The role of phosphorylation of Ulk1 by GSK-3b in myocardial autophagy and aging

GSK-3b磷酸化Ulk1在心肌自噬和衰老中的作用

• 批准号: 8719900
• 负责人: PEIYONG ZHAI
• 金额: $ 18.78万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8719900
• 关键词: 1-Phosphatidylinositol 3-Kinase; Adenoviruses; Adverse effects; Affect; Aging; Amino Acid Sequence; Animal Organ; Animals; Apoptosis; Attenuated; Autophagocytosis; Autophagosome; Autopsy; Biological Assay; Cardiac; Cardiac Myocytes; Catheterization; Cell Aging; Cells; Cessation of life; Characteristics; Complex; Consensus; Defect; Echocardiography; Family; Fibroblasts; Fibrosis; Focal Adhesion Kinase 1; Functional disorder; Genes; Glucose; Glycogen; Glycogen Synthase Kinases; Growth; Heart; Heart Diseases; Heart failure; Homeostasis; Housekeeping; Human; Image; Immunoblotting; Impairment; In Vitro; Insulin Receptor; Knock-in Mouse; Knock-out; Knockout Mice; Knowledge; Lead; Left; Life; Lysosomes; Mammals; Mass Spectrum Analysis; Measurement; Membrane; Metabolism; Mitotic; Modeling; Mus; Myocardial; Necrosis; Neonatal; Organ Weight; Organelles; Phenotype; Phosphorylation; Phosphotransferases; Play; Population; Prevalence; Process; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins c-akt; Rattus; Regulation; Research Project Grants; Rest; Role; Scheme; Serine; Signal Pathway; Signal Transduction; Stress; Testing; Transgenic Mice; Vacuole; Ventricular; Work; age related; aged; animal tissue; deprivation; glycogen synthase kinase 3 beta; in vivo; insight; macromolecule; novel; premature; pressure; research study; senescence; treatment strategy

DESCRIPTION (provided by applicant): Glycogen syntheses kinase-3b (GSK-3b) is a ubiquitously expressed serine/heroine protein kinase that has multiple functions in cells, including growth, death and metabolism. GSK-3b is active under resting conditions and inhibited via phosphorylation at Serine9 (S9) by upstream protein kinase B/Akt. It has been shown that S9 phosphorylation of GSK-3b is highly elevated in the heart of old animals and in stress-induced cellular senescence. Previous work done by the applicant revealed that inhibition of GSK-3b inhibits autophagy in the heart under stress conditions. Accumulating lines of evidence suggest that a decrease in autophagy activity may play a major role in accumulation of altered macromolecules and organelles, causing aging-related abnormalities. Our hypotheses are: 1) GSK-3b regulates autophagy by phosphorylating Unc-51 like kinase 1 (Ulk1), and 2) GSK-3b modulates cardiac aging through Ulk1-dependent regulation of autophagy. There are four specific aims in this proposal to test our hypotheses. In Aim 1, it will be determined if GSK-3b phosphorylates Ulk1 and if so, which residue of Ulk1 is phosphorylated by GSK-3b. An in vitro kinase assay and mass spectrometry will be used in this aim. In Aim 2, it will be determined if phosphorylation of Ulk1 by GSK-3b participates in modulation of autophagy. Glucose deprivation of neonatal rat cardiac myocytes will be used as a model to stimulate autophagy. We have generated adenovirus harboring mRFP-GFP-LC3 to study autophagic flux. In Aim 3, it will be determined, in constitutively active GSK-3bS9A knock-in mice and GSK- 3bS9A/mRFP-GFP-LC3 bigenic mice, if aging-associated changes, including autophagy, in the heart are attenuated. Postmortem measurements of organ weight, left ventricular catheterization, pressure-volume loop analysis, echocardiography, measurements of cardiac fibrosis, cardiac myocyte size, apoptosis and necrosis will be applied to characterize the aging-related changes in the heart. Immunoblotting of aging marker will also be carried out. Immunoblotting of p62 and LC3, and measurements of red and yellow puncta in images taken from cardiac sections of mRFP-GFP-LC3 transgenic mice and the bigenic mice will be employed to evaluate autophagy and autophagic flux in vivo. In Aim 4, it will be determined, in GSK-3bS9A/Ulk1+/- bigenic mice, if knock-out of Ulk1 attenuates the effects of GSK-3b on aging-associated changes in the heart. Information obtained from the proposed studies will provide insights into the role of GSK-3ss and autophagy in aging and help to develop strategies specifically targeting GSK-3b and autophagy to modulate the adverse effects of aging.

描述（由申请人提供）：糖原合成激酶-3b（GSK-3B）是一种普遍表达的丝氨酸/女英雄蛋白激酶，在细胞中具有多个功能，包括生长，死亡和代谢。 GSK-3B在静息条件下活跃，并通过上游蛋白激酶B/AKT在丝氨酸9（S9）处抑制。已经表明，在老动物的心脏和应激诱导的细胞衰老中，GSK-3B的S9磷酸化高度升高。申请人所做的先前工作表明，在压力条件下，抑制GSK-3B在心脏中抑制自噬。积累的证据线表明，自噬活性的降低可能在改变大分子和细胞器的累积中起主要作用，从而导致与衰老有关的异常。我们的假设是：1）GSK-3B通过磷酸化UNC-51（如激酶1（ULK1）和2）GSK-3B来调节自噬来调节自噬调节的心脏老化。该提案中有四个特定的目标来检验我们的假设。在AIM 1中，将确定GSK-3B是否磷酸化ULK1，如果是，则ULK1残基被GSK-3B磷酸化。该目标将使用体外激酶测定法和质谱法。在AIM 2中，将确定GSK-3B对ULK1的磷酸化是否参与自噬的调节。新生儿大鼠心肌细胞的葡萄糖剥夺将被用作刺激自噬的模型。我们已经产生了带有MRFP-GFP-LC3的腺病毒，以研究自噬通量。在AIM 3中，将在组成型活性的GSK-3BS9A敲入小鼠和GSK-3BS9A/MRFP-GFP-LC3临后小鼠中确定，如果患有衰老相关的变化（包括自噬）在心脏中被减弱。验尸测量器官重量，左心室导管，压力卷回路分析，超声心动图，心脏纤维化的测量值，心肌大小，凋亡和坏死将用于表征心脏中与衰老相关的变化。还将进行衰老标记的免疫印迹。 p62和LC3的免疫印迹，以及从MRFP-GFP-LC3转基因小鼠的心脏部分拍摄的红色和黄色点的测量值，将使用bige虫小鼠在体内评估自噬和自噬细胞。在AIM 4中，它将在GSK-3BS9A/ULK1 +/-胆汁顽固小鼠中确定，如果ULK1的敲除可以减轻GSK-3B对与衰老相关的心脏变化的影响。从拟议的研究中获得的信息将提供有关GSK-3S和自噬在衰老中的作用的见解，并有助于制定专门针对GSK-3B和自噬来调节衰老的不良影响的策略。