Supplemental Data LKB 1 Is the Upstream Kinase in the AMP-Activated Protein Kinase Cascade

Supplemental Experimental Procedures ized by enhanced chemiluminescence (Pierce SuperSignal West Femto kit). Preparation and Assay of AMPK and AMPKK Recombinant AMPK ( 1 1 1) was expressed in E. coli and purified Cell Culture Differentiation of H-2K cells was as described previously [S6]. After as previously described [S1]. Other AMPK complexes ( 1 2 1, 2 1 1, 2 2 1, 1[D157A] 1 1, and 2[D157A] 1 1) were extreatment as described in the figure legend for Figures 2C and 2D, the culture medium was removed and 0.5 ml ice-cold lysis buffer pressed in E. coli and purified using the same procedure. AMPK activity was determined by phosphorylation of the synthetic SAMS (50 mM Hepes [pH 7.5], 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulphonyl peptide [S2] in the presence of 0.2 mM AMP. AMPKK was purified from rat liver through the Q-Sepharose step as described [S3]. fluoride, 10% glycerol, and 1% Triton X-100) immediately added to the cells. Insoluble material was removed by centrifugation, and the AMPKK was further purified by gel filtration using a Superdex-200 column (Amersham Biosciences) on an AKTA Purifier System. A supernatant fraction used for subsequent analysis. single peak of AMPKK activity was recovered, with an estimated molecular mass between 220–190 kDa, as judged by comparison Supplemental References with the elution of molecular mass standards. AMPKK activity was determined by activation of recombinant AMPK as described [S1]. S1. Neumann, D. , Woods, A. , Carling, D. , Wallimann, T. , and SchlattAMPK activity in cell lysates was measured in immunoprecipitates ner, U. (2003). Mammalian AMP-activated protein kinase: funcisolated using rabbit anti-pan antibodies [S4]. One unit of AMPKK tional, heterotrimeric complexes by co-expression of subunits activity is the amount that increased the activity of recombinant in Escherichia coli. Protein Expr. Purif. 30, 230–237. AMPK by 1 nmol/min/mg. S2. Davies, S.P., Carling, D. , and Hardie, D.G. (1989). Tissue distribution of AMP-activated protein kinase, and lack of activation Expression of Recombinant LKB1 by cyclic AMP-dependent protein kinase, studied using a speWild-type mouse LKB1 or catalytically inactive LKB1 harboring a cific and sensitive peptide assay. Eur. J. Biochem. 186, 123–128. mutation of aspartic acid residue 194 to alanine (D194A), both conS3. Hawley, S.A. , Davison, M.D., Woods, A. , Davies, S.P., Beri, R.K. , taining an N-terminal FLAG epitope tag, were expressed in mammaCarling, D. , and Hardie, D.G. (1996). Characterisation of the lian cells as described [S5]. Recombinant LKB1 was isolated by AMP-activated protein kinase kinase from rat liver and identifiimmunoprecipitation with anti-FLAG resin (Sigma) and assayed as cation of threonine-172 as the major site at which it phosphorydescribed below. In some cases, cells were transfected with lates AMP-activated protein kinase. J. Biol. Chem. 271, 27879– pCDNA3 (Invitrogen) encoding -galactosidase as a control. 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 AMPLKB1 Assay activated protein kinase subunit and subunit-assembly of LKB1 was immunoprecipitated from partially purified rat liver the heterotrimeric complex in vitro. J. Biol. Chem. 271, 10282– AMPKK by incubation with sheep anti-LKB1 antibodies (Upstate) in 10290. the presence of biotinylated donkey anti-sheep antibody (Sigma) S5. Hong, S.P., Leiper, F.C. , Woods, A. , Carling, D. , and Carlson, M. and streptavidin-agarose (Sigma) for 1 hr at 4 C. Endogenous LKB1 (2003). Activation of yeast Snf1 and mammalian AMP-activated in cultured cells was immunoprecipitated by incubation with goat protein kinase by upstream kinases. Proc. Natl. Acad. Sci. USA anti-LKB1 antibodies (Santa Cruz) prebound to protein G-Sepharose 100, 8839–8843. for 2 hr at 4 C. In both cases, the immune-complexes were collected S6. Fryer, L.G. , Patel, A.P., and Carling, D. (2002). The anti-diabetic by brief centrifugation and washed three times with 1 ml of ice-cold drugs rosiglitazone and metformin stimulate AMP-activated assay buffer (50 mM Hepes, [pH 7.4] containing 1% [v/v] Triton protein kinase through distinct pathways. J. Biol. Chem. 277, X-100). LKB1 activity present in the immune complexes was deter25226–25232. mined by its ability to activate recombinant AMPK. Immunoprecipitates were incubated with recombinant AMPK ( 1 1 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.