AKAP Regulation of PKA Targeting in the Heart

AKAP 对心脏 PKA 靶向的调节

• 批准号: 7541501
• 负责人: Meredith Bond
• 金额: $ 5.11万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-16 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7541501
• 关键词: A kinase anchoring protein; AKAP13 gene; Address; Adrenergic Agents; Adrenergic Receptor; Adult; Affect; Affinity; Alanine; Aspartate; Binding; Binding Proteins; Calcium/calmodulin-dependent protein kinase; Cardiac; Cardiac Myocytes; Catalytic Domain; Cells; Chinese Hamster Ovary Cell; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Development; Dilated Cardiomyopathy; Disruption; Down-Regulation; Echocardiography; Elevation; Endoplasmic Reticulum; Gene Transfer; Heart; Heart failure; Human; Isoproterenol; Link; Localized; Location; Measurement; Modeling; Mutate; Myosin ATPase; Object Attachment; Pathway interactions; Peptides; Phosphorylation; Principal Investigator; Protein Binding; Protein Kinase; Rattus; Regulation; Ryanodine Receptor Calcium Release Channel; Serine; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; Surface Plasmon Resonance; Testing; Therapeutic; Troponin I; Work; adrenergic; base; in vivo; insight; knock-down; mutant; novel; novel strategies; programs; protein expression; protein kinase A kinase; research study; response

Decreased cardiac response to /?-adrenergic stimulation is linked to development of heart failure. During heart failure, down-regulation of the /?-adrenergic receptor takes place, however downstream alterations in the pathway, i.e. regulation of substrate phosphorylation by cAMP-dependent protein kinase (PKA), are also involved. The major hypothesis tested in this proposal is that regulation of interaction between Rll and A-kinase anchoring proteins (AKAPs) provides a novel mechanism of regulating the cardiac response to activation of the (3-adrenergic signaling pathway. PKA, primarily PKA Type II (PKA II), is targeted to its substrates by high affinity binding via the regulatory subunits, Rll, of PKA, to AKAPs. AKAPs, in turn bind to the PKA substrate. This facilitates PKA substrate phosphorylation by increasing the local concentration of PKA catalytic subunits, C, upon an elevation in cAMP. We showed by surface plasmon resonance (SPR) and by Rll overlay, that Rll phosphorylation by C on serine 96 (S96) increases AKAP affinity for Rll. Also, expression of mutant Rll. (Rllsgeo) which mimics phosphorylated Rll (RII-P), showed increased co-localization with AKAP15/18, and also with mAKAP, as compared with RllsgeA, which mimics unphosphorylated Rll. These results are consistent with our SPR and Rll overlay data with Rll vs RII-P which also show increased binding of RII-P vs Rll to AKAPs. We also demonstrated that phosphorylation of Rll, and PKA substrates (Tnl, MBP-C and PLB), is decreased in failing human hearts. We predict that reduced Rll phosphorylation in failing hearts results in decreased AKAP affinity for PKA, decreased AKAP-targeted PKA activity and decreased phosphorylation of substrates to which AKAP:PKA is targeted, i.e.a-subunit of L-type Ca2* channels and the Ryanodine Receptor of the SR (RyR). We recently demonstrated that expression of a competing Rll binding peptide, Ht31, by adenoviral (Ad) gene transfer into adult cardiac myocytes disrupts PKA: AKAP binding, decreasing PKA- dependent phosphorylation of myofibrillar PKA substrates (Tnl and MBP-C). We observed an increased contractile response to isoproterenol vs controls, but no significant effect on Ca2* cycling. We hypothesize that regulation of binding of PKA to AKAPs (by altered Rll phosphorylation or disruption of RlhAKAP interaction) is a novel mechanism to regulate cardiac function. We also hypothesize that altered Rll targeting contributes to decreased contractility in heart failure. We will address three Specific Aims: (1): to investigate the effect of altered Rll phosphorylation on targeting of PKA, and other signaling proteins, in order to test whether Rll phosphorylation increases Rll: AKAP interaction in cardiac cells: (2) to investigate the effect of altered PKA targeting on localized PKA activity, substrate phosphorylation, cardiac myocyte function and co-localization of PKA and AKAP15/18 and with mAKAP; (3) to determine whether altered PKA targeting via AKAPs regulates cardiac contractility by Ad gene transfer into failing rat hearts in vivo. These studies will provide new insights into the functional significance of AKAPPKA targeting in the heart. In the long term, results of these studies may offer novel approaches to heart failure therapy.

心脏对/？-肾上腺素能刺激反应减弱与心力衰竭的发生有关。在心力衰竭期间， /？-肾上腺素能受体下调，但下游途径的改变，即 CAMP依赖的蛋白激酶(PKA)对底物磷酸化的调节也参与其中。少校 这一建议中检验的假设是RLL和A-激酶锚定蛋白之间相互作用的调节 (AKAPs)提供了一种新的机制来调节心脏对激活(3-肾上腺素能)的反应 信号通路。PKA，主要是PKA II(PKA II)，通过高亲和力结合到其底物上，通过 PKA的调节性亚基RL1到AKAP。AKAP进而与PKA底物结合。这有利于PKA衬底 在cAMP升高时，通过增加PKA催化亚基C的局部浓度来实现磷酸化。我们 表面等离子体共振(SPR)和RLL重叠显示，RLL被丝氨酸96上的C磷酸化(S96) 增加AKAP对RLL的亲和力。此外，还研究了突变体rll的表达。(Rllsgeo)，它模仿磷酸化的Rll(RII-P)， 与RllsgeA相比，与AKAP15/18和mAKAP的共定位增加，RllsgeA模仿 未磷酸化的RL1。这些结果与我们的SPR和RLL叠加数据相一致，RLL与RII-P的叠加数据也表明 RII-P与RLL与AKAP的结合增加。我们还证明了RL1和PKA底物的磷酸化 (TNL、MBP-C和PLB)，在衰竭的心脏中减少。我们预测，在失败的情况下，RLL磷酸化减少 心脏导致AKAP对PKA的亲和力降低，AKAP靶向PKA活性降低， AKAP：PKA靶向底物的磷酸化，即L型钙通道的A亚单位和 Ryanodine受体(RyR)。我们最近证明了一种竞争的RLL结合肽的表达， Ht31通过腺病毒(Ad)基因转移到成人心肌细胞中，破坏PKA：AKAP结合，降低PKA- 依赖于肌原纤维蛋白激酶A底物(TnL和MBP-C)的磷酸化。我们观察到收缩增加了 对异丙肾上腺素的反应与对照组相比，但对Ca~(2+)循环没有显著影响。我们假设，对 PKA与AKAP的结合(通过改变RL1磷酸化或破坏RlhAKAP相互作用)是一种新的 调节心脏功能的机制。我们还假设，改变RLL目标有助于 心力衰竭时的收缩能力降低。我们将解决三个具体目标：(1)：调查改变的影响 RLL磷酸化对PKA等信号蛋白的靶向作用，以检测RLL是否磷酸化 增加RLL：AKAP在心肌细胞中的相互作用：(2)研究改变PKA靶向性对定位PKA的影响 PKA和AKAP15/18的活性、底物磷酸化、心肌细胞功能和共定位 MAKAP；(3)确定通过AKAP靶向改变的PKA是否通过Ad基因转移调节心肌收缩能力 变成了体内衰竭的大鼠心脏。这些研究将为AKAPPKA的功能意义提供新的见解 瞄准心脏。从长远来看，这些研究的结果可能会为心力衰竭的治疗提供新的方法。