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Article: 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

TitleAnalysis 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
Authors
Issue Date1996
PublisherBlackwell Publishing Ltd. The Journal's web site is located at http://www.blackwellpublishing.com/journals/EJB
Citation
European Journal Of Biochemistry, 1996, v. 237 n. 3, p. 800-808 How to Cite?
AbstractThe 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 us 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 Ni2+-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.
Persistent Identifierhttp://hdl.handle.net/10722/149554
ISSN
2006 Impact Factor: 3.579
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorChing, YPen_US
dc.contributor.authorDavies, SPen_US
dc.contributor.authorHardie, DGen_US
dc.date.accessioned2012-06-26T05:55:14Z-
dc.date.available2012-06-26T05:55:14Z-
dc.date.issued1996en_US
dc.identifier.citationEuropean Journal Of Biochemistry, 1996, v. 237 n. 3, p. 800-808en_US
dc.identifier.issn0014-2956en_US
dc.identifier.urihttp://hdl.handle.net/10722/149554-
dc.description.abstractThe 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 us 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 Ni2+-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.en_US
dc.languageengen_US
dc.publisherBlackwell Publishing Ltd. The Journal's web site is located at http://www.blackwellpublishing.com/journals/EJBen_US
dc.relation.ispartofEuropean Journal of Biochemistryen_US
dc.subject.meshAmp-Activated Protein Kinasesen_US
dc.subject.meshAmino Acid Sequenceen_US
dc.subject.meshAnimalsen_US
dc.subject.meshBase Sequenceen_US
dc.subject.meshBinding Sites - Geneticsen_US
dc.subject.meshCho Cellsen_US
dc.subject.meshCatalysisen_US
dc.subject.meshCricetinaeen_US
dc.subject.meshDna Primers - Geneticsen_US
dc.subject.meshEscherichia Coli - Geneticsen_US
dc.subject.meshGenetic Vectorsen_US
dc.subject.meshHydroxymethylglutaryl Coa Reductases - Genetics - Metabolismen_US
dc.subject.meshKineticsen_US
dc.subject.meshMolecular Sequence Dataen_US
dc.subject.meshMultienzyme Complexes - Metabolismen_US
dc.subject.meshMutagenesis, Site-Directeden_US
dc.subject.meshPhosphorylationen_US
dc.subject.meshPoint Mutationen_US
dc.subject.meshProtein Kinases - Metabolismen_US
dc.subject.meshProtein-Serine-Threonine Kinasesen_US
dc.subject.meshRecombinant Proteins - Genetics - Metabolismen_US
dc.subject.meshSubstrate Specificityen_US
dc.titleAnalysis 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 methoden_US
dc.typeArticleen_US
dc.identifier.emailChing, YP:ypching@hku.hken_US
dc.identifier.authorityChing, YP=rp00469en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.pmid8647128-
dc.identifier.scopuseid_2-s2.0-0029665214en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0029665214&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume237en_US
dc.identifier.issue3en_US
dc.identifier.spage800en_US
dc.identifier.epage808en_US
dc.identifier.isiWOS:A1996UK46700035-
dc.publisher.placeUnited Kingdomen_US
dc.identifier.scopusauthoridChing, YP=7005431277en_US
dc.identifier.scopusauthoridDavies, SP=7403126044en_US
dc.identifier.scopusauthoridHardie, DG=24373207900en_US

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