title: Chemoproteomic analysis of intertissue and interspecies isoform diversity of AMP-activated protein kinase (AMPK).
abstract: AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that senses and governs changes in the cellular energy balance represented by concentrations of AMP, ADP, and ATP. Each of its three chains (α, β, and γ) exists as either two or three subtypes, theoretically allowing up to 12 different forms of the complete enzyme. Tissue specificity in the distribution of AMPK subtypes is believed to underpin a range of biological functions for AMPK, a central regulator of metabolic function and response. It is of particular interest for drug discovery purposes to compare AMPK isoforms that are most prevalent in human liver and muscle with isoforms present in key preclinical species. To complement immunocapture/immunodetection methods, which for AMPK are challenged by sequence similarities and difficulties of obtaining accurate relative quantitation, AMPK was captured from lysates of a range of cells and tissues using the ActivX ATP probe. This chemical probe covalently attaches desthiobiotin to one or more conserved lysyl residues in the ATP-binding sites of protein kinases, including AMPK, while also labeling a wide range of ATP-utilizing proteins. Affinity-based recovery of labeled proteins followed by gel-based fractionation of the captured sample was followed by proteomic characterization of AMPK polypeptides. In agreement with transcript-based analysis and previous indications from immunodetection, the results indicated that the predominant AMPK heterotrimer in human liver is α1β2γ1 but that dog and rat livers mainly contain the α1β1γ1 and α2β1γ1 forms, respectively. Differences were not detected between the AMPK profiles of normal and diabetic human liver tissues.
authors.affiliation: From Pfizer Worldwide Research, Groton, Connecticut 06340 and.
authors.forename: Jiang
authors.initials: J
authors.lastname: Wu
authors.forename: Dinesh
authors.initials: D
authors.lastname: Puppala
authors.forename: Xidong
authors.initials: X
authors.lastname: Feng
authors.forename: Mara
authors.initials: M
authors.lastname: Monetti
authors.forename: Amanda Lee
authors.initials: AL
authors.lastname: Lapworth
authors.forename: Kieran F
authors.initials: KF
authors.lastname: Geoghegan
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004285:Dogs; D057809:HEK293 Cells; D022781:Hepatocytes / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009928:Organ Specificity; D020836:Protein Structure, Quaternary; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013045:Species Specificity
chemical_list: D007527:Isoenzymes; D012333:RNA, Messenger; D055372:AMP-Activated Protein Kinases
keywords: AMP-activated Kinase (AMPK); Chemical Biology; Chemical Modification; Drug Discovery; Mass Spectrometry (MS)