Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-inked polyubiquitin chains

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

    Research output: Contribution to journalArticle

    136 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)249-260
    Number of pages12
    JournalBiochemical Journal
    Volume411
    Early online date6 Feb 2008
    DOIs
    Publication statusPublished - 15 Apr 2008

    Keywords

    • Deubiquitination
    • LKB1
    • Protein kinase
    • Ubiquitination
    • Ubiquitin-specific protease

    Cite this

    Al-Hakim, Abdallah K. ; Zagorska, Anna ; Chapman, Louise ; Deak, Maria ; Peggie, Mark ; Alessi, Dario R. / Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-inked polyubiquitin chains. In: Biochemical Journal. 2008 ; Vol. 411. pp. 249-260.
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    Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-inked polyubiquitin chains. / Al-Hakim, Abdallah K.; Zagorska, Anna; Chapman, Louise; Deak, Maria; Peggie, Mark; Alessi, Dario R.

    In: Biochemical Journal, Vol. 411, 15.04.2008, p. 249-260.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-inked polyubiquitin chains

    AU - Al-Hakim, Abdallah K.

    AU - Zagorska, Anna

    AU - Chapman, Louise

    AU - Deak, Maria

    AU - Peggie, Mark

    AU - Alessi, Dario R.

    PY - 2008/4/15

    Y1 - 2008/4/15

    N2 - 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.

    AB - 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.

    KW - Deubiquitination

    KW - LKB1

    KW - Protein kinase

    KW - Ubiquitination

    KW - Ubiquitin-specific protease

    U2 - 10.1042/BJ20080067

    DO - 10.1042/BJ20080067

    M3 - Article

    VL - 411

    SP - 249

    EP - 260

    JO - Biochemical Journal

    JF - Biochemical Journal

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    ER -