Synemin is an A-Kinase Anchoring Protein in the Heart

描述(申请人提供)：心肌细胞中肌丝的组织和 心肌细胞的表型对施加在细胞上的压力有反应。通过中间纤维的细胞骨架网络(IF)，应力和应变通过肌节的胞腔和Z盘蛋白复合体传递到细胞外基质(ECM)和细胞外基质(ECM)。我们已经证明，与结蛋白或波形蛋白形成杂聚体的IF、SYNMENT也是一种A-激酶锚定蛋白(AKAP)。AKAP通过与调节亚单位(R)高亲和力结合来调节细胞内cAMP依赖的蛋白激酶(PKA)的分布， 特别是PKA的RII，从而靶向靠近其底物的PKA。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。我们确认了 RII在同步素C-末端尾部的结合位点，同步素的一个区域，它将IF与Z-盘以及与胞间细胞器和胞间细胞器交织在一起。IF和IF的磷酸化 相关蛋白(IFAP)调节IF的聚合。这种磷酸化为IF构成信号通路的内在成分的观点提供了强有力的支持。我们假设，同步素锚定的PKA调节肌原纤维和细胞骨架底物的磷酸化，进而调节心脏收缩和心肌肥厚期间的细胞骨架和肌丝组织。这些假说将在3个特定目标中进行检验：在特定目标1中，我们将通过(I)鉴定SynMinin结合蛋白和蛋白质的大分子复合体的成分来确定Synmin作为支架蛋白的作用。 (二)调查综合体的动态调节情况。这将通过免疫沉淀同步蛋白结合蛋白的无胶蛋白质组学分析来完成。在特定的目标2中，我们将确定同步蛋白在靶向PKA中的作用，以便磷酸化相邻的PKA底物。实验将在新生和成人心肌细胞中进行，表达不结合RII的同步素的脯氨酸衍生物(同步素-P)。然后我们将确定失去PKA靶向对底物磷酸化的影响。在特定的目标3中，我们将检测在分离的乳鼠和成年大鼠心肌细胞和在体大鼠心脏中Synminin靶向PKA的作用。我们将(I)比较心肌细胞的表型(通过RNAi敲除同步蛋白后，或通过腺病毒基因转移抑制同步蛋白P的表达)，然后评估(Ii)在分离的心肌细胞中的收缩反应，(Iii)在活体大鼠心脏中对腺病毒基因转移同步蛋白-P的反应。总体而言，这些研究将提供新的洞察力，以同步素靶向PKA在生理和肥大应激过程中调节细胞骨架/肌原纤维底物的磷酸化和收缩反应中的作用。

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.