The role of phosphorylation of Ulk1 by GSK-3b in myocardial autophagy and 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/海洛因蛋白激酶，在细胞中具有多种功能，包括生长、死亡和代谢。 GSK-3b 在静息条件下具有活性，并通过上游蛋白激酶 B/Akt 的丝氨酸 9 (S9) 磷酸化受到抑制。研究表明，GSK-3b 的 S9 磷酸化在老年动物的心脏和应激诱导的细胞衰老中高度升高。申请人之前所做的工作表明，抑制GSK-3b会抑制应激条件下心脏中的自噬。越来越多的证据表明，自噬活性的降低可能在改变的大分子和细胞器的积累中发挥重要作用，从而导致与衰老相关的异常。我们的假设是：1) GSK-3b 通过磷酸化 Unc-51 样激酶 1 (Ulk1) 来调节自噬，2) GSK-3b 通过 Ulk1 依赖性自噬调节来调节心脏衰老。该提案有四个具体目标来检验我们的假设。在目标 1 中，将确定 GSK-3b 是否磷酸化 Ulk1，如果是，则确定 Ulk1 的哪个残基被 GSK-3b 磷酸化。体外激酶测定和质谱分析将用于此目的。在目标 2 中，将确定 GSK-3b 对 Ulk1 的磷酸化是否参与自噬的调节。新生大鼠心肌细胞的葡萄糖剥夺将被用作刺激自噬的模型。我们生成了含有 mRFP-GFP-LC3 的腺病毒来研究自噬通量。在目标 3 中，将确定在组成型活性 GSK-3bS9A 敲入小鼠和 GSK-3bS9A/mRFP-GFP-LC3 双基因小鼠中，心脏中与衰老相关的变化（包括自噬）是否减弱。死后器官重量测量、左心室导管插入术、压力-容积环分析、超声心动图、心脏纤维化测量、心肌细胞大小、细胞凋亡和坏死将用于表征心脏中与衰老相关的变化。还将进行衰老标记物的免疫印迹。 p62 和 LC3 的免疫印迹以及从 mRFP-GFP-LC3 转基因小鼠和双基因小鼠心脏切片拍摄的图像中红色和黄色斑点的测量将用于评估体内自噬和自噬流。在目标 4 中，将确定在 GSK-3bS9A/Ulk1/- 双基因小鼠中，敲除 Ulk1 是否会减弱 GSK-3b 对心脏衰老相关变化的影响。从拟议研究中获得的信息将深入了解 GSK-3ss 和自噬在衰老中的作用，并有助于制定专门针对 GSK-3b 和自噬的策略，以调节衰老的不利影响。 公共卫生相关性：老龄化人口中心脏病的患病率急剧增加。本申请中提出的研究项目将获得信息，以帮助制定调节与衰老相关的心脏异常的策略。

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. PUBLIC HEALTH RELEVANCE: The prevalence of heart diseases increases dramatically in aging populations. The research project proposed in this application will obtain information to help developing strategies to modulate the aging-related abnormalities in the heart.