PHOSPHORYLATION AND ACTIVATION OF BETA-ADRENERGIC-RECEPTOR KINASE BY PROTEIN-KINASE-C
PHOSPHORYLATION AND ACTIVATION OF BETA-ADRENERGIC-RECEPTOR KINASE BY PROTEIN-KINASE-C
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DOI：
10.1074/jbc.270.31.18660
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发表时间：
1995-08-04
影响因子：
4.8
通讯作者：
DEBLASI, A
中科院分区：
文献类型：
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作者：
CHUANG, TT;LEVINE, H;DEBLASI, A
本研究的目的是测试第二信使和/或其下游成分对β-肾上腺素能受体激酶（β方舟）活性的可能修饰。利用人单核白细胞（MNL），我们发现钙离子载体可以以蛋白激酶C（PKC）依赖的方式提高β方舟活性约80%。PKC激活剂佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）产生类似作用的能力证实了这一点，表明β方舟活性的PKC依赖性调节。用纯化蛋白进行的体外实验表明，PKC可以直接磷酸化β ARK 1，β ARK 1的表观Km为6 nM。当β ARK 1被PKC磷酸化时，其磷酸化视紫红质的能力增加了61%。PMA处理后，从过表达该激酶的MNL和Sf 9细胞免疫沉淀的β ARK 1的磷酸化水平提高了约2-3倍。β-肾上腺素能受体（β AR）同源脱敏实验证实了PKC依赖性β方舟活性增加的功能意义。暴露于10 μ M异丙肾上腺素（37 ℃ 5分钟）诱导的β AR脱敏从对照组的42 +/- 10%增加到PMA预处理的MNL中的68 +/- 8%。PARK抑制剂肝素（160 μ g/ml）阻止PMA对β AR脱敏的增强作用。这些结果表明，β方舟活性可以通过PKC磷酸化而增加，从而表明β方舟可以被预处理以调节随后的细胞对受体活化的反应性，从而为细胞提供了一种可以异源调节特异性同源脱敏的机制。
The aim of this study was to test the possible modification of beta-adrenergic receptor kinase (beta ARK) activity by second messengers and/or their downstream components. Using human mononuclear leukocytes (MNL), we found that calcium ionophores could elevate beta ARK activity by about 80% in a protein kinase C (PKC)-dependent manner. This was confirmed by the ability of the PKC activator phorbol 12-myristate 13-acetate (PMA) to produce a similar effect, suggesting a PKC dependent modulation of beta ARK activity. In vitro experiments with purified proteins showed that PKC could directly phosphorylate beta ARK1 with an apparent K-m for beta ARK1 of 6 nM. The ability of beta ARK1 to phosphorylate rhodopsin was 61% greater when it was phosphorylated by PKC. The level of phosphorylation of beta ARK1 immunoprecipitated from MNL and Sf9 cells overexpressing this kinase was enhanced by about 2-3-fold after PMA treatment. Functional significance of PKC-dependent increase in beta ARK activity was demonstrated by beta-adrenergic receptor (beta AR) homologous desensitization experiments in MNL. beta AR desensitization, as induced by exposure to 10 mu M isoproterenol (5 min at 37 degrees C), was increased from 42 +/- 10% in control to 68 +/- 8% in PMA-pretreated MNL. PARK inhibitor heparin (160 mu g/ml) prevented the augmenting effect of PMA on beta AR desensitization. These results show that beta ARK activity can be increased through phosphorylation by PKC, thus indicating that beta ARK can be preconditioned to modulate the subsequent cellular responsiveness to receptor activation, providing the cell with a mechanism by which specific homologous desensitization can be regulated heterologously.