TY - JOUR
T1 - Protein kinase substrate recognition studied using the recombinant catalytic domain of AMP-activated protein kinase and a model substrate
AU - Scott, John W.
AU - Norman, David G.
AU - Hawley, Simon A.
AU - Kontogiannis, Luke
AU - Hardie, D. Grahame
N1 - Funding Information:
This study was supported by a Programme Grant (to D.G.H.) and a Prize Studentship (to J.W.S.) from the Wellcome Trust. We are very grateful to David Carling for gifts of plasmids expressing AMPK and the CCL13 cells.
PY - 2002/3/22
Y1 - 2002/3/22
N2 - 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.
AB - 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.
KW - Acetyl-CoA carboxylase
KW - AMP-activated protein kinase
KW - Kinase-substrate interaction
KW - Molecular modelling
KW - Site-directed mutagenesis
UR - http://www.scopus.com/inward/record.url?scp=0036290846&partnerID=8YFLogxK
U2 - 10.1006/jmbi.2001.5316
DO - 10.1006/jmbi.2001.5316
M3 - Article
C2 - 11902845
AN - SCOPUS:0036290846
SN - 0022-2836
VL - 317
SP - 309
EP - 323
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -