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Bacterially expressed heterotrimeric (α1, β1, and γ1) wild-type, catalytically inactive, and constitutively active forms of AMP-activated protein kinase (AMPK) were used to study phosphorylation by an upstream AMPK kinase preparation. Here, we report the identification of two new phosphorylation sites in the α-subunit, viz. Thr258 and Ser485 (Ser491 in the α2-subunit) by mass spectrometry, in addition to the previously characterized Thr172 site. Also, autophosphorylation sites in the β1-subunit were identified as Ser96, Ser101, and Ser108. Mutagenesis of Thr172, Thr258, and Ser485 to acidic residues to mimic phosphorylation in the recombinant proteins indicated that Thr172 was involved in AMPK activation, whereas Thr258 and Ser485 were not. Transfection of the non-phosphorylatable S485A and T258A mutants in CCL13 cells subjected to stresses known to activate AMPK either by increasing the AMP:ATP ratio (slow lysis) or without changing adenine nucleotide concentrations (hyperosmolarity) resulted in no significant differences in AMPK activation. All three sites within the α-subunit were phosphorylated in vivo, as seen in AMPK immunoprecipitated from anoxic rat liver. In transfected CCL13 cells, the level of Ser485 phosphorylation did not change upon AMPK activation. The newly identified phosphorylation sites could play a subtle role in the regulation of AMPK, e.g. in subcellular localization or substrate recognition. Bacterially expressed heterotrimeric (α1, β1, and γ1) wild-type, catalytically inactive, and constitutively active forms of AMP-activated protein kinase (AMPK) were used to study phosphorylation by an upstream AMPK kinase preparation. Here, we report the identification of two new phosphorylation sites in the α-subunit, viz. Thr258 and Ser485 (Ser491 in the α2-subunit) by mass spectrometry, in addition to the previously characterized Thr172 site. Also, autophosphorylation sites in the β1-subunit were identified as Ser96, Ser101, and Ser108. Mutagenesis of Thr172, Thr258, and Ser485 to acidic residues to mimic phosphorylation in the recombinant proteins indicated that Thr172 was involved in AMPK activation, whereas Thr258 and Ser485 were not. Transfection of the non-phosphorylatable S485A and T258A mutants in CCL13 cells subjected to stresses known to activate AMPK either by increasing the AMP:ATP ratio (slow lysis) or without changing adenine nucleotide concentrations (hyperosmolarity) resulted in no significant differences in AMPK activation. All three sites within the α-subunit were phosphorylated in vivo, as seen in AMPK immunoprecipitated from anoxic rat liver. In transfected CCL13 cells, the level of Ser485 phosphorylation did not change upon AMPK activation. The newly identified phosphorylation sites could play a subtle role in the regulation of AMPK, e.g. in subcellular localization or substrate recognition. The AMP-activated protein kinase (AMPK) 1The abbreviations used are: AMPK, AMP-activated protein kinase; AMPKK, AMP-activated protein kinase kinase; HPLC, high pressure liquid chromatography; PP, protein phosphatase; ESI-MS/MS, electrospray ionization tandem mass spectrometry.1The abbreviations used are: AMPK, AMP-activated protein kinase; AMPKK, AMP-activated protein kinase kinase; HPLC, high pressure liquid chromatography; PP, protein phosphatase; ESI-MS/MS, electrospray ionization tandem mass spectrometry. is a serine/threonine protein kinase that is highly conserved in higher eukaryotes, yeast, and plants (1Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1141) Google Scholar, 2Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 82-855Crossref Scopus (1275) Google Scholar, 3Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.P. Witters L.A. Trends Biochem. Sci. 1999; 24: 22-25Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). AMPK is activated by changes in the AMP:ATP ratio, although activation of AMPK has been observed in response to stimuli that do not change intracellular adenine nucleotide concentrations (4Fryer L.G. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 277: 25226-25232Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). Under stress conditions, for example, during anoxia or exercise-induced skeletal muscle contraction, the ATP:ADP ratio falls, and there is a subsequent rise in intracellular AMP (5Hardie D.G. Hawley S.A. Bioessays. 2001; 23: 1112-1119Crossref PubMed Scopus (675) Google Scholar, 6Winder W.W. J. Appl. Physiol. 2001; 91: 1017-1028Crossref PubMed Scopus (325) Google Scholar), which activates AMPK. AMPK can also be activated by hyperosmotic stress (4Fryer L.G. