Dissecting the Role of 5′-AMP for Allosteric Stimulation, Activation, and Deactivation of AMP-activated Protein Kinase

AMP-activated protein kinase (AMPK) is a heterotrimeric protein kinase that is crucial for cellular energy homeostasis of eukaryotic cells and organisms. Here we report on the activation of AMPK α1β1γ1 and α2β2γ1 by their upstream kinases (Ca2+/calmodulin-dependent protein kinase kinase-β and LKB1-MO25α-STRADα), the deactivation by protein phosphatase 2Cα, and on the extent of stimulation of AMPK by its allosteric activator AMP, using purified recombinant enzyme preparations. An accurate high pressure liquid chromatography-based method for AMPK activity measurements was established, which allowed for direct quantitation of the unphosphorylated and phosphorylated artificial peptide substrate, as well as the adenine nucleotides. Our results show a 1000-fold activation of AMPK by the combined effects of upstream kinase and saturating concentrations of AMP. The two AMPK isoforms exhibit similar specific activities (6 μmol/min/mg) and do not differ significantly by their responsiveness to AMP. Due to the inherent instability of ATP and ADP, it proved impossible to assay AMPK activity in the absolute absence of AMP. However, the half-maximal stimulatory effect of AMP is reached below 2 μm. AMP does not appear to augment phosphorylation by upstream kinases in the purified in vitro system, but deactivation by dephosphorylation of AMPK α-subunits at Thr-172 by protein phosphatase 2Cα is attenuated by AMP. Furthermore, it is shown that neither purified NAD+ nor NADH alters the activity of AMPK in a concentration range of 0–300 μm, respectively. Finally, evidence is provided that ZMP, a compound formed in 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside-treated cells to activate AMPK in vivo, allosterically activates purified AMPK in vitro, but compared with AMP, maximal activity is not reached. These data shed new light on physiologically important aspects of AMPK regulation. AMP-activated protein kinase (AMPK) is a heterotrimeric protein kinase that is crucial for cellular energy homeostasis of eukaryotic cells and organisms. Here we report on the activation of AMPK α1β1γ1 and α2β2γ1 by their upstream kinases (Ca2+/calmodulin-dependent protein kinase kinase-β and LKB1-MO25α-STRADα), the deactivation by protein phosphatase 2Cα, and on the extent of stimulation of AMPK by its allosteric activator AMP, using purified recombinant enzyme preparations. An accurate high pressure liquid chromatography-based method for AMPK activity measurements was established, which allowed for direct quantitation of the unphosphorylated and phosphorylated artificial peptide substrate, as well as the adenine nucleotides. Our results show a 1000-fold activation of AMPK by the combined effects of upstream kinase and saturating concentrations of AMP. The two AMPK isoforms exhibit similar specific activities (6 μmol/min/mg) and do not differ significantly by their responsiveness to AMP. Due to the inherent instability of ATP and ADP, it proved impossible to assay AMPK activity in the absolute absence of AMP. However, the half-maximal stimulatory effect of AMP is reached below 2 μm. AMP does not appear to augment phosphorylation by upstream kinases in the purified in vitro system, but deactivation by dephosphorylation of AMPK α-subunits at Thr-172 by protein phosphatase 2Cα is attenuated by AMP. Furthermore, it is shown that neither purified NAD+ nor NADH alters the activity of AMPK in a concentration range of 0–300 μm, respectively. Finally, evidence is provided that ZMP, a compound formed in 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside-treated cells to activate AMPK in vivo, allosterically activates purified AMPK in vitro, but compared with AMP, maximal activity is not reached. These data shed new light on physiologically important aspects of AMPK regulation. AMP-activated protein kinase (AMPK) 2The abbreviations used are: AMPK, 5′-AMP-activated protein kinase; AMPKK, AMPK kinase; AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; CaMKK, Ca2+/calmodulin-dependent protein kinase kinase; Cr, creatine; GST, glutathione S-transferase; LKB1, serine/threonine kinase 11 (STK11); MO25, mouse protein 25; PCr, phospho-creatine; PP2Cα, protein phosphatase-2C α isoform; SAMS, synthetic peptide HMRSAMSGLHLVKRR; STRAD, STE20-related adaptor protein; ZMP, AICAR-monophosphate; HPLC, high pressure liquid chromatography. and its homologues in insects, plants, and yeast are fuel sensors of the eukaryotic cell and master regulators of energy metabolism (1Carling D. Biochimie (Paris). 