Synemin is an A-Kinase Anchoring Protein in the Heart

描述（由申请人提供）：心肌细胞中肌丝的组织， 心肌细胞的表型对施加在细胞上的应力有反应。通过中间丝（IF）的细胞骨架网络，应力和应变经由肋节和肌节的Z盘的蛋白质复合物传递到细胞外基质（ECM）和从细胞外基质（ECM）传递。我们已经表明，IF，synemin，与结蛋白或波形蛋白形成杂聚物，也是一种A-激酶锚定蛋白（AKAP）。AKAP通过与调节亚基（R）高亲和力结合来调节cAMP依赖性蛋白激酶（PKA）在细胞中的分布， 特别是RII，从而靶向PKA接近其底物。大多数AKAP是多功能的支架蛋白，作为几个信号通路的整合节点。我们确定 RII在synemin C-末端尾上的结合位点，synemin的一个区域，其将IF与Z-盘、肋节和细胞内细胞器交联。IF和IF的磷酸化 相关蛋白（IFAP）调节IFs的聚合。这种磷酸化为IFs构成信号通路的内在成分的观点提供了强有力的支持。我们推测，synemin锚定PKA调节磷酸化的肌原纤维和细胞骨架的基板，这反过来又调节细胞骨架和肌丝组织在心脏收缩和心脏肥大。这些假设将在3个具体目标中进行测试：在具体目标1中，我们将通过以下方式确定synemin作为支架蛋白的作用：（i）鉴定synemin结合蛋白的大分子复合物的组分， (ii)研究复合体的动态调节。这将通过免疫沉淀的synemin结合蛋白的无凝胶蛋白质组学分析进行。在具体目标2中，我们将确定synemin在靶向PKA中的作用，以磷酸化邻近的PKA底物。实验将在表达synemin的脯氨酸化衍生物（synemin-P）的新生儿和成人心肌细胞中进行，该衍生物不结合RII。然后我们将确定PKA靶向丧失对底物磷酸化的影响。在具体目标3中，我们将研究synemin靶向PKA在分离的新生和成年大鼠心肌细胞和大鼠心脏在体内的作用。我们将（i）比较心肌细胞表型（通过RNAi敲低synemin，或通过腺病毒基因转移表达synemin-P），然后评价synemin-P的腺病毒基因转移后（ii）分离的肌细胞和（iii）体内大鼠心脏中的收缩反应。总的来说，这些研究将为synemin靶向PKA在调节细胞骨架蛋白中的作用提供新的见解。肌原纤维底物磷酸化和收缩反应在生理和肥大的压力。

DESCRIPTION (provided by applicant): The organization of myofilaments in cardiac myocytes and the phenotypes of myocardial cells are responsive to stresses exerted on the cell. By means of a cytoskeletal network of intermediate filaments (IF), stress and strain are transmitted to and from the extracellular matrix (ECM) via protein complexes of the costameres and the Z-discs of the sarcomeres. We have shown that the IF, synemin, which forms heteropolymers with desmin or vimentin, is also an A-kinase anchoring protein (AKAP). AKAPs regulate cAMP-dependent protein kinase (PKA) distribution in cells by binding with high affinity to the regulatory subunit (R), especially RII, of PKA, thus targeting PKA close to its substrate. Most AKAPs are multifunctional scaffolding proteins, acting as nodes of integration for several signaling pathways. We identified a binding site for RII on the synemin C-terminal tail, a region of synemin, which cross-links IFs, with the Z-disc, and with the costamere and intracellular organelles. Phosphoylation of IFs and IF associated proteins (IFAPs) regulate polymerization of IFs. This phosphorylation provides strong support for the idea that IFs constitute intrinsic components of signaling pathways. We hypothesize that synemin-anchored PKA regulates phosphorylation of myofibrillar and cytoskeletal substrates, which in turn regulates cytoskeletal and myofilament organization in the heart during contraction and during cardiac hypertrophy. These hypotheses will be tested in 3 Specific Aims: In Specific Aim 1, we will determine the role of synemin as a scaffolding protein by (i) identifying the components of the macromolecular complex of synemin binding proteins and (ii) investigating the dynamic regulation of the complex. This will be performed by gel-free proteomic analysis of immunoprecipitated synemin binding proteins. In Specific Aim 2, we will determine the role of synemin in targeting PKA in order to phosphoylate adjacent PKA substrates. Experiments will be carried out in neonatal and adult cardiac myocytes expressing the prolinated derivative of synemin, (synemin-P), which does not bind RII. We will then determine the effect of loss of PKA targeting on substrate phosphoylation. In Specific Aim 3, we will examine the role of synemin targeted PKA in isolated neonatal and adult rat cardiac myocytes and in rat heart in vivo. We will (i) compare cardiac myocytes phenotype (upon synemin knockdown by RNAi, or by synemin-P expression by adenoviral gene transfer), then evaluate contractile response (ii) in isolated myocytes and (iii) in rat hearts in vivo after adenoviral gene transfer of synemin-P. Overall, these studies will provide new insights into the roles of synemin-targeted PKA in regulating cytoskeletal/myofibrillar substrate phosphoylation and contractile response during physiological and hypertrophic stress.