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 277: 25226-25232Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar), leptin (7Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1675) Google Scholar), and adiponectin (8Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3453) Google Scholar); by the antidiabetic drugs metformin (4Fryer L.G. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 277: 25226-25232Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar, 9Hawley S. Gadalla A. Olsen G. Hardie D.G. Diabetes. 2002; 51: 2420-2425Crossref PubMed Scopus (576) Google Scholar) and rosiglitazone (4Fryer L.G. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 277: 25226-25232Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar); and by the compound 5-amino-4-imidazolecarboxamide ribonucleoside, which, upon entering cells, is converted into an AMP analog (10Henin N. Vincent M.-F. Van den berghe G. Biochim. Biophys. Acta. 1996; 1290: 197-203Crossref PubMed Scopus (94) Google Scholar). Once activated, AMPK acts to down-regulate ATP-consuming pathways such as fatty acid synthesis by phosphorylating and inactivating acetyl-CoA carboxylase (11Davies S.P. Sim A.T.R. Hardie D.G. Eur. J. Biochem. 1990; 187: 183-190Crossref PubMed Scopus (219) Google Scholar) and protein synthesis by promoting the phosphorylation of eukaryotic elongation factor-2 (12Horman S. Browne G.J. Krause U. Patel J.V. Vertommen D. Bertrand L. Lavoinne A. Hue L. Proud C.G. Rider M.H. Curr. Biol. 2002; 12: 1419-1423Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). AMPK also activates pathways involved in ATP production. For example, in heart, AMPK activation stimulates glycolysis by increasing glucose uptake (13Russell III, R.R. Bergeron R. Shulman G.I. Young L.H. Am. J. Physiol. 1999; 277: H643-H649PubMed Google Scholar) and by activating 6-phosphofructo-2-kinase (14Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.-F. Van den B erghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar). In yeast, the AMPK homolog Snf1 kinase is activated by low glucose concentrations and increases the transcription of genes for growth on alternative carbon sources (2Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 82-855Crossref Scopus (1275) Google Scholar). Therefore, AMPK can be regarded as both an energy and a nutrient sensor. AMPK is a heterotrimer consisting of a catalytic α-subunit and two regulatory subunits, β and γ. Each subunit exists as multiple isoforms (α1, α2, β1, β2, γ1, γ2, and γ3), giving 12 different possible combinations of holoenzyme with different tissue distribution and subcellular localization (2Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 82-855Crossref Scopus (1275) Google Scholar, 3Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.P. Witters L.A. Trends Biochem. Sci. 1999; 24: 22-25Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). AMPK is allosterically stimulated by AMP and is itself regulated by phosphorylation via an upstream AMPK kinase (AMPKK) (2Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 82-855Crossref Scopus (1275) Google Scholar, 3Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.P. Witters L.A. Trends Biochem. Sci. 1999; 24: 22-25Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). The major regulatory phosphorylation site has been identified as Thr172 within the activation loop between the DFG and APE motifs of the α-subunits (15Hawley S.A. Davison M. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. J. Biol. Chem. 1996; 271: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar, 16Mitchelhill K.I. Michell B.J House C.M. Stapleton D. Dyck J. Gamble J. Ullrich C. Witters L.A. Kemp B.E. J. Biol. Chem. 1997; 272: 24475-24479Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The upstream kinase has been partially purified from rat liver and was reported to contain an 58-kDa catalytic subunit in a 195-kDa complex (15Hawley S.A. Davison M. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. J. Biol. Chem. 1996; 271: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar). AMPK can also be phosphorylated at Thr172 by the surrogate AMPKK, Ca2+/calmodulin-dependent protein kinase I (17Hamilton S.R. O'Donnell Jr., J.B. Hammet A. Stapleton D. Habinowski S.A. Means A.R. Kemp B.E. Witters L.A. Biochem. Biophys. Res. Commun. 2002; 293 (B.): 892-898JCrossref PubMed Scopus (54) Google Scholar), leading to activation (17Hamilton S.R. O'Donnell Jr., J.B. Hammet A. Stapleton D. Habinowski S.A. Means A.R. Kemp B.E. Witters L.A. Biochem. Biophys. Res. Commun. 2002; 293 (B.): 892-898JCrossref PubMed Scopus (54) Google Scholar, 18Hawley S.A. Selbert M.A. Goldstein E.G. Edelman A.M. Carling D. Hardie D.G. J. Biol. Chem. 1995; 270: 27186-27191Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). AMP has been reported to activate AMPKK (18Hawley S.A. Selbert M.A. Goldstein E.G. Edelman A.M. Carling D. Hardie D.G. J. Biol. Chem. 1995; 270: 27186-27191Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar), although others have reported that AMPKK is constitutively active (17Hamilton S.R. O'Donnell Jr., J.B. Hammet A. Stapleton D. Habinowski S.A. Means A.R. Kemp B.E. Witters L.A. Biochem. Biophys. Res. Commun. 2002; 293 (B.): 892-898JCrossref PubMed Scopus (54) Google Scholar). Site-directed mutagenesis showed that phosphorylation at Thr172 accounts for most of the activation of AMPK by AMPKK, but that other sites were implicated (19Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (493) Google Scholar). The native β1-subunit was found to be phosphorylated in vivo at Ser24/Ser25, Ser108, and Ser182 (16Mitchelhill K.I. Michell B.J House C.M. Stapleton D. Dyck J. Gamble J. Ullrich C. Witters L.A. Kemp B.E. J. Biol. Chem. 1997; 272: 24475-24479Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Moreover, mutation of Ser108 to alanine leads to the inhibition of AMPK, whereas the S24A/S25A and S182A mutants have no effect on activity, but lead to the nuclear redistribution of the holoenzyme (20Warden S.M. Richardson C. O'Donnell Jr., J.B. Stapleton D. Kemp B.E. Witters L.A. Biochem. J. 2001; 354: 275-283Crossref PubMed Scopus (212) Google Scholar). AMPK autophosphorylates mainly on the β-subunits (21Chen Z. Heierhorst J. Mann R.J. Mitchelhill K.I. Michell B.J. Witters L.A. Lynch G.S. Kemp B.E. Stapleton D. FEBS Lett. 1999; 460: 343-348Crossref PubMed Scopus (114) Google Scholar), but these sites have not been identified. Here, we have used recently available bacterially expressed AMPK heterotrimers (22Neumann, D., Woods, A., Carling, D., Wallimann, T., and Schlattner, U. (2003) Protein Expression Purif., in pressGoogle Scholar) together with mass spectrometry to investigate phosphorylation of the kinase by an AMPKK preparation. By comparing the phosphorylation of the wild-type complex with that of a catalytically inactive mutant complex (D157A in the α1-subunit) (19Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (493) Google Scholar), we were able to distinguish between autophosphorylation sites and AMPKK phosphorylation sites. This to in addition to Thr172, two new AMPKK phosphorylation sites in the α-subunit and three autophosphorylation sites in the was from and were from and All other were from or and were from α-subunit was from α-subunit was by Hardie of The was in the an for to was from Expression and of and of wild-type and mutant in were as (22Neumann, D., Woods, A., Carling, D., Wallimann, T., and Schlattner, U. (2003) Protein Expression Purif., in pressGoogle Scholar). The of mutants and were from (19Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (493) Google Scholar) as for the of wild-type (22Neumann, D., Woods, A., Carling, D., Wallimann, T., and Schlattner, U. (2003) Protein Expression Purif., in pressGoogle Scholar). All other were into by which by of the mutation together with a new site Biochem. Scopus Google Scholar). the mutation or in wild-type the site was with a whereas the mutant was from the mutant with to and the mutant from the mutant by to were by and in The AMPK of was to the newly and the of the in an to the of the used for site of a site and of a of a site and of a in a new of Bacterially AMPK by recombinant AMPK was in the or of a of AMPKK partially purified from rat liver to the as previously (15Hawley S.A. Davison M. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. J. Biol. Chem. 1996; 271: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar). The in and with or without AMP and were for at AMPK was by to acid that were with and was to the and the was subjected to in of AMPK from with Protein AMPK purified from anoxic rat liver to the as previously D. Hardie D.G. Eur. J. Biochem. PubMed Scopus Google Scholar) was without and was to a of and a of protein and purified from as previously for muscle P. S. P. PubMed Scopus Google Scholar) was to a of recombinant from R. A. J. Carling D. G. F. Beri R. Biochem. J. 1996; Scholar) was at a of were for at addition of by and to the α-subunit and were from and in in M.H. M. Van J. K. S. J. H. J. Eur. J. Biochem. 1995; PubMed Scopus Google Scholar, K. M. Van J. M. S. J. 1996; PubMed Scopus Google Scholar). The phosphorylated proteins were of the and with of or as M.H. M. Van J. K. S. J. H. J. Eur. J. Biochem. 1995; PubMed Scopus Google Scholar). were by at a of M.H. M. Van J. K. S. J. H. J. Eur. J. Biochem. 