AKAP Regulation of PKA Targeting in the Heart

AKAP 对心脏 PKA 靶向的调节

• 批准号: 7674543
• 负责人: Meredith Bond
• 金额: $ 28.31万
• 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 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7674543
• 关键词: A kinase anchoring protein; AKAP13 gene; Address; Adrenergic Agents; 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; Down-Regulation; Echocardiography; Endoplasmic Reticulum; Gene Transfer; Heart; Heart failure; Human; Isoproterenol; Link; Location; Measurement; Modeling; Mutate; Myosin ATPase; Pathway interactions; Peptides; Phosphorylation; Principal Investigator; Protein Binding; Protein Kinase; Rattus; Regulation; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Serine; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; Surface Plasmon Resonance; Testing; Therapeutic; Troponin I; Work; adrenergic; base; beta-adrenergic receptor; in vivo; insight; knock-down; mutant; novel; novel strategies; programs; protein expression; protein kinase A kinase; research study; response

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.

描述（由申请方提供）：对β-肾上腺素能刺激的心脏反应降低与心力衰竭的发生有关。在心力衰竭期间，β-肾上腺素能受体发生下调，但也涉及该途径的下游改变，即cAMP依赖性蛋白激酶（PKA）对底物磷酸化的调节。在该提议中测试的主要假设是RII和A-激酶锚定蛋白（AKAP）之间的相互作用的调节提供了调节对β-肾上腺素能信号传导途径的激活的心脏响应的新机制。PKA，主要是PKA II型（PKA II），通过经由PKA的调节亚基RII与AKAP的高亲和力结合而靶向其底物。AKAP又与PKA底物结合。这通过在cAMP升高时增加PKA催化亚基C的局部浓度来促进PKA底物磷酸化。我们通过表面等离子体共振（SPR）和RII覆盖显示，丝氨酸96（S96）上的C对RII的磷酸化增加了AKAP对RII的亲和力。 此外，突变体RII的表达。与模拟未磷酸化RII的R11 S96 A相比，模拟磷酸化RII（RII-P）的R11 S96 D显示出与AKAP 15/18以及与mAKAP的共定位增加。这些结果与我们的SPR和RII与RII对RII-P的重叠数据一致，其也显示RII-P对RII与AKAP的结合增加。我们还证明了RII和PKA底物（TnI、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RII磷酸化的降低导致AKAP对PKA的亲和力降低、AKAP靶向的PKA活性降低和AKAP：PKA靶向的底物（即L型Ca 2+通道的α亚基和SR的兰尼碱受体（RyR））的磷酸化降低。我们最近证明，通过腺病毒（Ad）基因转移到成人心肌细胞中的竞争性Rll结合肽Ht 31的表达破坏PKA：AKAP结合，降低肌原纤维PKA底物（TnI和MBP-C）的PKA依赖性磷酸化。我们观察到与对照组相比，对异丙肾上腺素的收缩反应增加，但对Ca 2+循环无显著影响。我们假设PKA与AKAP结合的调节（通过改变RII磷酸化或破坏RII：AKAP相互作用）是调节心脏功能的新机制。我们还假设改变的RII靶向有助于心力衰竭中收缩性降低。我们将提出三个具体目的：（1）：研究改变的RII磷酸化对PKA和其它信号传导蛋白的靶向的影响，以测试RII磷酸化是否增加心脏细胞中的RII：AKAP相互作用：（2）研究PKA靶向改变对PKA活性、底物磷酸化心肌细胞功能和PKA和AKAP 15/18以及与mAKAP的共定位;（3）确定是否通过将Ad基因转移到体内衰竭的大鼠心脏中来改变经由AKAP的PKA靶向调节心脏收缩性。这些研究将为AKAP：PKA在心脏中的靶向功能意义提供新的见解。从长远来看，这些研究的结果可能为心力衰竭治疗提供新的方法。