AKAP Regulation of PKA Targeting in the Heart

描述（由申请人提供）：心脏对-肾上腺素能刺激的反应降低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，但该途径的下游改变，即camp依赖性蛋白激酶（PKA）对底物磷酸化的调节也参与其中。本研究的主要假设是Rll和a激酶锚定蛋白（AKAPs）之间相互作用的调节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型（PKA II），通过PKA的调控亚基Rll与akap高亲和力结合，靶向其底物。反过来，akap与PKA底物结合。当cAMP升高时，PKA催化亚基C的局部浓度增加，从而促进PKA底物磷酸化。通过表面等离子体共振（SPR）和Rll覆盖，我们发现C对丝氨酸96 （S96）的Rll磷酸化增加了AKAP对Rll的亲和力。突变体Rll的表达。与模拟未磷酸化Rll的RllS96A相比，模拟磷酸化Rll （RII-P）的RllS96D与AKAP15/18和mAKAP的共定位增加。这些结果与我们的SPR和Rll覆盖数据一致，Rll与RII-P也显示RII-P与Rll与AKAPs的结合增加。我们还证明，Rll和PKA底物（Tnl， MBP-C和PLB）的磷酸化在衰竭的人类心脏中降低。我们预测，衰竭心脏中Rll磷酸化的降低导致AKAP对PKA的亲和力降低，AKAP靶向的PKA活性降低，AKAP:PKA靶向的底物磷酸化降低，即l型Ca2+通道的α亚基和SR的Ryanodine受体（RyR）。我们最近证明，通过腺病毒（Ad）基因转移到成人心肌细胞中，一种竞争性Rll结合肽Ht31的表达会破坏PKA: AKAP的结合，降低PKA依赖的肌纤维PKA底物（Tnl和MBP-C）的磷酸化。我们观察到与对照组相比，异丙肾上腺素增加了收缩反应，但对Ca2+循环没有显著影响。我们假设PKA与AKAP结合的调节（通过改变Rll磷酸化或破坏RlI:AKAP相互作用）是一种调节心功能的新机制。我们还假设Rll靶向改变有助于心力衰竭患者收缩力下降。我们将研究三个特定目的：(1)：研究Rll磷酸化改变对PKA和其他信号蛋白靶向的影响，以测试Rll磷酸化是否会增加心脏细胞中Rll: AKAP的相互作用；(2)研究PKA靶向改变对局部PKA活性、底物磷酸化、心肌细胞功能以及PKA和AKAP15/18以及与mAKAP共定位的影响；(3)确定通过AKAPs靶向改变的PKA是否通过Ad基因转移到衰竭大鼠心脏中来调节心脏收缩力。这些研究将为AKAP:PKA靶向在心脏中的功能意义提供新的见解。从长远来看，这些研究的结果可能会为心力衰竭治疗提供新的方法。

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.