Chuang T T, LeVine H, De Blasi A
Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche Mario Negri, Santa Maria Imbaro, Italy.
J Biol Chem. 1995 Aug 4;270(31):18660-5. doi: 10.1074/jbc.270.31.18660.
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 Km 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 ws demonstrated by beta-adrenergic receptor (beta AR) homologous desensitization experiments in MNL. beta AR desensitization, as induced by exposure to 10 microM isoproterenol (5 min at 37 degrees C), was increased from 42 +/- 10% in control to 68 +/- 8% in PMA-pretreated MNL. beta ARK inhibitor heparin (160 micrograms/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.
本研究的目的是检测第二信使和/或其下游成分对β-肾上腺素能受体激酶（βARK）活性的可能修饰作用。利用人单核白细胞（MNL），我们发现钙离子载体能够以蛋白激酶C（PKC）依赖的方式使βARK活性升高约80%。PKC激活剂佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）产生类似效应，证实了这一点，提示βARK活性存在PKC依赖的调节作用。用纯化蛋白进行的体外实验表明，PKC能够直接磷酸化βARK1，其对βARK1的表观Km为6 nM。当βARK1被PKC磷酸化后，其磷酸化视紫红质的能力增强了61%。经PMA处理后，从MNL和过表达该激酶的Sf9细胞中免疫沉淀的βARK1的磷酸化水平提高了约2 - 3倍。MNL中的β-肾上腺素能受体（βAR）同源脱敏实验证明了PKC依赖的βARK活性增加的功能意义。暴露于10 μM异丙肾上腺素（37℃ 5分钟）诱导的βAR脱敏，在对照中为42±10%，在PMA预处理的MNL中增加到68±8%。βARK抑制剂肝素（160 μg/ml）可阻止PMA对βAR脱敏的增强作用。这些结果表明，βARK活性可通过PKC磷酸化而增加，这表明βARK可被预处理以调节细胞随后对受体激活的反应性，为细胞提供了一种可通过异源方式调节特异性同源脱敏的机制。
