AMPK (AMP-activated protein kinase)-related kinases regulate cell polarity as well as proliferation and are activated by the LKB1-tumour suppressor kinase. In the present study we demonstrate that the AMPK-related kinases, NUAK1 (AMPK-related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4), are polyubiquitinated in vivo and interact with the deubiquitinating enzyme USP9X (ubiquitin specific protease-9). Knockdown of USP9X increased polyubiquitination of NUAK1 and MARK4, whereas overexpression of USP9X inhibited ubiquitination. USP9X, catalysed the removal of polyubiquitin chains from wild-type NUAK1, but not from a non-USP9X-binding mutant. Topological analysis revealed that ubiquitin monomers attached to NUAK1 and MARK4 are linked by Lys29 and/or Lys33 rather than the more common Lys48/Lys63. We find that AMPK and other AMPK-related kinases are also polyubiquitinated in cells. We identified non-USP9X-binding mutants of NUAK1 and MARK4 and find that these are hyper-ubiquitinated and not phosphorylated at their T-loop residue targeted by LKB1 when expressed in cells, suggesting that polyubiquitination may inhibit these enzymes. The results of the present study demonstrate that NUAK1 and MARK4 are substrates of USP9X and provide the first evidence that AMPK family kinases are regulated by unusual Lys29/Lys33-linked polyubiquitin chains.
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Research Article|
March 27 2008
Control of AMPK-related kinases by USP9X and atypical Lys29/Lys33-linked polyubiquitin chains
Abdallah K. Al-Hakim;
Abdallah K. Al-Hakim
1MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Anna Zagorska;
Anna Zagorska
1MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Louise Chapman;
Louise Chapman
1MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Maria Deak;
Maria Deak
1MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Mark Peggie;
Mark Peggie
1MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Dario R. Alessi
Dario R. Alessi
1
1MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
1To whom correspondence should be addressed (email d.r.alessi@dundee.ac.uk).
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Publisher: Portland Press Ltd
Received:
January 08 2008
Revision Received:
February 04 2008
Accepted:
February 06 2008
Accepted Manuscript online:
February 06 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 411 (2): 249–260.
Article history
Received:
January 08 2008
Revision Received:
February 04 2008
Accepted:
February 06 2008
Accepted Manuscript online:
February 06 2008
Connected Content
This is a commentary on:
Regulation of the AMPK-related protein kinases by ubiquitination
Citation
Abdallah K. Al-Hakim, Anna Zagorska, Louise Chapman, Maria Deak, Mark Peggie, Dario R. Alessi; Control of AMPK-related kinases by USP9X and atypical Lys29/Lys33-linked polyubiquitin chains. Biochem J 15 April 2008; 411 (2): 249–260. doi: https://doi.org/10.1042/BJ20080067
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