Calmodulin-dependent protein kinase kinase-beta activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP.
Calmodulin-dependent protein kinase kinase-beta activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP.
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DOI：
10.1042/bj20091372
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发表时间：
2010-01-27
期刊：
影响因子：
--
通讯作者：
Hardie DG
中科院分区：
文献类型：
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作者：
Fogarty S;Hawley SA;Green KA;Saner N;Mustard KJ;Hardie DG
通过Thr 172磷酸化激活AMPK（AMP活化蛋白激酶）由至少两种不同的上游激酶催化，即肿瘤抑制因子LKB 1和CaMKKβ（Ca 2 +/钙调蛋白依赖性蛋白激酶激酶-β）。Thr 172周围的序列在AMPK的两种催化亚基同种型和12种AMPK相关激酶之间高度保守，并且LKB 1已被证明在所有它们的上游起作用。在本文中，我们报告了在完整细胞或无细胞试验中，没有一种AMPK相关激酶可以被CaMKKβ磷酸化或激活，尽管我们确实观察到CaMKKα对BRSK 1（脑特异性激酶1）的缓慢磷酸化和激活。尽管最近的报道，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，在HeLa细胞（缺乏LKB 1）中增加AMP浓度对基础AMPK磷酸化没有影响，但增强了增加细胞内Ca 2+激活AMPK的试剂的能力。这与AMP对Thr 172磷酸化的作用是由于去磷酸化的抑制一致的，并且证实AMP的作用不依赖于所利用的上游激酶。
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 CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase-β). 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 CaMKKβ, although we did observe a slow phosphorylation and activation of BRSK1 (brain-specific kinase 1) by CaMKKα. Despite recent reports, we could not find any evidence that the α and/or β subunits of AMPK formed a stable complex with CaMKKβ. 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.