Abstract
The insulin/IGF-1 (insulin-like growth factor 1)-activated protein kinase Akt (also known as protein kinase B) phosphorylates Ser487 in the ‘ST loop’ (serine/threonine-rich loop) within the C-terminal domain of AMPK-α1 (AMP-activated protein kinase-α1), leading to inhibition of phosphorylation by upstream kinases at the activating site, Thr172. Surprisingly, the equivalent site on AMPK-α2, Ser491, is not an Akt target and is modified instead by autophosphorylation. Stimulation of HEK (human embryonic kidney)-293 cells with IGF-1 caused reduced subsequent Thr172 phosphorylation and activation of AMPK-α1 in response to the activator A769662 and the Ca2+ ionophore A23187, effects we show to be dependent on Akt activation and Ser487 phosphorylation. Consistent with this, in three PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null tumour cell lines (in which the lipid phosphatase PTEN that normally restrains the Akt pathway is absent and Akt is thus hyperactivated), AMPK was resistant to activation by A769662. However, full AMPK activation could be restored by pharmacological inhibition of Akt, or by re-expression of active PTEN. We also show that inhibition of Thr172 phosphorylation is due to interaction of the phosphorylated ST loop with basic side chains within the αC-helix of the kinase domain. Our findings reveal that a previously unrecognized effect of hyperactivation of Akt in tumour cells is to restrain activation of the LKB1 (liver kinase B1)–AMPK pathway, which would otherwise inhibit cell growth and proliferation.
Highlights
AMPK (AMP-activated protein kinase) is a ubiquitously expressed sensor of cellular energy status [1], which exists in essentially all eukaryotic cells as heterotrimeric complexes comprising a catalytic α subunit and regulatory β and γ subunits
There may be epigenetic or Abbreviations: ACC, acetyl-CoA carboxylase; AICAR, 5-amino-4-imidazolecarboxamide riboside; AMPK, AMP-activated protein kinase; BRSK, brainspecific kinase; CaMKK, calmodulin-dependent kinase kinase β; DMEM, Dulbecco’s modified Eagle’s medium; GSK3, glycogen synthase kinase 3; HEK, human embryonic kidney; IGF-1, insulin-like growth factor 1; LKB1, liver kinase B1; MEF, mouse embryonic fibroblast; MO25α, mouse protein25α; mTORC1, mammalian target of rapamycin complex 1; NEAA, non-essential amino acid; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PTEN, phosphatase and tensin homologue deleted on chromosome 10; S6K1, S6 kinase 1; ST loop, serine/threonine-rich loop; STRADα, Ste20-related adapter protein-α; WT, wild-type
It has been reported previously that prior phosphorylation of Ser485 on rat AMPK-α1 by Akt, within a rat α1β1γ 1 complex, caused a 40 % reduction in the rate of subsequent phosphorylation of Thr172 and activation by LKB1 in cell-free assays, an effect that was abolished by a non-phosphorylatable S485A substitution [27]
Summary
AMPK (AMP-activated protein kinase) is a ubiquitously expressed sensor of cellular energy status [1], which exists in essentially all eukaryotic cells as heterotrimeric complexes comprising a catalytic α subunit and regulatory β and γ subunits. AMPK has a cytostatic effect on proliferating cells, suggesting that it might exert some of the tumour suppressor effects of its upstream kinase, LKB1. This was supported by recent studies of a mouse B-cell lymphoma model, in which AMPK appeared to act as a negative regulator both of the rapid glycolysis (Warburg effect) and the high growth rate of the tumours [20]. In the HeLa cell, derived from a cervical cancer that had undergone a large deletion in STK11 [23], increases in AMP and ADP do not enhance Thr172 phosphorylation [4] because the basal activity of CaMKKβ is too low to support this unless intracellular Ca2+ is elevated [24]. There may be epigenetic or Abbreviations: ACC, acetyl-CoA carboxylase; AICAR, 5-amino-4-imidazolecarboxamide riboside; AMPK, AMP-activated protein kinase; BRSK, brainspecific kinase; CaMKK, calmodulin-dependent kinase kinase β; DMEM, Dulbecco’s modified Eagle’s medium; GSK3, glycogen synthase kinase 3; HEK, human embryonic kidney; IGF-1, insulin-like growth factor 1; LKB1, liver kinase B1; MEF, mouse embryonic fibroblast; MO25α, mouse protein25α; mTORC1, mammalian (or mechanistic) target of rapamycin complex 1; NEAA, non-essential amino acid; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A (cAMP-dependent protein kinase); PTEN, phosphatase and tensin homologue deleted on chromosome 10; S6K1, S6 kinase 1; ST loop, serine/threonine-rich loop; STRADα, Ste20-related adapter protein-α; WT, wild-type
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