Protein kinase substrate recognition studied using the recombinant catalytic domain of AMP-activated protein kinase and a model substrate

John W. Scott, David G. Norman, Simon A. Hawley, Luke Kontogiannis, D. Grahame Hardie (Lead / Corresponding author)

    Research output: Contribution to journalArticlepeer-review

    143 Citations (Scopus)

    Abstract

    We have expressed a truncated form of the α1 kinase domain of AMP-activated protein kinase (AMPK) in Escherichia coli as a glutathione-S-transferase fusion (GST-KD). A T172D mutant version did not require prior phosphorylation and was utilized for most subsequent studies. We have also created a recombinant substrate (GST-ACC) by expressing 34 residues around the major phosphorylation site (Ser79) on rat acetyl-CoA carboxylase-1/α (ACC1) as a GST fusion. This was an excellent substrate that was phosphorylated with similar kinetic parameters to ACC1 by both native AMPK and the bacterially expressed kinase domain. We also constructed a structural model for the binding of the ACC1 sequence to the kinase domain, based on crystal structures for related protein kinases. The model was tested by making a total of 25 mutants of GST-ACC and seven mutants of GST-KD, and measuring kinetic parameters with different combinations. The results reveal that AMPK and ACC1 interact over a much wider region than previously realized (>20 residues). The features of the interaction can be summarised as follows: (i) an amphipathic helix from P - 16 to P - 5 on the substrate binds in a hydrophobic groove on the large lobe of the kinase; (ii) basic residues at P - 6 and P - 4 bind to two acidic patches (D215/D216/D217 and E103/D100/E143, respectively), on the large lobe; (iii) a histidine at P + 3 interacts with D56 on the small lobe; (iv) the side-chain of P + 4 leucine could not be precisely positioned, but a new finding was that asparagine or glutamine could replace a hydrophobic residue at this position. These interactions position the serine residue to be phosphorylated in close proximity to the γ-phosphate group of ATP. Although based on modelling rather than a determined structure, this represents one of the most detailed studies of the interaction between a kinase and its substrate achieved to date.

    Original languageEnglish
    Pages (from-to)309-323
    Number of pages15
    JournalJournal of Molecular Biology
    Volume317
    Issue number2
    DOIs
    Publication statusPublished - 22 Mar 2002

    Keywords

    • Acetyl-CoA carboxylase
    • AMP-activated protein kinase
    • Kinase-substrate interaction
    • Molecular modelling
    • Site-directed mutagenesis

    ASJC Scopus subject areas

    • Structural Biology
    • Molecular Biology

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