A novel mass spectrometry-based assay for GSK-3beta activity.
BACKGROUND
As a component of the progression from genomic to proteomic analysis, there is a need for accurate assessment of protein post-translational modifications such as phosphorylation. Traditional kinase assays rely heavily on the incorporation of gamma-P32 radiolabeled isotopes, monoclonal anti-phospho-protein antibodies, or gel shift analysis of substrate proteins. In addition to the expensive and time consuming nature of these methods, the use of radio-ligands imposes restrictions based on the half-life of the radionucleotides and pose potential health risks to researchers. With the shortcomings of traditional assays in mind, the aim of this study was to develop a high throughput, non-radioactive kinase assay for screening Glycogen Synthase Kinase-3beta (GSK-3beta) activity.


RESULTS
Synthetic peptide substrates designed with a GSK-3beta phosphorylation site were assayed with both recombinant enzyme and GSK-3beta immunoprecipitated from NIH 3T3 fibroblasts. A molecular weight shift equal to that of a single phosphate group (80 Da.) was detected by surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) in a GSK-3beta target peptide (2B-Sp). Not only was there a dose-dependent response in molecular weight shift to the amount of recombinant GSK-3beta used in this assay, this shift was also inhibited by lithium chloride (LiCl), in a dose-dependent manner.


CONCLUSIONS
We present here a novel method to sensitively measure peptide phosphorylation by GSK-3beta that, due to the incorporation of substrate controls, is applicable to either purified enzyme or cell extracts. Future studies using this method have the potential to elucidate the activity of GSK-3beta in vivo, and to screen enzyme activity in relation to a variety of GSK-3beta related disorders.
Bowley
Erin
E
Cell and Molecular Biology Laboratory, Hand and Upper Limb Centre, Lawson Health Research Institute, St. Joseph's Health Centre, London, Ontario, Canada. bowley@hotmail.com
Mulvihill
Erin
E
Howard
Jeffrey C
JC
Pak
Brian J
BJ
Gan
Bing Siang
BS
O'Gorman
David B
DB
D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D007700:Kinetics; D018021:Lithium Chloride / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D010455:Peptides; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride