Regulation of Histone Deacetylases by mAKAP Signalosomes

心脏对慢性应激的反应包括激活心肌细胞信号转导网络，该网络在 疾病促进病理性心脏重塑和心力衰竭。在这些细胞和 病理生理改变是决定心脏表型的基因转录的改变。IIa类 组蛋白去乙酰基酶是转录抑制因子，其核输出与诱导 病理性重构。这些HDAC由多个在功能上相反的翻译后管理 修饰，包括分别促进核出口和进口的PKD和PKA的磷酸化。 支架蛋白mAKAPβ在病理性心脏所需的心肌细胞中协调信号传递 改建。而mAKAPβ结合的PKD是HDAC5核输出所必需的，以响应α-肾上腺素能 受体刺激培养的乳鼠心室肌细胞，mAKAPβ结合的PKA可赋予β肾上腺素能 (βAR)抑制这一过程。然而，βAR信号促进IIa类HDAC在细胞核中的滞留， 仅在急性βAR刺激时。疾病中存在的慢性βAR刺激导致HDAC5核 导出，也通过mAKAPβ依赖的机制。在这个项目中，我们将研究双向控制 MAKAPβ信号体的IIa类hdac磷酸化和核输出。我们建议将此转换为 信号转导的部分原因是PKA诱导的蛋白磷酸酶2A(PP2A)和盐诱导的 MAKAPβ信号体中的激酶I(SIK1)活性具体目标1：对MAKAPβ-IIa级HDAC的要求 病理重塑中的复合体。在这个目标中，我们将表征mAKAPβ的结构和功能- 含HDAC5和HDAC靶标MEF2D的络合物及其对儿茶酚胺的重要性- 体外诱导成人心肌细胞肥大。使用腺相关病毒将干扰肽传递到 在活体心肌细胞中，我们将测试抑制复合体的形成是否会防止病理 压力超负荷所致的重塑和心力衰竭。具体目标2：阐明 β依赖的β-肾上腺素能抑制HDAC5核输出。使用特定的mAKAP突变体 蛋白质和锚定干扰蛋白，我们将剖析依赖PKA的HDAC5的相关作用 MAKAPβ的磷酸化和PP2A激活抑制GqPCR诱导的HDAC5核输出。特定的 目的3：HDAC核输出和病理重塑对SIK1的要求。我们现在揭晓， HDAC激酶SIK1与mAKAPβ结合，mAKAPβ结合的PKA是诱导SIK1进入心肌细胞所必需的。 我们将研究hDAC5核是否需要SIK1及其由mAKAPβ结合的PKA的磷酸化 用条件性基因敲除法体外提取SIK1并检测其与在体成人心肌细胞的相关性 老鼠模型。这些目的将阐明mAKAPβ信号体是如何双向协调IIa型HDAC型的 在肌细胞中的功能。此外，该项目还将揭示靶向mAKAPβ信号小体如何调控 HDAC在预防心脏重塑和心力衰竭方面有治疗上的好处。

The cardiac response to chronic stress involves the activation of a myocyte signal transduction network that in disease promotes pathological cardiac remodeling and heart failure. Underlying these cellular and pathophysiological changes is the altered transcription of genes that determine cardiac phenotype. Class IIa histone deacetylases are transcriptional repressors whose nuclear export is associated with the induction of pathological remodeling. These HDACs are regulated by multiple, functionally opposing post-translational modifications, including phosphorylation by PKD and PKA that promote nuclear export and import, respectively. The scaffold protein mAKAPβ orchestrates signaling in the cardiac myocyte required for pathological cardiac remodeling. Whereas mAKAPβ-bound PKD was required for HDAC5 nuclear export in response to α-adrenergic receptor stimulation of cultured neonatal rat ventricular myocytes, mAKAPβ-bound PKA conferred β-adrenergic (βAR) inhibition of that process. βAR signaling promoting class IIa HDAC retention in the nucleus is, however, only upon acute βAR stimulation. Chronic βAR stimulation as present in disease resulted in HDAC5 nuclear export, also by a mAKAPβ-dependent mechanism. In this project, we will investigate the bidirectional control of class IIa HDAC phosphorylation and nuclear export by mAKAPβ signalosomes. We propose that this switch in signaling is due in part to the presence of PKA-inducible protein phosphatase 2A (PP2A) and salt-inducible kinase I (SIK1) activity in mAKAPβ signalosomes. Specific Aim 1: Requirement for mAKAPβ - class IIa HDAC complexes in pathological remodeling. In this Aim we will characterize the structure and function of mAKAPβ- complexes containing HDAC5 and the HDAC target MEF2D, as well as their importance for catecholamine- induced hypertrophy of adult myocytes in vitro. Using adeno-associated virus to deliver the disruptor peptide to the cardiac myocyte in vivo, we will test whether inhibited complex formation will prevent the pathological remodeling and heart failure induced by pressure overload. Specific Aim 2: Elucidation of the mechanism for mAKAPβ-dependent β-adrenergic inhibition of HDAC5 nuclear export. Using specific mAKAP mutant proteins and anchoring disruptor proteins, we will dissect the relative roles of PKA-dependent HDAC5 phosphorylation and PP2A activation at mAKAPβ in inhibiting GqPCR-induced HDAC5 nuclear export. Specific Aim 3: Requirement for SIK1 in HDAC nuclear export and pathological remodeling. We now reveal that the HDAC kinase SIK1 binds mAKAPβ and that mAKAPβ-bound PKA is required for SIK1 induction in myocytes. We will study whether SIK1 and its phosphorylation by mAKAPβ-bound PKA is required for HDAC5 nuclear export in vitro and test the relevance of SIK1 to the adult cardiac myocyte in vivo using a conditional knock-out mouse model. These Aims will elucidate how mAKAPβ signalosomes bidirectionally coordinate type IIa HDAC function in myocytes. In addition, this project will reveal how targeting of mAKAPβ signalosome regulation of HDACs can be therapeutically beneficial in the prevention of cardiac remodeling and heart failure.