PT-1 selectively activates AMPK-γ1 complexes in mouse skeletal muscle, but activates all three γ subunit complexes in cultured human cells by inhibiting the respiratory chain.
PT-1 selectively activates AMPK-γ1 complexes in mouse skeletal muscle, but activates all three γ subunit complexes in cultured human cells by inhibiting the respiratory chain.
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DOI：
10.1042/bj20141142
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发表时间：
2015-05-01
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影响因子：
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通讯作者：
Richter EA
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文献类型：
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作者：
Jensen TE;Ross FA;Kleinert M;Sylow L;Knudsen JR;Gowans GJ;Hardie DG;Richter EA
AMP活化蛋白激酶（AMPK）以异源三聚体复合物形式存在，其中催化亚基（α1/α2）与两个β亚基（β1/β2）之一和三个γ亚基（γ1/γ2/γ3）之一结合。选择性激活特定亚型的能力将是一种有用的研究工具，也是对抗癌症和2型糖尿病等疾病的有希望的策略。我们报道AMPK激活剂PT-1选择性地增加了孵育的小鼠肌肉中含有γ1而不是γ3的复合物的活性。PT-1增加了自噬调节激酶ULK 1在Ser 555上的AMPK依赖性磷酸化，但不增加γ3 AMPK底物如TBC 1D 1 Ser 231或ACC 2 Ser 212磷酸化，PT-1也不刺激葡萄糖转运。然而，令人惊讶的是，在表达人γ1、γ2或γ3的HEK-293细胞中，PT-1同等地激活所有三种复合物。我们无法重现先前的研究结果，表明PT-1通过激酶和α亚基的自抑制结构域之间的直接结合激活AMPK。相反，我们发现PT-1通过抑制呼吸链和增加细胞AMP：ATP和/或ADP：ATP比率间接激活AMPK。与该机制一致，PT-1未能激活表达AMPK-γ1的AMP不敏感R299 G突变体的HEK-293细胞中的AMPK。我们认为，PT-1不能激活肌肉中含有γ3的复合物并不是这种复合物的内在特征，而是因为PT-1不能增加γ3复合物所在的特定亚细胞区室中的细胞AMP：ATP比率。
AMP-activated protein kinase (AMPK) occurs as heterotrimeric complexes in which a catalytic subunit (α1/α2) is bound to one of two β subunits (β1/β2) and one of three γ subunits (γ1/γ2/γ3). The ability to selectively activate specific isoforms would be a useful research tool, and a promising strategy to combat diseases such as cancer and type 2 diabetes. We report that the AMPK activator PT-1 selectively increased the activity of γ1- but not γ3-containing complexes in incubated mouse muscle. PT-1 increased the AMPK-dependent phosphorylation of the autophagy-regulating kinase ULK1 on Ser555 but not proposed γ3 AMPK substrates such as TBC1D1 Ser231 or ACC2 Ser212 phosphorylation, nor did PT-1 stimulate glucose transport. Surprisingly, however, in HEK-293 cells expressing human γ1, γ2 or γ3, PT-1 activated all three complexes equally. We were unable to reproduce previous findings suggesting that PT-1 activates AMPK by direct binding between the kinase and auto-inhibitory domains of the α subunit. We show instead that PT-1 activates AMPK indirectly by inhibiting the respiratory chain and increasing cellular AMP:ATP and/or ADP:ATP ratios. Consistent with this mechanism, PT-1 failed to activate AMPK in HEK-293 cells expressing an AMP-insensitive R299G mutant of AMPK-γ1. We propose that the failure of PT-1 to activate γ3-containing complexes in muscle is not an intrinsic feature of such complexes, but is because PT-1 does not increase cellular AMP:ATP ratios in the specific subcellular compartment(s) in which γ3 complexes are located.