2005; 87: 87-91Crossref PubMed Scopus (181) Google Scholar, 2Hardie D.G. Hawley S.A. Scott J.W. J. Physiol. (Lond.). 2006; 574: 7-15Crossref Scopus (655) Google Scholar, 3Kahn B.B. 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M. Carling D. Cell Metab. 2005; 2: Full Text Full Text PDF PubMed Scopus Google Scholar). The of stimulation of AMPK by AMP in vitro was of extent (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. (Bronx N. Y.). 2003; 2: 28Google Scholar, A. Neumann D. Schlattner U. Wallimann T. M. Carling D. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, S.A. Mustard K.J. L. J. Hardie D.G. Cell Metab. 2005; 2: Full Text Full Text PDF PubMed Scopus Google Scholar, A. R. M. M. Carling D. Cell Metab. 2005; 2: Full Text Full Text PDF PubMed Scopus Google Scholar), a that to with a and high responsiveness in to the energy of cell In cells and AMPK is by but of the effects of AMPK using the compound D.G. Hawley S.A. Scott J.W. J. Physiol. (Lond.). 2006; 574: 7-15Crossref Scopus (655) Google Scholar). is a that is by cells and to AMP that the effects of AMP on the AMPK Hawley S.A. Hardie D.G. J. Biochem. 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Physiol. 2006; PubMed Scopus Google Scholar, M. R.A. U. Neumann D. Schlattner U. M.H. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, M.H. Schlattner U. Neumann D. M. M.H. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). AMPK in is but by upstream kinases that are capable of AMPK α-subunits at Thr-172 D. Woods A. Carling D. Wallimann T. Schlattner U. 2003; PubMed Scopus Google Scholar). AMPK activity is by of artificial peptide the and of the phosphorylated peptide by Carling D. Hardie D.G. J. Biochem. PubMed Scopus Google Scholar). The peptide is on the AMPK of and been used for AMPK activity the peptide the synthetic peptide is have been S. Hardie D.G. PubMed Scopus Google Scholar). of a of the AMPK have been used to the of phosphorylation by AMPK J.W. D.G. Hawley S.A. L. Hardie D.G. J. Biol. PubMed Scopus Google Scholar), and a assay using was for AMPK activity in A. T. 2006; PubMed Google Scholar). In we a method and the of AMP in AMPK regulation. AMPK and AMPK as D. Woods A. Carling D. Wallimann T. Schlattner U. 2003; PubMed Scopus Google Scholar). AMPK on and to kinase activity and as by as for enzyme of the enzyme in at of AMPK by Upstream and activity was with as and which was The assay of 2 AMP, SAMS, as well as AMPK for as in the A the of for was a and was as M. R. PubMed Scopus Google Scholar). The and of the heterotrimeric was in M. R. U. and T. in AMPK in on a at and at the and the was by the in liquid by at of activation by the of the was to by of the of and a The was with a of of a concentration of and with high in by and at of to a concentration of protein concentrations using protein assay with as a ADP, and NADH and on with a in using with two of was at and The was used to ATP of AMP ATP used was by the method and for its AMP which was below and and on a with a in at and in in liquid at and to to using Thr-172 of and with and by on a using The method for AMPK activity on artificial peptide substrate, that is with AMPK in of The of is by as the at the of the AMPK, with adenine nucleotides. AMP is allosteric is a of the kinase and ATP is a high concentrations of and AMP of ATP to the kinase by a ATP is to stimulation of AMPK by AMP by to the allosteric Hawley S.A. Hardie D.G. J. Biochem. PubMed Scopus Google Scholar). accurate of the concentrations of adenine SAMS, and of the AMPK of adenine is with chromatography. The with a of was to with at and the of phosphorylation its to the a allowed for of AMPK activity and two on the different a of two of adenine as well as and The of the for activity of AMPK is in of activity assay at different and for The data show the of the and respectively. AMP was at concentrations to AMPK A in compared with was with different of AMPK a of to by inherent activity of AMPK to of ATP as it to with AMPK in the absence of not ATP not shown in the of and the of the AMPK and of and AMPK Furthermore, the concentrations of and in a are and the of the used and adenine to the but in of was a data is and used for of specific of purified heterotrimeric AMPK α1β1γ1 and α2β2γ1 was with recombinant of of the two upstream kinases of the was used at saturating activation with a of upstream kinase to AMPK to with the heterotrimeric a of was to The specific activity of AMPK activation and stimulation by AMP was in the range of of of at was similar for isoforms of AMPK and reported with recombinant AMPK, using recombinant AMPK by purified upstream kinase (5Woods A. Vertommen D. Neumann D. Turk R. Bayliss J. Schlattner U. Wallimann T. Carling D. Rider M.H. J. Biol. Chem. 2003; 278: 28434-28442Abstract Full Text Full Text PDF PubMed Scopus (183) Google by recombinant A. R. M. M. Carling D. Cell Metab. 2005; 2: Full Text Full Text PDF PubMed Scopus Google Scholar). AMPK have been purified to and similar high specific activities of using the method and as a D. Witters L.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the method with the used method of AMPK activity we a purified of AMPK with a specific activity at as by using the and we its activity by to the kinase assay AMP, and we using assay AMP, and reached a of that the method the specific activities by a of in to the used assay that the specific activity of purified recombinant AMPK is to activity using Furthermore, the of AMPK in significant of specific activity that AMPK been by upstream kinases high specific activities in vitro in the absence of AMP of AMPK as shown in by activation by AMPKK, of in α-subunits as a of phosphorylation of AMPK at Thr-172 by as been A. R. M. M. Carling D. Cell Metab. 2005; 2: Full Text Full Text PDF PubMed Scopus Google Scholar). The of the in activation by that β-subunits to in In the β-subunits of the and not in and are not activation by not that the of the is by However, a of the not we do not have for The of AMPK which may different of the to a of AMPK The of the activation with AMPK α2β2γ1 is shown in of at different of with upstream kinases to that the phosphorylation activity was reached the of as by the of the in is with a of the stimulatory effect by AMP of AMPK with Upstream by does not the activities of the upstream kinases (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. (Bronx N. Y.). 2003; 2: 28Google Scholar, A. Neumann D. Schlattner U. Wallimann T. M. Carling D. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, S.A. Mustard K.J. L. J. Hardie D.G. Cell Metab. 2005; 2: Full Text Full Text PDF PubMed Scopus Google Scholar, A. R. M. M. Carling D. Cell Metab. 2005; 2: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the of of AMP to AMPK the enzyme a for upstream kinases is a of used the kinase of AMPK and allosteric effects by AMP, in with the two heterotrimeric isoforms of AMPK their and for allosteric regulation. AMPK was in the and absence of AMP with the two upstream kinases and by the Thr-172 with the two AMPK α1β1γ1 and α2β2γ1 isoforms as shown in of the different AMPK with of AMP was that of AMP to the allosteric does not augment phosphorylation at the of AMPK by upstream at the of the activation assay was in AMP was which to stimulation of However, in in vitro system, by of AMPK α-subunit the of in with the of AMPK and for the two to the not Thr-172 phosphorylation in the in the absence of AMP not and are compared and a was in with AMPK which the that AMPK is a for upstream and compared with AMPK A effect of the that is different in the and and are to of AMPK by of AMPK stimulation by AMP and the concentration range AMP its stimulatory effect are for in of the using the method ATP and by a significant of AMP as on and ATP ATP was purified by HPLC, but ATP of AMP to with a at ATP at and AMP, at the of AMPK activity and of AMP to in the assay with and AMPK α1β1γ1 and respectively. In the absence of its substrate, the AMPK not significant of AMP not which the that enzyme kinase have the of AMP the of the the of the to AMP AMP as the concentration of AMP with the of ATP by The AMP of each was the the AMP concentration at the of each assay at the using ATP and the recombinant of AMPK activity in absence of AMP to the it was not to assay with AMP. to AMP the assay using not the concentration of AMP not below not of AMPK activity in the absence of AMP it is that the in AMP is specific activities of AMPK of the activity data using the that is a results in a with high and are and However, we that the of a AMPK activity and AMP not for AMPK, a of 2 was on evidence J.W. Hawley S.A. M. D.G. Hardie D.G. J. 2004; PubMed Scopus Google Scholar). using a of 2 and the is and are and stimulation of AMPK by AMP below 2 AMPK activity