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J. Cell Sci. 118 (23): 5661-5673


Research Article

14-3-3 cooperates with LKB1 to regulate the activity and localization of QSK and SIK

Abdallah K. Al-Hakim1,*, Olga Göransson1, Maria Deak1, Rachel Toth1, David G. Campbell1, Nick A. Morrice1, Alan R. Prescott2, and Dario R. Alessi1

1 MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK
2 Division of Cell Biology and Immunology, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK

* Author for correspondence (e-mail: a.alhakim{at}dundee.ac.uk)

Accepted for publication 31 August 2005.

Abstract: The LKB1 tumour suppressor kinase phosphorylates and activates a number of protein kinases belonging to the AMP-activated protein kinase (AMPK) subfamily. We have used a modified tandem affinity purification strategy to identify proteins that interact with AMPK{alpha}, as well as the twelve AMPK-related kinases that are activated by LKB1. The AMPKß and AMPK{gamma} regulatory subunits were associated with AMPK{alpha}, but not with any of the AMPK-related kinases, explaining why AMP does not influence the activity of these enzymes. In addition, we identified novel binding partners that interacted with one or more of the AMPK subfamily enzymes, including fat facets/ubiquitin specific protease-9 (USP9), AAA-ATPase-p97, adenine nucleotide translocase, protein phosphatase 2A holoenzyme and isoforms of the phospho-protein binding adaptor 14-3-3. Interestingly, the 14-3-3 isoforms bound directly to the T-loop Thr residue of QSK and SIK, after these were phosphorylated by LKB1. Consistent with this, the 14-3-3 isoforms failed to interact with non-phosphorylated QSK and SIK, in LKB1 knockout muscle or in HeLa cells in which LKB1 is not expressed. Moreover, mutation of the T-loop Thr phosphorylated by LKB1, prevented QSK and SIK from interacting with 14-3-3 in vitro. Binding of 14-3-3 to QSK and SIK, enhanced catalytic activity towards the TORC2 protein and the AMARA peptide, and was required for the cytoplasmic localization of SIK and for localization of QSK to punctate structures within the cytoplasm. To our knowledge, this study provides the first example of 14-3-3 binding directly to the T-loop of a protein kinase and influencing its catalytic activity and cellular localization.

Key Words: AMPK • MARK/Par1 • Cell polarity • Mass spectrometry • Tandem affinity purification


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