Cheung P C, Salt I P, Davies S P, Hardie D G, Carling D
Cellular Stress Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, U.K.
Biochem J. 2000 Mar 15;346 Pt 3(Pt 3):659-69.
The AMP-activated protein kinase (AMPK) cascade plays an important role in the regulation of energy homeostasis within the cell. AMPK is a heterotrimer composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma). We have isolated and characterized two isoforms of the gamma subunit, termed gamma2 and gamma3. Both gamma2 (569 amino acids) and gamma3 (492 amino acids) have a long N-terminal domain which is not present in the previously characterized isoform, gamma1. As with gamma1, mRNA encoding gamma2 is widely expressed in human tissues, whereas significant expression of gamma3 mRNA was only detected in skeletal muscle. Using isoform-specific antibodies, we determined the AMPK activity associated with the different gamma isoforms in a number of rat tissues. In most tissues examined more than 80% of total AMPK activity was associated with the gamma1 isoform, with the remaining activity being accounted for mainly by the gamma2 isoform. Exceptions to this were testis and, more notably, brain where all three isoforms contributed approximately equally to activity. There was no evidence for any selective association between the alpha1 and alpha2isoforms and the various gamma isoforms. However, the AMP-dependence of the kinase complex is markedly affected by the identity of the gamma isoform present, with gamma2-containing complexes having the greatest AMP-dependence, gamma3 the lowest, and gamma1 having an intermediate effect. Labelling studies, using the reactive AMP analogue 8-azido-[(32)P]AMP, indicate that the gamma subunit may participate directly in the binding of AMP within the complex.
AMP 激活的蛋白激酶（AMPK）级联在细胞内能量稳态的调节中起重要作用。AMPK 是一种异源三聚体，由一个催化亚基（α）和两个调节亚基（β和γ）组成。我们已经分离并鉴定了γ亚基的两种异构体，分别称为γ2和γ3。γ2（569个氨基酸）和γ3（492个氨基酸）都有一个长的 N 端结构域，而先前鉴定的异构体γ1中不存在该结构域。与γ1一样，编码γ2的 mRNA 在人体组织中广泛表达，而γ3 mRNA 仅在骨骼肌中检测到显著表达。使用异构体特异性抗体，我们测定了多种大鼠组织中与不同γ异构体相关的 AMPK 活性。在大多数检测的组织中，超过80%的总 AMPK 活性与γ1异构体相关，其余活性主要由γ2异构体负责。睾丸是个例外，更值得注意的是大脑，在那里所有三种异构体对活性的贡献大致相同。没有证据表明α1和α2异构体与各种γ异构体之间存在任何选择性关联。然而，激酶复合物对 AMP 的依赖性明显受所存在的γ异构体的特性影响，含γ2的复合物对 AMP 的依赖性最大，γ3最低，γ1具有中等影响。使用反应性 AMP 类似物8-叠氮基-[（32）P]AMP 进行的标记研究表明，γ亚基可能直接参与复合物中 AMP 的结合。