to the AMP significant of AMP. we at it is that of the results in for half-maximal that AMPK to concentrations of AMP. 2 AMP the for half-maximal and are significantly The stimulatory effect of AMP on AMPK activity is to AMP was to AMP AMPK activity and and the extent of stimulation may on activation of AMPK by upstream kinases However, of AMP to the is as by the stimulatory effect of AMP AMP and the specific activities at In a of of the is stimulatory effect by AMP. we the as we do not the AMP may well AMPK activity of the data a high stimulatory of AMP with half-maximal effect at stimulation of AMPK by AMP at μm, AMPK activity is to maximal at AMP with AMPK isoforms AMPK in the and of AMPK activity in the absence of AMP, the of AMP the we AMPK activity in the absence of upstream of recombinant AMPK to activation by is but The specific activities in the absence of AMP for AMPK These to of AMP at saturating concentrations not AMP in NAD+ for of cellular which is by was recently to AMPK activity in to R. L. L. D. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). AMPK and AMPK to in a by NADH AMPK a stimulatory effect of NAD+ was in using NAD+ a of NAD+ by HPLC, a significant which we to AMP, as is a of AMP and NAD+ with AMP to using of the that AMP with the that is in NAD+ NAD+ concentrations of and μm, the AMP in the AMPK assay was to at and μm, respectively. the NAD+ was to AMPK activity In the absence of AMP at concentrations the AMPK Furthermore, NAD+ a in a AMP at and at in AMP concentration of NAD+ that NAD+ is in at the stimulation by NAD+ of AMPK a direct allosteric R. L. L. D. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google is to NADH AMPK was by HPLC, and as shown in A concentration range of to NADH effect on the AMPK α1β1γ1 AMP of the AMPK by phosphorylated of AMPK by protein and the for the activity in Hardie D.G. Scholar). The of is for dephosphorylation of AMPK at Thr-172 in cells J. Hardie D.G. J. Biochem. PubMed Scopus Google Scholar). at saturating concentrations recombinant AMPK, in the as well as in the absence of AMP not However, the of phosphatase allowed for a of with and AMPK and at AMP with Thr-172 and the results are shown in with of quantitation by The of α2β2γ1 dephosphorylation is significant and a similar of is with is not reached. In dephosphorylation of does not show AMP The enzyme activities phosphatase are shown in activities with AMP to the phosphatase the activity of AMPK by the kinase was by the AMP AMP does not but the dephosphorylation of AMPK by is attenuated by of AMP to the allosteric of the of of AMPK by compound been used to activate AMPK in cell and in N. Chem. 2006; PubMed Scopus Google Scholar). is by cells and to a of adenine In vivo, AMPK activation a direct allosteric Hawley S.A. Hardie D.G. J. Biochem. PubMed Scopus Google Scholar). Here we the allosteric effect of on recombinant AMPK in to not AMPK to In with AMP for stimulation of the of was significantly at μm, and at high concentrations of stimulation by AMP was reached. However, it that AMPK activity of AMP. it is not to the stimulatory effect of AMPK is important kinase a for the of the cellular energy by a high cellular ATP to AMP D.G. Scott J.W. 2003; PubMed Scopus Google Scholar). In to AMPK is to its activity to of cellular energy that of AMPK and D. Schlattner U. Wallimann T. Biochem. 2003; PubMed Google Scholar). Here we new data the activation of AMPK by AMP and the of the allosteric on AMPK the upstream kinase and phosphatase we used purified recombinant AMPK, of AMPK in D. Woods A. Carling D. Wallimann T. Schlattner U. 2003; PubMed Scopus Google Scholar). A that may using recombinant AMPK and recombinant for of enzyme may a of the used to to the in in J. 2005; PubMed Scopus Google Scholar), and the important for protein in M. 2004; PubMed Scopus Google Scholar). have used recombinant AMPK its with AMPK that been purified cell AMPK is that are not capable of Thr-172 of the phosphorylation at Thr-172 is below the with the Thr-172 we that AMPK is by of new phosphorylation sites using the (5Woods A. Vertommen D. Neumann D. Turk R. Bayliss J. Schlattner U. Wallimann T. Carling D. Rider M.H. J. Biol. Chem. 2003; 278: 28434-28442Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). In AMPK is at sites (4Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (499) Google Scholar, A. Vertommen D. Neumann D. Turk R. Bayliss J. Schlattner U. Wallimann T. Carling D. Rider M.H. J. Biol. 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Full Text Full Text PDF PubMed Scopus Google Scholar). to eukaryotic do not it is to of AMPK on the used for the of AMPK isoforms as in cells and Here we show that AMPK is 1000-fold by the combined effect of activation by its upstream with its allosteric AMP. upstream kinases activate AMPK to a similar and the two isoforms of AMPK α1β1γ1 and α2β2γ1 exhibit similar specific as well as to AMP. high of AMPK AMP at stimulation of AMPK was reached at 2 AMP, AMP is half-maximal stimulation at significantly with we stimulation by AMP (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. (Bronx N. Y.). 2003; 2: 28Google Scholar, A. Neumann D. Schlattner U. Wallimann T. M. Carling D. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, S.A. Mustard K.J. L. J. Hardie D.G. Cell Metab. 2005; 2: Full Text Full Text PDF PubMed Scopus Google Scholar, A. R. M. M. Carling D. Cell Metab. 2005; 2: Full Text Full Text PDF PubMed Scopus Google Scholar), but a stimulatory of AMP is the AMP is These the that AMPK is a LKB1-MO25-STRAD nor is by AMP (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. (Bronx N. Y.). 2003; 2: 28Google Scholar, A. Neumann D. Schlattner U. Wallimann T. M. Carling D. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, S.A. Mustard K.J. L. J. Hardie D.G. Cell Metab. 2005; 2: Full Text Full Text PDF PubMed Scopus Google Scholar, A. R. M. M. Carling D. Cell Metab. 2005; 2: Full Text Full Text PDF PubMed Scopus Google Scholar). However, phosphorylation of AMPK by a purified LKB1-MO25-STRAD was by AMP (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. (Bronx N. Y.). 2003; 2: 28Google Scholar), and in AMP not activation of AMPK by the A. Neumann D. Schlattner U. Wallimann T. M. Carling D. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). to results AMP direct effect on nor does of AMP to the allosteric of the AMPK appear to augment phosphorylation at Thr-172 of the α-subunit by the upstream kinases and In we reported that phosphorylation of recombinant AMPK by a purified of upstream kinases is by AMP J. Neumann D. R. Wallimann T. Hurley R.L. Witters L.A. 2005; PubMed Scopus Google Scholar). but it is that is in M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). AMPK phosphorylation in in purified not but the of of AMPK by its phosphatase is significantly attenuated by concentrations of AMP, and the not is with AMPK results reported Hardie D.G. PubMed Scopus Google Scholar), the that the as a for of the dephosphorylation by AMP concentrations phosphorylation of the α-subunit of AMPK at a prerequisite for significant AMPK to the cellular AMP AMP two direct for of AMPK activity allosteric stimulation of activity and of deactivation by M. Hardie D.G. Carling D. J. PubMed Scopus Google of AMPK activity by to a direct allosteric the effect of been recently by D.G. J. Physiol. 2006; PubMed Scopus Google Scholar). we have been to a direct allosteric effect on AMPK activity on its upstream kinases by in the concentration range of D. and T. A direct of AMPK with reported by the M. Hardie D.G. Carling D. J. PubMed Scopus Google Scholar), as AMPK in a cell by a enzyme in of high and energy T. M. D. Biochem. J. PubMed Scopus Google Scholar, Schlattner U. M. A. Wallimann T. J. Physiol. (Lond.). 2006; Scopus Google Scholar). However, a phosphorylation of kinase by AMPK M. Hardie D.G. Carling D. J. PubMed Scopus Google not In we the of NAD+ and NADH to allosterically AMPK activity as by R. L. L. D. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the to of NAD+ and identified AMP, a significant allosteric activation of However, neither NAD+ nor NADH direct effect on AMPK activity at concentrations to μm. the that AMPK as a for the cellular been used to activate AMPK in cells and N. Chem. 2006; PubMed Scopus Google Scholar). is by cells and to ZMP, which is to activate AMPK direct allosteric Here we direct allosteric effects of on AMPK in Our results in to AMP, the stimulatory of is and high concentrations to AMPK, that of AMP by in is is that activation of AMPK, not maximal for a cellular the which the compound additional that to activation of AMPK by in In the using a in vitro with purified AMPK, upstream and to a of physiologically important aspects of AMPK and regulation. AMP does not augment phosphorylation of AMPK in vitro, the of AMP in to the of Thr-172 phosphorylation of AMPK in in additional may are to for of D. Carling for and for with of the