Homogeneous assays for adenosine 5'-monophosphate-activated protein kinase.
Allosteric activation of 5(')-AMP-activated protein kinase (AMPK) is currently of interest as an approach for the treatment of metabolic disorders because AMPK plays multiple roles in glucose and lipid metabolism. The availability of ultrafast, ultrasensitive, and robust assays suited to high-throughput screening (HTS) is key to obtaining small-molecule AMPK activators. In the absence of high-affinity and selective antiphospho Ser/Thr antibodies for AMPK substrates, we have developed two homogeneous AMPK assays with the commercially available antibody Anti-pS(133)-CREB and an engineered peptide ACC-CREBp. Anti-pS(133)-CREB antibody was raised against the phospho-CREB peptide derived from cAMP response element binding protein (CREB). ACC-CREBp was a variant (Arg to Pro) of ACC-CREB, a hybrid peptide consisting of a 9-amino-acid peptide from rat acetyl-CoA carboxylase (ACC), CREB peptide, and the addition of two hydrophobic Leu residues. ACC-CREBp showed increased suitability as a substrate for AMPK, eliminated phosphorylation by PKA, and preserved antibody binding. The homogeneous time-resolved fluorescence and AlphaScreen AMPK assays were developed using both Anti-pS(133)-CREB antibody and ACC-CREBp that are either labeled with a fluorescent probe or linked to a photoactivated bead, respectively. Thus, ACC-CREBp phosphorylation can be measured as a signal change resulting from the formation of antibody-antigen complex. This approach of adapting known antibody and antigenic peptide pairs to monitor alternate Ser/Thr kinases may be of general use.
D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004789:Enzyme Activation; D007118:Immunoassay / Q000379:methods*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000032:analysis* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D017186:Titrimetry
D000906:Antibodies; D017362:Cyclic AMP Response Element-Binding Protein; D009097:Multienzyme Complexes; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase