Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
Biochem J. 2010 Jan 27;426(1):109-18. doi: 10.1042/BJ20091372.
Activation of AMPK (AMP-activated protein kinase) by phosphorylation at Thr172 is catalysed by at least two distinct upstream kinases, i.e. the tumour suppressor LKB1, and CaMKKbeta (Ca2+/calmodulin-dependent protein kinase kinase-beta). The sequence around Thr172 is highly conserved between the two catalytic subunit isoforms of AMPK and the 12 AMPK-related kinases, and LKB1 has been shown to act upstream of all of them. In the present paper we report that none of the AMPK-related kinases tested could be phosphorylated or activated in intact cells or cell-free assays by CaMKKbeta, although we did observe a slow phosphorylation and activation of BRSK1 (brain-specific kinase 1) by CaMKKalpha. Despite recent reports, we could not find any evidence that the alpha and/or beta subunits of AMPK formed a stable complex with CaMKKbeta. We also showed that increasing AMP concentrations in HeLa cells (which lack LKB1) had no effect on basal AMPK phosphorylation, but enhanced the ability of agents that increase intracellular Ca2+ to activate AMPK. This is consistent with the effect of AMP on phosphorylation of Thr172 being due to inhibition of dephosphorylation, and confirms that the effect of AMP is independent of the upstream kinase utilized.
AMPK（AMP 激活的蛋白激酶）的激活是通过 Thr172 位点的磷酸化来实现的，该磷酸化由至少两种不同的上游激酶催化，即肿瘤抑制因子 LKB1 和 CaMKKβ（Ca2+/钙调蛋白依赖性蛋白激酶激酶-β）。在 AMPK 的两个催化亚基同工型和 12 种 AMPK 相关激酶之间，Thr172 周围的序列高度保守，并且已经证明 LKB1 在上游作用于所有这些激酶。在本文中，我们报告说，在完整细胞或无细胞测定中，CaMKKβ 不能使测试的任何一种 AMPK 相关激酶磷酸化或激活，尽管我们确实观察到 CaMKKα可以缓慢磷酸化和激活 BRSK1（脑特异性激酶 1）。尽管有最近的报道，但我们没有发现任何证据表明 AMPK 的α和/或β亚基与 CaMKKβ 形成稳定的复合物。我们还表明，增加 HeLa 细胞（缺乏 LKB1）中的 AMP 浓度对基础 AMPK 磷酸化没有影响，但增强了增加细胞内 Ca2+的试剂激活 AMPK 的能力。这与 AMP 对 Thr172 磷酸化的作用是由于抑制去磷酸化的作用一致，并证实了 AMP 的作用独立于所利用的上游激酶。