1995; PubMed Scopus Google Scholar). were D. Rider M.H. Y. J. Van J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) by ionization tandem mass spectrometry in an mass were in and to and The was at and the energy was to the for were identified in by the of low and the phosphorylated was in In were purified for M.H. Van J. Vertommen D. A. J. Hue L. FEBS Lett. PubMed Scopus Google Scholar). For in vivo phosphorylation site AMPK was immunoprecipitated from anoxic rat liver with or A. J. Hardie D.G. Carling D. FEBS Lett. 1996; PubMed Scopus Google Scholar) and subjected to in Protein were from the and as for were by on a as D. Rider M.H. Y. J. Van J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). For phosphorylation site the mass was in with an at a This of a by a high and a of the most from the energy was at and of on a on the observed in was from the a was observed in the of the was in the and of protein indicated in the were by in and a The was in and with low The was with of in the for with the was for at with the to with the were For of AMPK the were by a was of Bacterially recombinant AMPK was activated with the of AMPKK in consisting of in and ATP for at the or of as in the were in and and for AMPK by phosphorylation of the S.P. Carling D. Hardie D.G. Eur. J. Biochem. PubMed Scopus Google Scholar) of purified AMPK from anoxic rat liver purified to the as previously D. Hardie D.G. Eur. J. Biochem. PubMed Scopus Google Scholar) was also by phosphorylation of the Transfection of CCL13 and of AMPK was a to the CCL13 cells were with of the wild-type or T258A and the and into by C. H. Biol. PubMed Scopus Google Scholar). The an G.I. G. Biol. PubMed Scopus Google Scholar), and the an were by of two or lysis) T. Carling D. Biochem. J. 2002; PubMed Google Scholar). AMPK was in by from G.I. G. Biol. PubMed Scopus Google Scholar). The was to the of of the as by of with were for at with to protein were by at for and with to AMPK was as expressed as of into the of of of AMPK by both autophosphorylation and AMPKK was by from into the and of wild-type, catalytically inactive and constitutively active the and the of the AMPK the mutant was catalytically inactive was by the that there was no phosphorylation of AMPK in the of of the mutant with AMPKK to phosphorylation of the α-subunit, which was to AMP For the there was no phosphorylation of the in the of AMPKK, that phosphorylation of the β-subunits of the wild-type AMPK complex was low in the of In the of AMPKK, both the and β-subunits were The mutant autophosphorylation of the α-subunit to a of the in the of AMPKK and autophosphorylation of the was in the of an of the The α-subunit of the mutant was phosphorylated by AMPKK, the of sites on the α-subunit for AMPKK other phosphorylation The of phosphorylation in was For example, for phosphorylation by AMPKK, was of of The for such a low is not of for AMPKK in the AMPK expressed wild-type and catalytically inactive AMPK heterotrimers were with purified AMPKK and as The and were by and in for with or The were by of the and rise to major in with in the rise to two major not were AMPK was without AMPKK and sites for the upstream kinase differences in the were observed between wild-type and catalytically inactive although were in the and contain autophosphorylation sites. were in with different of both AMPK and AMPKK, that the distribution and of between were Each was for by of by a of mass to upon low energy with a addition to a of the in The of to the of via in of the phosphorylated J. J. Am. 1998; Scholar). the at was in the of the was and the phosphorylated was identified as Ser485 The in the was also found to be phosphorylated in of Ser485 was also by of Ser485 as an in phosphorylation site in the by major in from the of AMPK phosphorylated by AMPKK was by and found to contain in the of the is of was observed low energy to an at the of the is The and have a mass of to the of and and to the or of the of AMPK by vivo in a new a of which upon low energy to a of not This was to Thr172 as the phosphorylated in phosphorylated Thr172, was observed in the in and to a to the that was by the phosphorylated Thr172 in in the was not seen the or were with and be by by from a a of mass by upon low energy This mass was to a in the α-subunits in that two phosphorylated residues and and two in the mass could not the Thr258 in a that not be to be on the the of in Therefore, Thr258 is most to be the phosphorylated in of in the AMPK expressed wild-type AMPK heterotrimer was and the β1-subunit was purified and as rise to major in were not observed with catalytically inactive AMPK with or without AMPKK, that of with a phosphorylated in a β1-subunit in the and identified as the not of This mass