Raquel Sancho Solis 1 , Ying Ge 1,2* and Jeffery W. Walker 3†
1 Department of Physiology, 2 Human Proteomics Program, School of Medicine and Public
Health, University of Wisconsin-Madison, WI 53706, and 3 Department of Physiology, University of Arizona, Tucson, AZ 85724
Running Title: AMPK Phosphorylation Sites on Troponin I
*Address correspondence to: Ying Ge, Ph.D., 1300 University Ave., SMI 130, Madison, WI,
53706. Email: yge@physiology.wisc.edu; Tel: 608-263-9212; Fax: 608-265-5512.
† This work is dedicated to the memory of Prof. Jeffery W. Walker (1954-2010).
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EXPERIMENTAL PROCEDURES
Materials .
All reagents were obtained from Sigma Chemical Co (St Louis, MO) unless noted otherwise. SAMS peptide and LKB1/STRAD/MO25 complex were from Millipore
(Framingham, MA). Primers were from Operon (Huntsville, Alabama). Complete  protease inhibitor cocktail was from Roche (Mannheim, Germany). AMPK  phosphothreonine-172 polyclonal antibody and AMPK  polyclonal antibody were from Cell Signaling (Beverly, MA).
Secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
Expression and purification of recombinant human AMPK  1 constructs.
cDNA for Nterminal His-tagged AMPK  1 (His-AMPK  1) was obtained from Genecopoeia (Germantown,
Maryland). Mutations were introduced using site-directed mutagenesis kits (Stratagene, La Jolla,
CA). His-AMPK  1(1-312) was created by introducing a STOP codon after amino acid 312 on
His-AMPK  1. A constitutively active His-AMPK  1(1-312) T172D was created by mutating
T 172 to Aspartate (D) on the His-AMPK  1(1-312) construct. A similar constitutively active kinase domain, His-AMPK  1(1-308) T172D, was created by mutating T 172 to D and by introducing a STOP codon after amino acid 308. Expression plasmids were transformed into
BL21(DE3) Escherichia coli cells and grown at 37  C in LB medium containing 100  g/ml ampicillin until A
600
reached 0.4. The culture was then induced with 0.4 mM IPTG and cells harvested 4 h later by centrifugation. Purification was carried out at 4  C. Constructs were purified with Profinity IMAC Ni-charged resin (BioRad, Hercules, CA) following the manufacturer's directions. Concentration and purity was assessed by SDS-PAGE using BSA as a standard.
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Activity of purified human AMPK  1 constructs .
Enzymatic activity of purified AMPK  1 constructs was evaluated by their ability to phosphorylate the SAMS peptide, a specific substrate for AMPK.
1 Purified AMPK  1 constructs (50-1000 nM) were incubated in the presence or absence of 15 nM LKB1/STRAD/MO25 complex in kinase buffer (60 mM HEPES-NaOH pH
7.5, 3 mM MgCl
2
, 3  M Na
3
VO
4
, 1.2 mM DTT, 500  M ATP and protease inhibitor cocktail) for 15 minutes at 37  C, followed by addition of 250  M SAMS peptide and further incubation for varied times. Peptide phosphorylation was assessed using HPLC. Thirty microliter aliquots were injected onto an ion exchange HPLC column, eluted at 5 ml/min with a linear gradient of 0-
1 M NaCl and monitored by UV absorbance at 220 nm. Peak identities were confirmed by
MALDI-TOF mass spectrometry as described previously.
2
Western blots.
Purified His-AMPK  1(1-312) (400 nM) was incubated with 15 nM
LKB1/STRAD/MO25 complex for varied times at 37  C in kinase buffer (see above). Samples were subjected to 12% SDS-PAGE, transferred to nitrocellulose membrane and immunoblotted with an AMPK  polyclonal antibody to phosphothreonine-172. Loading control was obtained by stripping the membrane and probing with an AMPK  polyclonal antibody.
Data Fitting .
Data from time course phosphorylation experiments were fitted using Origin v
7.5 Software (Origin lab corporation, Northampton, MA).
RESULTS
A plasmid containing the cDNA of human AMPK  1 with an N-terminal His-tag was used to create the AMPK constructs. A His-AMPK  1(1-312) construct was created by introducing a STOP codon after amino acid 312. A constitutively active His-AMPK  1(1-312)
T172D was created by mutating Thr 172 to Aspartate (D) on the His-AMPK  1(1-312) construct.
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A similar constitutively active construct, His-AMPK  1(1-308) T172D, was created by introducing a STOP codon after amino acid 308 and mutating Thr 172 to D. All mutations were introduced using site-directed mutagenesis and verified by DNA sequencing. The plasmids were transformed into Escherichia coli and affinity-purified using the His-tag. Concentration and purity were assessed by SDS-PAGE using BSA as a standard. His-AMPK  1(1-312), His-
AMPK  1(1-312) T72D and His-AMPK  1(1-308) T72D were purified to near homogeneity
(Figure 1A). Enzymatic activity was evaluated by the ability of the constructs to phosphorylate the SAMS peptide, a specific substrate for AMPK.
1 His-AMPK  1(1-312), His-AMPK  1(1-
312) T172D and His-AMPK  1(1-308) T172D were essentially inactive. Upon incubation with the upstream kinase LKB1/STRAD/MO25, the activity of His-AMPK  1(1-312) greatly increased while His-AMPK  1(1-312) T72D and His-AMPK  1(1-308) T172D remained inactive (Figure 2B), consistent with results from other studies.
3-5 It is difficult to compare the observed activity of His-AMPK  1(1-312) to published activities for similar constructs because of the great variety in assay conditions and different units for the quantification of activity.
Reasons for the difficulties in creating a pseudophosphorylated constitutively active AMPK construct remain unresolved. However, it is possible that, for the specific case of AMPK, the introduced Aspartate residue is unable to fully mimic a phosphate moiety, failing to promote the conformational changes needed for activity of the kinase domain. Alternatively, there may still be some autoinhibition present in the first 308 amino acids of the alpha subunit. Finally, it is possible that the constructs containing Aspartate do not fold properly or are unstable and denature quickly under the conditions employed.
To determine the incubation time required to completely phosphorylate the AMPK kinase domain, His-AMPK  1(1-312) was incubated with LKB1/STRAD/MO25 and aliquots removed
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at various times. Phosphorylation of Thr 172 was assessed using a phosphospecific antibody.
Antibody signals within the linear range of the film were quantified by densitometry, plotted and fitted to first order exponentials. Under the conditions employed, His-AMPK  1(1-312) was fully phosphorylated within a 5 min incubation time (Figure 2).
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1.
REFERENCES
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3.
4.
5.
Davies SP, Carling D,Hardie DG (1989) Tissue distribution of the AMP-activated protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive peptide assay. Eur J Biochem 186:123-128.
Chung KY,Walker JW (2007) Interaction and inhibitory cross-talk between endothelin and ErbB receptors in the adult heart. Mol Pharmacol 71:1494-1502.
Pang T, Xiong B, Li JY, Qiu BY, Jin GZ, Shen JK,Li J (2007) Conserved alpha-helix acts as autoinhibitory sequence in AMP-activated protein kinase alpha subunits. J Biol Chem
282:495-506.
Hamilton SR, O'Donnell JB, Jr., Hammet A, Stapleton D, Habinowski SA, Means AR,
Kemp BE,Witters LA (2002) AMP-activated protein kinase kinase: detection with recombinant AMPK alpha1 subunit. Biochem Biophys Res Commun 293:892-898.
Crute BE, Seefeld K, Gamble J, Kemp BE,Witters LA (1998) Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase. J Biol Chem 273:35347-
35354.
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FIGURES LEGENDS
Supplemental Figure 1 : Purification and activity of His-AMPK constructs. A. Representative gel of AMPK constructs purified from Escherichia coli.
B. Activities of purified His-AMPK constructs assessed by phosphorylation of the SAMS peptide. Values are means  SEM (n = 3-
6). Activities for  1(1-312) T172D and  1 (1-308) T172D were identical in the presence and absence of LKB1/STRAD/MO25 and data was pooled.
Supplemental Figure 2 : Phosphorylation of Thr 172 in purified His-AMPK  1(1-312). His-
AMPK  1(1-312) was treated with LKB1/STRAD/MO25. Phosphorylation was assessed by
Western blotting analysis using a phosphospecific antibody. Top panel shows representative blots for AMPK  phosphothreonine-172 and AMPK  (load control). Bottom graph shows the exponential fitting to the data from 3 separate experiments.
Supplemental Figure 3 : Localization of cTnI Ser 149 as a phosphorylated site in cTnI.
Representative spectra of z 
66
8+ (A) and z 
53
6+ (B) ion fragments from ECD of cTnI 1P or 2P from samples treated with active (left) or inactive (right) AMPK. p z and pp z represent mono- and bisphosphorylated z  fragment ions. Circles represent the theoretical abundance isotopic distribution of the isotopic peaks corresponding to the assigned mass. Calc'd, calculated most abundant mass; Expt'l, experimental most abundant mass.
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Figure 1.
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Figure 2.
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Figure 3.
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