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Abstract
The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2- (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2β2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.
Original language | English |
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Pages (from-to) | 587-599 |
Number of pages | 13 |
Journal | Biochemical Journal |
Volume | 481 |
Issue number | 8 |
Early online date | 22 Apr 2024 |
DOIs | |
Publication status | Published - Apr 2024 |
Keywords
- AMPK
- LKB1
- CBS repeats
- AMP
- ADP
- adenine nucleotides
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology
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Dive into the research topics of 'AMP-activated protein kinase can be allosterically activated by ADP but AMP remains the key activating ligand'. Together they form a unique fingerprint.Projects
- 1 Finished
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Role of AMPK in Nutrient Sensing and in Cancer (Investigator Award Renewal)
Hardie, G. (Investigator)
1/10/17 → 31/03/24
Project: Research