Analysis of the specificity of the AMP-activated protein kinase by site-directed mutagenesis of bacterially expressed 3-hydroxy 3-methylglutaryl-CoA reductase, using a single primer variant of the unique-site-elimination method.
The specificity of protein kinases is usually examined using synthetic peptide substrates, either designed variants, or, more recently random peptide libraries. However not all protein kinases utilize synthetic peptides efficiently as substrates. Even among those that do, these approaches neglect effects caused by three-dimensional protein conformation, or the existence of determinants remote from the phosphorylation site. To follow up our previous peptide studies on the specificity of the AMP-activated protein kinase (AMPK) [Dale, S., Wilson, W. A., Edelman, A.M., & Hardie, D. G. (1995) FEBS Lett. 361, 191-195], we have expressed the C-terminal, catalytic domain of Chinese hamster hydroxymethylglutaryl-CoA reductase in Escherichia coli. The domain was expressed with an N-terminal His6 tag which allowed rapid purification on Nj(2+)-agarose. The purified protein retained full enzymic activity, and as with the native enzyme, was totally inactivated by phosphorylation by AMPK at a single site corresponding to Ser871. Using a novel modification of the unique-site elimination method (which allowed direct mutagenesis of the double-stranded expression vector using a single oligonucleotide primer) we expressed 18 mutations involving residues around Ser871. The results broadly confirmed the recognition motif previously proposed on the basis of peptide studies. Three of the mutants were better substrates for AMPK than the wild type, and one of these (K872A) had hydroxymethylglutaryl-CoA reductase kinetic parameters virtually indistinguishable from the wild type. This suggests that hydroxymethylglutaryl-CoA reductase may have been selected to be a sub-optimal substrate for AMPK.
Ching
Y P
YP
Department of Biochemistry, The University, Dundee, Scotland.
Davies
S P
SP
Hardie
D G
DG
D055372:AMP-Activated Protein Kinases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002384:Catalysis; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D006903:Hydroxymethylglutaryl CoA Reductases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases