in the AMP-Activated Protein Kinase Cascade. Angela Woods, Stephen R. Johnstone,. Kristina Dickerson, Fiona C. Leiper,. Lee G.D. Fryer, Dietbert Neumann,.
Supplemental Data
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LKB1 Is the Upstream Kinase in the AMP-Activated Protein Kinase Cascade Angela Woods, Stephen R. Johnstone, Kristina Dickerson, Fiona C. Leiper, Lee G.D. Fryer, Dietbert Neumann, Uwe Schlattner, Theo Wallimann, Marian Carlson, and David Carling Supplemental Experimental Procedures Preparation and Assay of AMPK and AMPKK Recombinant AMPK (␣11␥1) was expressed in E. coli and purified as previously described [S1]. Other AMPK complexes (␣12␥1, ␣21␥1, ␣22␥1, ␣1[D157A]1␥1, and ␣2[D157A]1␥1) were expressed in E. coli and purified using the same procedure. AMPK activity was determined by phosphorylation of the synthetic SAMS peptide [S2] in the presence of 0.2 mM AMP. AMPKK was purified from rat liver through the Q-Sepharose step as described [S3]. AMPKK was further purified by gel filtration using a Superdex-200 column (Amersham Biosciences) on an AKTA Purifier System. A single peak of AMPKK activity was recovered, with an estimated molecular mass between 220–190 kDa, as judged by comparison with the elution of molecular mass standards. AMPKK activity was determined by activation of recombinant AMPK as described [S1]. AMPK activity in cell lysates was measured in immunoprecipitates isolated using rabbit anti-pan  antibodies [S4]. One unit of AMPKK activity is the amount that increased the activity of recombinant AMPK by 1 nmol/min/mg. Expression of Recombinant LKB1 Wild-type mouse LKB1 or catalytically inactive LKB1 harboring a mutation of aspartic acid residue 194 to alanine (D194A), both containing an N-terminal FLAG epitope tag, were expressed in mammalian cells as described [S5]. Recombinant LKB1 was isolated by immunoprecipitation with anti-FLAG resin (Sigma) and assayed as described below. In some cases, cells were transfected with pCDNA3 (Invitrogen) encoding -galactosidase as a control. LKB1 Assay LKB1 was immunoprecipitated from partially purified rat liver AMPKK by incubation with sheep anti-LKB1 antibodies (Upstate) in the presence of biotinylated donkey anti-sheep antibody (Sigma) and streptavidin-agarose (Sigma) for 1 hr at 4⬚C. Endogenous LKB1 in cultured cells was immunoprecipitated by incubation with goat anti-LKB1 antibodies (Santa Cruz) prebound to protein G-Sepharose for 2 hr at 4⬚C. In both cases, the immune-complexes were collected by brief centrifugation and washed three times with 1 ml of ice-cold assay buffer (50 mM Hepes, [pH 7.4] containing 1% [v/v] Triton X-100). LKB1 activity present in the immune complexes was determined by its ability to activate recombinant AMPK. Immunoprecipitates were incubated with recombinant AMPK (␣11␥1, 12.5 g/ml) in assay buffer containing 0.1 mM ATP and 5 mM MgCl2 for 30 min at 37⬚C. Following brief centrifugation, AMPK activity in the supernatant was measured using the SAMS peptide assay [S2] in the presence of 0.2 mM AMP. In preliminary experiments, we determined that LKB1 activity was linear with respect to both protein concentration and time (results not shown). We define 1 unit of LKB1 activity as the amount required to increase the activity of recombinant AMPK by 1 nmol/min/mg. Western Blot Analysis LKB1 was detected using sheep anti-LKB1 antibody (Upstate; 1:1000 dilution). AMPK expression in mammalian cells was determined by detecting the  subunit using rabbit anti-pan  antiserum (1:20,000 dilution), since we were unable to detect AMPK ␣1 or ␣2 expression in crude cell lysates. Blots were developed with horseradish peroxidase linked secondary antibodies (Bio-Rad) and visual-
ized by enhanced chemiluminescence (Pierce SuperSignal West Femto kit). Cell Culture Differentiation of H-2Kb cells was as described previously [S6]. After treatment as described in the figure legend for Figures 2C and 2D, the culture medium was removed and 0.5 ml ice-cold lysis buffer (50 mM Hepes [pH 7.5], 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulphonyl fluoride, 10% glycerol, and 1% Triton X-100) immediately added to the cells. Insoluble material was removed by centrifugation, and the supernatant fraction used for subsequent analysis. Supplemental References S1. Neumann, D., Woods, A., Carling, D., Wallimann, T., and Schlattner, U. (2003). Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli. Protein Expr. Purif. 30, 230–237. S2. Davies, S.P., Carling, D., and Hardie, D.G. (1989). Tissue distribution of 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. S3. Hawley, S.A., Davison, M.D., Woods, A., Davies, S.P., Beri, R.K., Carling, D., and Hardie, D.G. (1996). Characterisation of the AMP-activated protein kinase kinase from rat liver and identification of threonine-172 as the major site at which it phosphorylates AMP-activated protein kinase. J. Biol. Chem. 271, 27879– 27887. S4. Woods, A., Cheung, P.C.F., Smith, F.C., Davison, M.D., Scott, J., Beri, R.K., and Carling, D. (1996). Characterization of AMPactivated protein kinase  subunit and ␥ subunit-assembly of the heterotrimeric complex in vitro. J. Biol. Chem. 271, 10282– 10290. S5. Hong, S.P., Leiper, F.C., Woods, A., Carling, D., and Carlson, M. (2003). Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases. Proc. Natl. Acad. Sci. USA 100, 8839–8843. S6. Fryer, L.G., Patel, A.P., and Carling, D. (2002). The anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct pathways. J. Biol. Chem. 277, 25226–25232.
