Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms.
Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms.
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DOI：
10.1042/bj20150910
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发表时间：
2016-01-15
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影响因子：
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通讯作者：
Hardie DG
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作者：
Ross FA;Jensen TE;Hardie DG
含有γ1、γ2或γ3亚基异构体的AMPK复合物在人细胞中表达产生。异三聚体AMPK复合物的γ亚基含有调节腺嘌呤核苷酸AMP、ADP和ATP的结合位点，它们显示出腺嘌呤核苷酸引起调节的所有三种作用的差异，即变构激活、促进磷酸化和抑制去磷酸化。我们通过生成稳定表达γ1、γ2或γ3亚型的flag标记版本的细胞，研究了含有不同γ亚型的复合物是否对腺嘌呤核苷酸表现出不同的反应。在生理ATP浓度（5 mM）下，含γ - 1和γ - 2复合物被AMP变构激活近10倍，EC50值比ATP浓度低一到两个数量级。相比之下，在这些条件下，γ - 3复合物几乎没有被AMP激活，尽管我们确实观察到在较低ATP浓度下有一些激活。尽管如此，当细胞与增加细胞AMP的线粒体抑制剂一起孵育时，由于Thr172磷酸化增加，所有三种复合物都被激活。对于γ - 1复合物，上游激酶LKB1(肝激酶B1；但在无细胞实验中，AMP显著促进了钙调素依赖性激酶（CaMKKβ）的表达，而ADP的促进程度更小，作用更弱。然而，AMP或ADP对γ2和γ3复合物的激活和磷酸化的影响很小或不显著。AMP或ADP的结合保护所有三种γ亚基复合物免受Thr172去磷酸化的失活；对于γ - 2复合物，ADP的效价与AMP相似，但对于γ - 1和γ - 3复合物，ADP的效价低于AMP。因此，含有不同γ亚基异构体的AMPK复合物对AMP、ADP或ATP变化的反应不同。这些差异可能会调整同种异构体的反应，以适应它们不同的生理作用。
AMPK complexes containing γ1, γ2 or γ3 subunit isoforms were generated by expression in human cells. They displayed differences in all three effects by which adenine nucleotides cause regulation, i.e. in allosteric activation, promotion of phosphorylation and inhibition of dephosphorylation The γ subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different γ isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged versions of the γ1, γ2 or γ3 isoform. When assayed at a physiological ATP concentration (5 mM), γ1- and γ2-containing complexes were allosterically activated almost 10-fold by AMP, with EC50 values one to two orders of magnitude lower than the ATP concentration. By contrast, γ3 complexes were barely activated by AMP under these conditions, although we did observe some activation at lower ATP concentrations. Despite this, all three complexes were activated, due to increased Thr172 phosphorylation, when cells were incubated with mitochondrial inhibitors that increase cellular AMP. With γ1 complexes, activation and Thr172 phosphorylation induced by the upstream kinase LKB1 [liver kinase B1; but not calmodulin-dependent kinase kinase (CaMKKβ)] in cell-free assays was markedly promoted by AMP and, to a smaller extent and less potently, by ADP. However, effects of AMP or ADP on activation and phosphorylation of the γ2 and γ3 complexes were small or insignificant. Binding of AMP or ADP protected all three γ subunit complexes against inactivation by Thr172 dephosphorylation; with γ2 complexes, ADP had similar potency to AMP, but with γ1 and γ3 complexes, ADP was less potent than AMP. Thus, AMPK complexes containing different γ subunit isoforms respond differently to changes in AMP, ADP or ATP. These differences may tune the responses of the isoforms to fit their differing physiological roles.