Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-linked polyubiquitin chains.
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 Lys(29) and/or Lys(33) rather than the more common Lys(48)/Lys(63). 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 Lys(29)/Lys(33)-linked polyubiquitin chains.
Al-Hakim
Abdallah K
AK
MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK.
Zagorska
Anna
A
Chapman
Louise
L
Deak
Maria
M
Peggie
Mark
M
Alessi
Dario R
DR
D000595:Amino Acid Sequence; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D025821:Polyubiquitin / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*
D012097:Repressor Proteins; C506234:USP9X protein, human; D025821:Polyubiquitin; D011494:Protein Kinases; C483047:MARK4 protein, human; C470845:NUAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D043222:Ubiquitin Thiolesterase; D008239:Lysine