Crebp
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.
PMID: 14511678 [PubMed - in process]