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

描述(申请人提供)：糖原合成酶-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、死亡和新陈代谢。GSK-3b在静息条件下是活性的，并通过上游蛋白激酶B/Akt在丝氨酸9(S9)的磷酸化而被抑制。已有研究表明，在老年动物心脏和应激诱导的细胞衰老过程中，GSK-3b的S9磷酸化水平高度升高。申请人之前所做的工作表明，在压力条件下，抑制GSK-3b可以抑制心脏的自噬。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主要作用，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依赖的自噬调节来调节心脏衰老。这项提议有四个具体目的来检验我们的假设。在目标1中，将确定GSK-3b是否使Ulk1磷酸化，如果是，Ulk1的哪个残基被GSK-3b磷酸化。在这一目标中，将使用体外激酶分析和质谱仪。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。新生大鼠心肌细胞葡萄糖剥夺可作为刺激自噬的模型。我们已经制备了携带MRFP-GFP-LC3的腺病毒来研究自噬通量。在目标3中，将确定在结构性活跃的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是否被减弱，包括自噬。死后测量器官重量、左心室插管、压力-容量环分析、超声心动图、心脏纤维化、心肌细胞大小、细胞凋亡和坏死的测量将被用于表征心脏与年龄相关的变化。还将进行衰老标记物的免疫印迹。免疫印迹法检测p62和Lc3蛋白，并测量MRFP-GFP-Lc3转基因小鼠和双基因小鼠心脏切片中的红色和黄色斑点，以评估体内的自噬和自噬通量。在目标4中，将在GSK-3bS9A/ulk1+/-双基因小鼠中确定Ulk1基因敲除是否减弱了GSK-3b对衰老相关心脏变化的影响。从拟议的研究中获得的信息将为深入了解GSK-3SS和自噬在衰老中的作用提供见解，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。

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.