Control of AMPK-related kinases by USP9X and atypical Lys29/Lys33-linked polyubiquitin chains

Abdallah K Al-Hakim , Anna Zagorska , Louise Chapman , Maria Deak , Mark Peggie , Dario R Alessi (1)

1 MRC Protein Phosphorylation Unit, Department of Biochemistry

AMPK-related kinases regulate cell polarity as well as proliferation and are activated by the LKB1-tumour suppressor kinase. We demonstrate that the AMPK-related kinases, NUAK1 and MARK4, are polyubiquitinated in vivo and interact with the deubiquitinating enzyme USP9X. Knockdown of USP9X increased polyubiquitination of NUAK1 and MARK4, whilst overexpression of USP9X inhibited ubiquitination. USP9X catalysed the removal of polyubiquitin chains from wild type NUAK1, but not 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 targetted by LKB1 when expressed in cells, suggesting that polyubiquitination may inhibit these enzymes. Our results 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.

Mots clés: Life Sciences

Abdallah K Al-Hakim, Anna Zagorska, Louise Chapman, Maria Deak, Mark Peggie, et al.. Control of AMPK-related kinases by USP9X and atypical Lys29/Lys33-linked polyubiquitin chains. Biochemical Journal, 2008, 411 (2), pp.249-260. ⟨10.1042/BJ20080067⟩. ⟨hal-00478950⟩