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， 疾病促进病理性心脏重塑和心力衰竭。在这些细胞和 病理生理变化是决定心脏表型的基因转录改变。IIa类 组蛋白去乙酰化酶是转录抑制因子，其核输出与诱导 病理性重塑这些HDAC受多个功能上相反的翻译后调节， 修饰，包括分别促进核输出和输入的PKD和PKA磷酸化。 支架蛋白mAKAPβ协调心肌细胞中病理性心脏病所需的信号传导 重塑而mAKAPβ结合的PKD是HDAC 5核输出所必需的， 受体刺激培养的新生大鼠心室肌细胞，mAKAPβ结合PKA赋予β-肾上腺素能 （βAR）抑制该过程。然而，βAR信号促进IIa类HDAC在细胞核中的保留， 仅在急性βAR刺激后。疾病中存在的慢性βAR刺激导致HDAC 5核 输出，也通过mAKAPβ依赖性机制。在这个项目中，我们将研究双向控制的 IIa类HDAC磷酸化和mAKAPβ信号体的核输出。我们建议， 信号传导部分是由于PKA诱导的蛋白磷酸酶2A（PP 2A）和盐诱导的蛋白磷酸酶2A（PP 2A）的存在。 mAKAPβ信号体中的激酶I（SIK 1）活性。具体目标1：mAKAPβ -IIa类HDAC的要求 病理性重塑中的复合物。本研究旨在对mAKAPβ- 含有HDAC 5和HDAC靶向MEF 2D的复合物，以及它们对儿茶酚胺的重要性。 体外诱导成年心肌细胞肥大。使用腺相关病毒将破坏肽递送至 在体内的心肌细胞，我们将测试是否抑制复合物的形成将防止病理性 重构和压力超负荷引起的心力衰竭。具体目标2：阐明 HDAC 5核输出的mAKAPβ依赖性β-肾上腺素能抑制。使用特定的mAKAP突变体 蛋白和锚定破坏蛋白，我们将剖析PKA依赖的HDAC 5 在抑制GqPCR诱导的HDAC 5核输出中，在mAKAPβ处的磷酸化和PP 2A活化。具体 目的3：在HDAC核输出和病理重塑中需要SIK 1。我们现在揭示， HDAC激酶SIK 1结合mAKAPβ，而mAKAPβ结合的PKA是肌细胞中SIK 1诱导所必需的。 我们将研究SIK 1及其通过mAKAPβ结合PKA的磷酸化是否是HDAC 5核转录所必需的。 体外输出，并使用条件性敲除检测SIK 1与体内成人心肌细胞的相关性 小鼠模型这些目的将阐明mAKAPβ信号体如何双向协调IIa型HDAC 在肌细胞中的功能。此外，该项目还将揭示靶向mAKAPβ信号体调节 HDAC在预防心脏重塑和心力衰竭方面可以是治疗有益的。

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.