A-Kinase Anchoring Protein Dysregulation during Alcohol-Associated Liver Disease

项目摘要 脂肪肝会增强酒精相关性肝病（ALD）的发展。AKAP 12（A-激酶锚定蛋白 12)是通过锚定蛋白激酶（PKA、PKC）、细胞周期蛋白-D1和热信号转导的支架蛋白 休克蛋白47。我们最近的蛋白质组学研究表明，AKAP 12与 在小鼠模型和人ALD中，酒精可增加激酶与磷酸酶的相互作用。这导致我们 研究酒精是否调节AKAP 12磷酸化。通过磷酸肽图谱，我们确定 酒精强烈抑制小鼠肝脏中AKAP 12的磷酸化。AKAP 12的酒精敏感磷酸化 位点主要是PKA预测的底物。突变这些磷酸化位点降低了AKAP 12的稳定性， 蛋白质，表明磷酸化稳定AKAP 12。AKAP 12相互作用组揭示了其相互作用 与已知调节脂质体内平衡的蛋白质如PKA、β 2-肾上腺素能受体（β 2-AR）和脂解 脂肪甘油三酯脂肪酶（ATGL）。因此，我们评估了AKAP 12的功能作用。 肝细胞中的相互作用体。以前的研究表明，AKAP 12介导的PKA支架将其募集到AKAP 2- 1， AR. AKAP 12、PKA和β 2-AR的信号体通过β 2-AR去磷酸化过程调节cAMP水平。 致敏/再致敏并且可能涉及AKAP 12磷酸化。β 2-AR/PKA/cAMP信号传导限制脂质 积累并增强脂肪分解。我们新的初步数据表明，酒精暴露失调， AKAP 12/PKA/PK 2-AR信号体的几种组分。酒精抑制PKA与 2002-AR。AKAP 12在S696-S698 PKA磷酸化位点高度磷酸化，该位点先前已发表于 促进AKAP 12-β 2-AR结合。该位点在酒精暴露后表现出磷酸化降低， 使该AKAP 12位点突变减少了其对PKA和AKAP 2-AR的支架。这种AKAP 12突变也 降低的β 2-AR蛋白稳定性。酒精干扰AKAP 12-β 2-AR相互作用，并促进AKAP 12和β 2-AR相互作用。 β 2-AR溶酶体降解。基于这些数据，我们假设AKAP 12磷酸化水平降低， 酒精暴露后可能干扰AKAP 12/PKA/β 2-AR信号体，导致cAMP的改变， 信号传导，这反过来又可能失调脂质稳态。支持这一假设的初步数据是： 1)增强内源性AKAP 12水平逆转酒精对PKA-AKAP 2-AR相互作用的抑制作用; 2） AKAP 12诱导抑制酒精介导的甘油三酯水平增加; 3）AKAP 12诱导逆转 酒精对脂肪酸氧化的抑制作用。AKAP 12的诱导也逆转了酒精对细胞增殖的抑制作用。 影响脂肪分解酶ATGL活性。AKAP 12/PKA/PKA 2-AR支架是否控制脂质 体内平衡是未知的。我们假设AKAP 12信号体的失调有利于脂质过氧化。 积累该提案将评估酒精敏感性AKAP 12磷酸化如何使AKAP 12不稳定， 损害其支架活性并导致脂质体内平衡失调。的治疗益处 将在ALD的小鼠模型中测试AKAP 12磷酸稳定化在改善脂质功能障碍中的作用。

PROJECT SUMMARY Fatty liver potentiates alcohol-associated liver disease (ALD) development. AKAP12 (A-kinase anchoring protein 12) is a scaffolding protein in signal transduction by anchoring protein kinases (PKA, PKC), cyclin-D1 and heat shock protein 47. Our recent proteomics studies showed that AKAP12 exhibited decreased interaction with kinases and increased interaction with phosphatases by alcohol in mouse models and human ALD. This led us to examine whether alcohol regulated AKAP12 phosphorylation. By phospho-peptide mapping we determined that alcohol strongly inhibited AKAP12 phosphorylation in mouse liver. AKAP12’s alcohol-sensitive phospho- sites were mainly PKA-predicted substrates. Mutating these phospho-sites reduced the stability of AKAP12 protein, suggesting that phosphorylation stabilized AKAP12. The AKAP12 interactome revealed its interactions with proteins known to regulate lipid homeostasis such as PKA, 2-adrenergic receptor (2-AR) and lipolytic enzymes, adipose-triglyceride lipase (ATGL). We therefore assessed the functional role of this AKAP12 interactome in hepatocytes. It was shown previously that AKAP12-mediated PKA scaffolding recruits it to 2- AR. The signalosome of AKAP12, PKA and 2-AR regulates cAMP levels through the process of 2-AR de- sensitization/re-sensitization and may involve AKAP12 phosphorylation. 2-AR/PKA/cAMP signaling limits lipid accumulation and enhances lipolysis. Our novel preliminary data indicates that alcohol exposure dysregulates several components of the AKAP12/PKA/2-AR signalosome. Alcohol inhibited the interaction between PKA and 2-AR. AKAP12 was highly phosphorylated at a S696-S698 PKA-phospho-site that was previously published to facilitate AKAP12-2-AR binding. This site exhibited decreased phosphorylation upon alcohol exposure and mutating this AKAP12 site reduced its scaffolding towards both PKA and 2-AR. This AKAP12 mutation also reduced 2-AR protein stability. Alcohol interfered with AKAP12-2-AR interaction and facilitated AKAP12 and 2-AR lysosomal degradation. Based on these data, we hypothesize that decreased AKAP12 phosphorylation following alcohol exposure might interfere with AKAP12/PKA/2-AR signalosome, causing alterations in cAMP signaling, which in turn could dysregulate lipid homeostasis. Preliminary data that support this hypothesis are: 1) Enhancing endogenous AKAP12 levels reverses the inhibitory effect of alcohol on PKA-2-AR interaction; 2) AKAP12 induction inhibits alcohol-mediated increase of triglyceride levels; 3) AKAP12 induction reverses alcohol’s suppressive effect on fatty acid oxidation. Induction of AKAP12 also reverses alcohol’s suppressive effect on the activity of lipolytic enzyme, ATGL. Whether the AKAP12/PKA/2-AR scaffold controls lipid homeostasis is unknown. We hypothesize that dysregulation of the AKAP12 signalosome favors lipid accumulation. This proposal will evaluate how alcohol-sensitive AKAP12 phosphorylation destabilizes AKAP12, impairs its scaffolding activities and contributes to dysregulation of lipid homeostasis. The therapeutic benefit of AKAP12 phospho-stabilization in ameliorating lipid dysfunction will be tested in mouse models of ALD.