Phosphorylation of the cAMP-dependent protein kinase (PKA) regulatory subunit modulates PKA-AKAP interaction, substrate phosphorylation, and calcium signaling in cardiac cells
Phosphorylation of the cAMP-dependent protein kinase (PKA) regulatory subunit modulates PKA-AKAP interaction, substrate phosphorylation, and calcium signaling in cardiac cells
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DOI：
10.1074/jbc.m802278200
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发表时间：
2008-08-29
影响因子：
4.8
通讯作者：
Bond, Meredith
中科院分区：
文献类型：
--
作者：
Manni, Sabrina;Mauban, Joseph H.;Bond, Meredith
蛋白激酶A锚定蛋白（AKAP）对cAMP依赖性蛋白激酶（PKA）的亚细胞区室化促进了局部蛋白磷酸化。然而，很少有人知道PKA靶向AKAP是如何在完整的细胞调节。PKA通过调节亚基的N-末端结构域与AKAP的两亲性螺旋区结合。体外研究表明，II型调节亚基（RII）的自磷酸化可以改变其对AKAP和催化亚基（PKA（cat））的亲和力。我们现在研究RII上丝氨酸96的磷酸化是否调节PKA靶向AKAP、下游底物磷酸化和原代培养心肌细胞中的钙循环。我们证明，虽然有基础磷酸化的RII亚基，持续最大激活PKA的结果在磷酸酶依赖性损失的RII磷酸化。为了研究RII磷酸化的功能效应，我们构建了包含模拟磷酸化（RIIS 96 D）、非磷酸化（RIIS 96 A）RII或野生型（WT）RII突变体的腺病毒载体，并对新生大鼠心肌细胞进行腺病毒感染。免疫共沉淀显示，与RIIS 96 A相比，更多的AKAP 15/18被磷酸模拟物RIIS 96 D拉下。受磷蛋白和ryanodine受体的磷酸化在表达RIIS 96 D的细胞中与表达RIIS 96 A的细胞相比显著增加。重组RII构建体的表达对胞浆钙瞬变有显著影响。我们提出了一个模型，说明了中央的作用，RII磷酸化在当地PKA活性的调节。我们的结论是，RII磷酸化调节PKA依赖的底物磷酸化，并可能对心脏功能的调制有重要意义。
Subcellular compartmentalization of the cAMP-dependent protein kinase (PKA) by protein kinase A-anchoring proteins (AKAPs) facilitates local protein phosphorylation. However, little is known about how PKA targeting to AKAPs is regulated in the intact cell. PKA binds to an amphipathic helical region of AKAPs via an N-terminal domain of the regulatory subunit. In vitro studies showed that autophosphorylation of type II regulatory subunit (RII) can alter its affinity for AKAPs and the catalytic subunit (PKA(cat)). We now investigate whether phosphorylation of serine 96 on RII regulates PKA targeting to AKAPs, downstream substrate phosphorylation and calcium cycling in primary cultured cardiomyocytes. We demonstrated that, whereas there is basal phosphorylation of RII subunits, persistent maximal activation of PKA results in a phosphatase-dependent loss of RII phosphorylation. To investigate the functional effects of RII phosphorylation, we constructed adenoviral vectors incorporating mutants which mimic phosphorylated (RIIS96D), nonphosphorylated (RIIS96A) RII, or wild-type (WT) RII and performed adenoviral infection of neonatal rat cardiomyocytes. Coimmunoprecipitation showed that more AKAP15/18 was pulled down by the phosphomimic, RIIS96D, than RIIS96A. Phosphorylation of phospholamban and ryanodine receptor was significantly increased in cells expressing RIIS96D versus RIIS96A. Expression of recombinant RII constructs showed significant effects on cytosolic calcium transients. We propose a model illustrating a central role of RII phosphorylation in the regulation of local PKA activity. We conclude that RII phosphorylation regulates PKA-dependent substrate phosphorylation and may have significant implications for modulation of cardiac function.