Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: a cautionary tale.
AMP-activated protein kinase (AMPK) is responsible for sensing of the cell's energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades. Mutations in the gene encoding the gamma 2 regulatory subunit have been shown to cause hypertrophic cardiomyopathy (HCM) with conduction abnormalities. As part of a study to examine the role of AMPK in the heart, we tested whether specific domains of the thick filament component cardiac myosin binding protein-C (cMyBP-C) were good in vitro AMPK substrates. The commercially available pET28a expression vector was used to generate a recombinant form of the cMyBP-C C8 domain as a fusion protein with a hexahistidine tag. In vitro phosphorylation with activated kinase showed that the purified fusion protein was a good AMPK substrate, phosphorylated at a similar rate to the control SAMS peptide and with phosphate incorporation specifically in serine residues. However, subsequent analysis of alanine replacement mutants and thrombin digestion revealed that the strong AMPK phosphorylation site was contained within the thrombin cleavage sequence encoded by the vector. As this sequence is common to many commercial pET vectors, caution is advised in the mapping of AMPK phosphorylation sites when this sequence is present.
Renz
Bernhard
B
Department of Cardiovascular Medicine, University of Oxford, West Wing Level 6, John Radcliffe Hospital, Oxford OX3 9DU, UK.
Davies
Joanna K
JK
Carling
David
D
Watkins
Hugh
H
Redwood
Charles
C
D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D000099163:Myosin Binding Protein C
D055372:AMP-Activated Protein Kinases; D002352:Carrier Proteins; D006639:Histidine; D009842:Oligopeptides; D011994:Recombinant Proteins; D013917:Thrombin; D000099163:Myosin Binding Protein C; C471213:His-His-His-His-His-His