Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: a cautionary tale

Abstract

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.

Fig. 1

Partial sequence of pET28a showing the translation start site, hexahistidine tag and thrombin recognition site. The serines encoded within the leader peptide are boxed; the serine within the thrombin cleavage site shown to be phosphorylated in this study is indicated by a shaded box.

Fig. 2

Phosphorylation of his6-C8 by AMPK. (A) One microgram of his₆-C8 was phosphorylated by AMPK in the presence of γ³²P-ATP as described in Materials and methods and separated by SDS–PAGE. Lane 1: Coomassie Blue-stained gel; lane 2: autoradiograph. (B) Time course of AMPK phosphorylation of 800 pmol SAMS (filled circles) and 800 pmol his₆-C8 (open circles). Phosphorylation reactions were set up as described; aliquots were taken at the indicated time points and spotted onto phosphocellulose paper. The radioactivity remaining on the paper after washing with 10% TCA was determined by scintillation counting. (C) Phosphoamino acid analysis of ³²P-labeled his₆-C8. Total amino acids were separated by electrophoresis at pH 1.9 (first) and pH 3.5 and ³²P-amino acids detected by autoradiography. Position of stained phosphoamino acid standards are marked.

Fig. 3

Thrombin digestion reveals that the phosphorylatable serine of his₆-C8 lies in the leader peptide not within C8 itself. (A and B) Thrombin digestion of AMPK-phosphorylated his₆-C8. A: Coomassie Blue-stained gel; B: autoradiograph. Lane 1: undigested AMPK-phosphorylated his₆-C8 (control); lane 2: AMPK-phosphorylated his₆-C8 after thrombin digestion. (C and D) AMPK phosphorylation subsequent to thrombin digestion of his6-C8 fusion protein. A: Coomassie Blue-stained gel; B: autoradiograph. Lane 1: undigested AMPK-phosphorylated his₆-C8 (control); lane 2: AMPK-phosphorylated his₆-C8 after thrombin digestion.