Multiple-site phosphorylation of the 280 kDa isoform of acetyl-CoA carboxylase in rat cardiac myocytes:: evidence that cAMP-dependent protein kinase mediates effects of β-adrenergic stimulation
Multiple-site phosphorylation of the 280 kDa isoform of acetyl-CoA carboxylase in rat cardiac myocytes:: evidence that cAMP-dependent protein kinase mediates effects of β-adrenergic stimulation
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DOI：
10.1042/0264-6021:3410347
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发表时间：
1999-07-15
影响因子：
4.1
通讯作者：
Brownsey, RW
中科院分区：
文献类型：
--
作者：
Boone, AN;Rodrigues, B;Brownsey, RW
描述了哺乳动物乙酰辅酶a羧化酶（EC 6.4.1.2）的两种主要形式acc - α和acc - β，并推导了其同工型的序列。acc - β是在心脏和骨骼肌中表达的主要亚型，丙二酰辅酶a在其中的主要作用可能是调节脂肪酸β -氧化。acc - β的调控特性尚不完全明确，但已知一些细胞应激在amp活化蛋白激酶（AMP-PK）的同时导致抑制。在这里，我们检测了完整大鼠心室肌细胞中acc - β的磷酸化状态。用β -肾上腺素受体激动剂异丙肾上腺素（异丙肾上腺素）处理肌细胞导致ACC- β磷酸化增加，并在2分钟内和50 nM激动剂中达到最大，异丙肾上腺素的作用通过在用[P-32]P-1孵育的细胞中P-32掺入ACC中而显着减少（约1 nM）来揭示。80%)， camp依赖性蛋白激酶（PKA）随后在体外磷酸化。色氨酸磷酸化肽分析表明，acc - β在体外与PKA或AMP-PK孵育时在多个位点被磷酸化。用异丙肾上腺素处理肌细胞影响了acc - β的所有主要磷酸化位点，这些磷酸化位点在体外被纯化的PKA识别，因此在细胞刺激后，随后的体外磷酸化大大减少。β -肾上腺素能刺激导致细胞丙二酰辅酶a浓度降低，但细胞匀浆和部分纯化蛋白质后，ACC的动力学性质没有变化。结果表明：(1)β -肾上腺素能刺激后，acc - β在完整心室肌细胞内的多个位点迅速磷酸化；(2)体外实验中，PKA和AMP-PK在多个位点磷酸化acc - β，包括每种激酶可到达的至少一个位点，以及激酶选择性位点；(3)PKA是一种具有生理意义的acc - β激酶。
Two major forms of mammalian acetyl-CoA carboxylase (EC 6.4.1.2), ACC-alpha and ACC-beta, have been described and the sequences of the isoforms deduced. ACC-beta is the predominant isoform expressed in heart and skeletal muscles, in which a major role of malonyl-CoA is probably to regulate fatty acid beta-oxidation. The regulatory properties of ACC-beta are incompletely defined but it is known that some cellular stresses lead to inhibition in parallel with the activation of AMP-activated protein kinase (AMP-PK). Here we examine the phosphorylation state of ACC-beta within intact rat cardiac ventricular myocytes. Treatment of myocytes with the beta-adrenergic agonist isoprenaline (isoproterenol) led to increased ACC-beta phosphorylation that was maximal within 2 min and with 50 nM agonist, Effects of isoprenaline were revealed by the incorporation of P-32 into ACC in cells incubated with [P-32]P-1 and also by a marked decrease (approx. 80%) in subsequent phosphorylation in vitro with cAMP-dependent protein kinase (PKA). Analysis of tryptic phosphopeptides revealed that ACC-beta was phosphorylated at multiple sites by incubation in vitro with PKA or AMP-PK. Treatment of myocytes with isoprenaline affected all the major phosphorylation sites of ACC-beta that were recognized in vitro by purified PKA, so that subsequent phosphorylation in vitro was greatly diminished after cell stimulation. beta-Adrenergic stimulation fed to decreases in cellular malonyl-CoA concentrations but no changes in kinetic properties of ACC were detected after cell homogenization and partial purification of proteins. The results suggest that: (1) ACC-beta is rapidly phosphorylated at multiple sites within intact cardiac ventricular myocytes after beta-adrenergic stimulation, (2) ACC-beta is phosphorylated in vitro by PKA and AMP-PK at multiple sites, including at least one site accessible to each kinase, as well as kinase-selective sites, and (3) PKA is a physiologically significant ACC-beta kinase.