Mitchelhill K I, Michell B J, House C M, Stapleton D, Dyck J, Gamble J, Ullrich C, Witters L A, Kemp B E
St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 Australia.
J Biol Chem. 1997 Sep 26;272(39):24475-9. doi: 10.1074/jbc.272.39.24475.
The AMP-activated protein kinase (AMPK) consists of catalytic alpha and noncatalytic beta and gamma subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and the rat liver AMPK alpha1 and alpha2 catalytic subunits are associated with beta1 and gamma1 noncatalytic subunits. We find that the isolated gamma1 subunit is N-terminally acetylated with no other posttranslational modification. The isolated beta1 subunit is N-terminally myristoylated. Transfection of COS cells with AMPK subunit cDNAs containing a nonmyristoylatable beta1 reduces, but does not eliminate, membrane binding of AMPK heterotrimer. The isolated beta1 subunit is partially phosphorylated at three sites, Ser24/25, Ser182, and Ser108. The Ser24/25 and Ser108 sites are substoichiometrically phosphorylated and can be autophosphorylated in vitro. The Ser-Pro site in the sequence LSSS182PPGP is stoichiometrically phosphorylated, and no additional phosphate is incorporated into this site with autophosphorylation. Based on labeling studies in transfected cells, we conclude that alpha1 Thr172 is a major, although not exclusive, site of both basal and stimulated alpha1 phosphorylation by an upstream AMPK kinase.
AMP 激活的蛋白激酶（AMPK）由催化性的α亚基以及非催化性的β和γ亚基组成，负责充当 AMP 水平的代谢传感器。每个亚基都有多个基因，大鼠肝脏中的 AMPK α1 和α2 催化亚基与β1 和γ1 非催化亚基相关联。我们发现，分离出的γ1 亚基在 N 端被乙酰化，没有其他翻译后修饰。分离出的β1 亚基在 N 端被肉豆蔻酰化。用含有不可肉豆蔻酰化的β1 的 AMPK 亚基 cDNA 转染 COS 细胞，可减少但不能消除 AMPK 异源三聚体与膜的结合。分离出的β1 亚基在三个位点 Ser24/25、Ser182 和 Ser108 处部分磷酸化。Ser24/25 和 Ser108 位点以亚化学计量磷酸化，并且在体外可被自身磷酸化。序列 LSSS182PPGP 中的 Ser-Pro 位点以化学计量磷酸化，自身磷酸化时该位点不会再掺入额外的磷酸。基于对转染细胞的标记研究，我们得出结论，α1 Thr172 是上游 AMPK 激酶进行基础和刺激的α1 磷酸化的主要位点，尽管不是唯一位点。
