AKAP Regulation of PKA Targeting in the Heart

描述(由申请人提供)：心脏对β-肾上腺素能刺激的反应减弱与心力衰竭的发展有关。在心力衰竭期间，β-肾上腺素能受体发生下调，但下游通路的改变，即cAMP依赖的蛋白激酶(PKA)对底物磷酸化的调节也参与其中。这一建议检验的主要假设是，调节RLL和A-激酶锚定蛋白(AKAPs)之间的相互作用提供了一种新的机制来调节心脏对β-肾上腺素能信号通路激活的反应。PKA，主要是PKA II(PKA II)，通过PKA的调节亚基R11与AKAP高亲和力结合到底物上。AKAP进而与PKA底物结合。这通过在cAMP升高时增加PKA催化亚基的局部浓度C来促进PKA底物的磷酸化。我们通过表面等离子体共振(SPR)和RLL重叠分析表明，RLL被丝氨酸96上的C(S96)磷酸化增加了AKAP对RLL的亲和力。此外，还研究了突变体rll的表达。与模拟非磷酸化RLL的RllS96A相比，与AKAP15/18和mAKAP的共定位显示出与模拟磷酸化RLL(RII-P)的RllS96D更高的共定位。这些结果与我们的SPR和RLL与RLL与RII-P的重叠数据一致，这也表明RII-P与RLL与AKAP的结合增加。我们还证明，在衰竭的心脏中，RLL和PKA底物(TNL、MBP-C和PLB)的磷酸化水平降低。我们预测，在衰竭的心脏中，Rll磷酸化降低会导致AKAP与PKA亲和力降低，AKAP靶向PKA活性降低，并减少AKAP：PKA靶向底物的磷酸化，即L型钙通道的阿尔法亚单位和SR的Ryanodine受体(RyR)。我们最近证实，通过腺病毒(Ad)基因转移到成人心肌细胞中，竞争的RLL结合肽Ht31的表达破坏了PKA：AKAP的结合，减少了PKA依赖的肌原纤维PKA底物(TNL和MBP-C)的磷酸化。我们观察到，与对照组相比，异丙肾上腺素的收缩反应增强，但对钙循环没有显著影响。我们推测，调节PKA与AKAP的结合(通过改变RL1磷酸化或破坏RLI：AKAP相互作用)是调节心脏功能的一种新机制。我们还假设，改变RLL靶向有助于心力衰竭患者收缩能力的降低。我们将针对三个具体目标：(1)研究改变的RLL磷酸化对靶向PKA及其他信号蛋白的影响，以测试RLL磷酸化是否增加心肌细胞中RLL：AKAP的相互作用；(2)研究改变的PKA靶向对局部PKA活性、底物磷酸化、心肌细胞功能以及PKA和AKAP15/18以及与mAKAP的共存的影响；(3)确定通过AKAPs靶向改变的PKA是否通过Ad基因转移调节衰竭大鼠心脏的收缩性能。这些研究将为AKAP的功能意义提供新的见解：心脏中的PKA靶向。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。

DESCRIPTION (provided by applicant): Decreased cardiac response to beta-adrenergic stimulation is linked to development of heart failure. During heart failure, down-regulation of the beta-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 beta-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. (RllS96D) which mimics phosphorylated Rll (RII-P), showed increased co-localization with AKAP15/18, and also with mAKAP, as compared with RllS96A, 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. alpha-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 RlI:AKAP 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 AKAP:PKA targeting in the heart. In the long term, results of these studies may offer novel approaches to heart failure therapy.