Characterization of AMP-activated protein kinase γ-subunit isoforms and their role in AMP binding
Characterization of AMP-activated protein kinase γ-subunit isoforms and their role in AMP binding
复制标题
DOI：
10.1042/0264-6021:3460659
复制
发表时间：
2000-03-15
影响因子：
4.1
通讯作者：
Carling, D
中科院分区：
文献类型：
--
作者：
Cheung, PCF;Salt, IP;Carling, D
AMP活化的蛋白激酶（AMPK）级联在细胞内能量稳态的调节中起重要作用。AMPK是由催化亚基（α）和两个调节亚基（β和γ）组成的异源三聚体。我们已经分离和表征了两种亚型的γ亚基，称为γ 2和γ 3。都是伽马2（569个氨基酸）和γ 3（492个氨基酸）具有长的N-末端结构域，其不存在于先前表征的同种型γ 1中。与γ 1一样，编码γ 2的mRNA在人组织中广泛表达，而γ 3 mRNA的显著表达仅在骨骼肌中检测到。使用同种型特异性抗体，我们测定了许多大鼠组织中与不同γ同种型相关的AMPK活性。在检查的大多数组织中，超过80%的总AMPK活性与γ 1亚型相关，其余活性主要由γ 2亚型引起，除此之外还有睾丸，更值得注意的是，脑，其中所有三种亚型对活性的贡献大致相等。没有证据表明鳗鱼和α 2亚型与各种γ亚型之间存在任何选择性关联。然而，激酶复合物的AMP依赖性明显受到存在的γ同种型的身份的影响，其中含有γ 2的复合物具有最大的AMP依赖性：γ 3最低，γ 1具有中间效果。使用反应性AMP类似物8-叠氮基-[P-32]AMP的标记研究表明，γ亚基可能直接参与复合物内AMP的结合。
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 (a) and two regulatory subunits (beta and gamma). We have isolated and characterized two isoforms of the gamma subunit, termed gamma 2 and gamma 3. Both gamma 2 (569 amino acids) and gamma 3 (492 amino acids) have a long N-terminal domain which is not present in the previously characterized isoform, gamma 1, As with gamma 1, mRNA encoding gamma 2 is widely expressed in human tissues, whereas significant expression of gamma 3 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 gamma 1 isoform, with the remaining activity being accounted for mainly by the gamma 2 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 eel and alpha 2 isoforms 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 gamma 2-containing complexes having the greatest AMP-dependence: gamma 3 the lowest, and gamma 1 having an intermediate effect. Labelling studies, using the reactive AMP analogue 8-azido-[P-32]AMP, indicate that the gamma subunit may participate directly in the binding of AMP within the complex.