can be to a in the β1-subunit in that three phosphorylated residues and to the identification of Ser108 as the phosphorylated both and Ser108, no phosphorylated could be in an of which upon low energy This mass can be to a phosphorylated in the β1-subunit in that two residues and two identified as the phosphorylated was observed in the phosphorylated Ser108 in a with no in of in in the AMPK phosphorylation sites identified in could also be in vivo, we AMPK or immunoprecipitated from anoxic rat liver by liquid This to that and Thr258 in the AMPK α-subunits and Ser108 in the β-subunits were phosphorylated in vivo the sites identified in or previously in the (16Mitchelhill K.I. Michell B.J House C.M. Stapleton D. Dyck J. Gamble J. Ullrich C. Witters L.A. Kemp B.E. J. Biol. Chem. 1997; 272: 24475-24479Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) were not to of the phosphorylated not have been such that phosphorylation at these sites could of of on AMPK by AMPKK, and AMP AMPK has to of the native holoenzyme purified from rat liver (22Neumann, D., Woods, A., Carling, D., Wallimann, T., and Schlattner, U. (2003) Protein Expression Purif., in pressGoogle Scholar) and is to the of The AMPK of bacterially expressed phosphorylation site mutants were both autophosphorylation and phosphorylation by that the wild-type AMPK complex was inactive the phosphorylated by AMPKK or to mimic phosphorylation at Thr172 by of a or of Thr258 and Ser485 to inactive phosphorylated by mutation of Thr172 to or not activation, as was not phosphorylation by The mutant was active wild-type AMPK that been activated with AMPKK, that the partially The mutant and the mutant were not active the mutant and were not activated upon phosphorylation by these that phosphorylation is for activation and that phosphorylation at Ser485 or Thr258 not lead to a significant activation. The AMPK of the and mutants were in the of AMP The of the complex was This is to the previously reported for the mutant expressed in cells (19Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (493) Google Scholar). of the phosphorylation site and did not change the for not Transfection of in CCL13 of AMPK is known to via both an and an hyperosmotic Ser485 and Thr258 for activation by either of these AMPK was in cells transfected with AMPK mutants with an alanine at these sites. of cells were and in such a as to the cells to either anoxic (slow lysis) or The of AMPK activation of the non-phosphorylatable S485A or T258A mutant by either anoxia or was not different from that of the were with either or to were The of phosphorylation of Thr172 the of AMPK activation, of AMPK was activated via AMP (slow lysis) or in Ser485 was phosphorylated AMPK was inactive in the Moreover, the of phosphorylation of Ser485 was in anoxia and to stress that Ser485 is constitutively phosphorylated and has no effect on AMPK activation. AMPK from anoxic rat was with either or a of and in a of not The were with or the there was no significant change in the with that site is to by the protein was with that bacterially expressed wild-type and were with AMPKK and with these proteins that were with AMPKK at the concentrations used these that AMPK is constitutively phosphorylated at Ser485 and that is to in AMPKK multiple sites within the α-subunit, we to the effect of AMP on the phosphorylation of sites. This was possible by the of for and that phosphorylation of Thr172 was stimulated by AMP of a whereas phosphorylation of Ser485 was by The we did not an effect of AMP on the phosphorylation by AMPKK as by could have been to the that a of the phosphorylation is Expression of AMPK in to of heterotrimeric of subunit (22Neumann, D., Woods, A., Carling, D., Wallimann, T., and Schlattner, U. (2003) Protein Expression Purif., in pressGoogle Scholar). the recombinant is inactive and exists in an is an to study Here, we the phosphorylation of bacterially expressed recombinant wild-type, catalytically inactive, and constitutively active forms of AMPK by an upstream AMPKK preparation. This to two new phosphorylation sites in the α-subunit and In addition to Thr172, the new AMPKK phosphorylation sites in the α-subunits were identified as Thr258 and Ser485 both in and in of the Thr258, and a not found Thr172 This that Thr258 and could be phosphorylated by the AMPKK that be from the that Thr172 for activation. The of AMPKK could also for the AMP we observed upon phosphorylation of a Thr172 AMPKK to but an AMP could be in of two to phosphorylation of of AMP to AMPK could a substrate for Thr172 or AMP could allosterically activate AMPKK as previously reported (18Hawley S.A. Selbert M.A. Goldstein E.G. Edelman A.M. Carling D. