Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade
Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade
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DOI：
10.1042/bj20061520
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发表时间：
2007-04-01
影响因子：
4.1
通讯作者：
Carling, David
中科院分区：
文献类型：
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作者：
Sanders, Matthew J.;Grondin, Pascal O.;Carling, David
AMP激活的蛋白激酶（AMPK）通过AMP以及催化α亚基内苏氨酸（Thr）172的磷酸化发生变构激活。在此我们表明，调节性γ亚基的突变会降低AMP对该激酶的变构激活作用。除了其变构效应外，AMP还显著降低蛋白磷酸酶2Cα（PP2Cα）对苏氨酸172的去磷酸化作用。此外，γ亚基的一种突变几乎完全消除了AMP对去磷酸化的抑制作用。我们使用这些蛋白质的重组制剂，无法检测到AMP对LKB1或Ca²⁺/钙调蛋白依赖性蛋白激酶激酶β（CaMKKβ）所致苏氨酸172磷酸化的任何影响。然而，使用从大鼠肝脏部分纯化的AMPK时，LKB1对苏氨酸172的磷酸化存在明显的AMP刺激作用，但这种作用会被添加蛋白磷酸酶抑制剂氟化钠（NaF）所阻断。对部分纯化的大鼠肝脏AMPK和LKB1进行蛋白质印迹分析，结果显示制剂中存在PP2Cα。我们认为，先前报道AMP促进苏氨酸172磷酸化的研究被误解了。AMP这种作用的一种合理解释是，它抑制了早期研究中所用激酶制剂中存在的PP2Cα的去磷酸化作用。综上所述，我们的结果表明AMP通过两种机制激活AMPK：直接变构激活以及保护苏氨酸172免于去磷酸化。基于我们的新发现，我们提出了一个关于LKB1和CaMKKβ在哺乳动物细胞中调节AMPK的简单模型。该模型解释了AMPK被两种不同信号激活的情况：一种是由CaMKK介导的Ca²⁺依赖性途径，另一种是由LKB1介导的AMP依赖性途径。
AMPK (AMP-activated protein kinase) is activated allosterically by AMP and by phosphorylation of Thr(172) within the catalytic a subunit. Here we show that mutations in the regulatory gamma subunit reduce allosteric activation of the kinase by AMP. In addition to its allosteric effect, AMP significantly reduces the dephosphorylation of Thr(172) by PP (protein phosphatase)2C alpha. Moreover, a mutation in the gamma subunit almost completely abolishes the inhibitory effect of AMP on dephosphorylation. We were unable to detect any effect of AMP on Thr(172) phosphorylation by either LKB1 or CaMKK beta (Ca2+/calmodulin-dependent protein kinase kinase beta) using recombinant preparations of the proteins. However, using partially purified AMPK from rat liver, there was an apparent AMP-stimulation of Thr(172) phosphorylation by LKB1, but this was blocked by the addition of NaF, a PP inhibitor. Western blotting of partially purified rat liver AMPK and LKB1 revealed the presence of PP2C alpha in the preparations. We suggest that previous studies reporting that AMP promotes phosphorylation of Thr(172) were misinterpreted. A plausible explanation for this effect of AMP is inhibition of dephosphorylation by PP2C alpha, present in the preparations of the kinases used in the earlier studies. Taken together, our results demonstrate that AMP activates AMPK via two mechanisms: by direct allosteric activation and by protecting Thr(172). from dephosphorylation. On the basis of our new findings, we propose a simple model for the regulation of AMPK in mammalian cells by LKB1 and CaMKK beta. This model accounts for activation of AMPK by two distinct signals: a Ca2+-dependent pathway, mediated by CaMKK and an AMP-dependent pathway, mediated by LKB1.