The use of phosphoproteomics to discover novel AMPK substrates
N Morrice, F Vandermoere and K Sakamoto
MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, DD1 5EH Dundee, UK
AMPK is a key enzyme that is activated by AMP during exercise, hypoxia and glucose deprivation. Most of the metabolic changes induced by AMPK, such as increased glucose uptake or decreased gluconeogenesis would be desirable outcomes for the treatment of type 2 diabetes. AMPK was therefore proposed to be a promising target for treatment with drugs like AICAR, metformin and phenformin. However these drugs that indirectly activate AMPK are either toxic either inefficiently incorporated in vivo. A new allosteric activator of AMPK, A-769662, has been recently characterized1.
Two different label-free approaches were used to investigate the changes in phosphorylation induced by this new activator in wild type and AMPK knock-out cells. The first approach used was a method based on calcium phosphate precipitation2 coupled with immobilized metal affinity chromatography and titanium dioxide phosphopeptide enrichment. Enriched fractions were then analysed by LC-MS on an LTQ-orbitrap with gas phase fractionation. The second approach used a pre-enrichment/fractionation step by Hydrophilic Interaction Chromatography3 followed by IMAC, then the same LC-MS strategy as described above.
Both approaches essentially enriched just phosphopeptides and we were able to quantify and identify more than three thousand phosphopeptides by each method. Based on the consensus sequence for AMPK phosphorylation we also found 25 potential new substrates of AMPK for which phosphorylation is induced by the A-769662 in wild type cells but not in AMPK knock-out cells. The new substrates included glucocosamine-fructose 6-phosphate amino transferase, a limiting enzyme of the hexoamines biosynthesis pathway that could be a key in glucose incorporation regulation by AMPK. This substrate has been validated as an in vivo substrate of AMPK and we are in the process of validating a number of other substrates.
We observed that only 15-20% of the ions were selected for msms on the LTQ-orbitrap and as such we were not identifying or quantifying all the phosphopeptides that had been enriched by either strategy. Therefore the number of potential AMPK substrates that could have been identified may well have been greater.
(1) Göransson O et al, Mechanism of action of A-769662, a valuable tool for activation of AMP-activated protein kinase. J Biol Chem. 2007 282: 32549 - 32560
(2) Zhang X et al, Highly Efficient Phosphopeptide Enrichment by Calcium Phosphate Precipitation Combined with Subsequent IMAC Enrichment. Mol Cell Proteomics. 2007 6: 2032-2042
(3) McNulty DE et al, Hydrophilic-interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection. Mol Cell Proteomics. 2008 (in press)
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