Dyck J R, Gao G, Widmer J, Stapleton D, Fernandez C S, Kemp B E, Witters L A
Endocrine-Metabolism Division, Department of Medicine, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
J Biol Chem. 1996 Jul 26;271(30):17798-803. doi: 10.1074/jbc.271.30.17798.
The mammalian 5'-AMP-activated protein kinase is a heterotrimer consisting of an alpha catalytic subunit and beta and gamma noncatalytic subunits, each of which is represented in a larger isoprotein family, related to the SNF1 kinase and its interacting proteins in yeast. In this study, we have used mammalian cell transfection to compare the activities of the two alpha subunit isoforms, alpha-1 and alpha-2, and to study the influence of the noncatalytic subunits on enzyme subunit association and activity. Expression of epitope-tagged protein subunits in COS7 cells indicates detectable but low level kinase activity for each of the two catalytic alpha subunits. Co-expression of alpha subunits with the beta or gamma subunits modestly increases kinase activity accompanied by the formation of alpha/beta or alpha/gamma heterodimers. Co-expression of all three subunits, however, is accompanied by a 50-110-fold increase in kinase activity with the formation of a heterotrimeric complex. In addition to binding of each noncatalytic subunit to the alpha subunit, the beta and gamma subunits bind to each other, likely resulting in a more stable heterotrimeric complex. The increase in kinase activity associated with expression of this heterotrimer is due both to an increase in enzyme-specific activity (units/enzyme mass) and to an apparent enhanced alpha subunit expression. Co-expression of a catalytically defective alpha subunit or the beta/gamma-binding COOH-terminal domain of the alpha subunit results in reduced heterotrimeric kinase activity. The synergistic positive regulatory roles for both the noncatalytic beta and gamma subunits of 5'-AMP-activated protein kinase contrasts with the Snf1p kinase, where only heterodimers of Snf1p and Snf4p seem to be required for maximum kinase activity.
哺乳动物的5'-AMP激活蛋白激酶是一种异源三聚体，由一个α催化亚基和β及γ非催化亚基组成，每个亚基在一个更大的同型蛋白家族中都有代表，该家族与酵母中的SNF1激酶及其相互作用蛋白相关。在本研究中，我们利用哺乳动物细胞转染来比较两种α亚基异构体α-1和α-2的活性，并研究非催化亚基对酶亚基缔合和活性的影响。在COS7细胞中表达表位标记的蛋白亚基表明，两种催化性α亚基中的每一种都具有可检测到但水平较低的激酶活性。α亚基与β或γ亚基共表达适度增加激酶活性，并伴随着α/β或α/γ异二聚体的形成。然而，所有三个亚基共表达伴随着激酶活性增加50 - 110倍，并形成异源三聚体复合物。除了每个非催化亚基与α亚基结合外，β和γ亚基相互结合，可能导致形成更稳定的异源三聚体复合物。与这种异源三聚体表达相关的激酶活性增加既归因于酶比活性（单位/酶质量）的增加，也归因于α亚基表达的明显增强。共表达催化缺陷的α亚基或α亚基的β/γ结合COOH末端结构域会导致异源三聚体激酶活性降低。5'-AMP激活蛋白激酶的非催化β和γ亚基的协同正调控作用与Snf1p激酶形成对比，在Snf1p激酶中，似乎只有Snf1p和Snf4p的异二聚体是最大激酶活性所必需的。
