剪接在类似的方式。独特的系统将揭示激酶调节的本质，重点关注 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，了解胰岛素作用对医疗保健具有巨大影响，因为它是疾病相关的第六大原因在美国死亡。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首要任务。胰岛素刺激其靶标后前体 mRNA 的加工组织是一个我们知之甚少的区域，在糖尿病状态下会发生改变。了解胰岛素受体信号传导途径，旨在定义胰岛素作用如何在细胞核中反映胰岛素反应组织将使我们能够确定胰岛素信号传导的特异性受体调节代谢功能，导致胰岛素作用抵抗。本提案设计研究胰岛素信号级联中的一种新激酶 Clk/Sty 及其核底物 SR 蛋白质，它修改 RNA 加工以改变基因表达。 SR 蛋白在胰岛素中发生改变反抗。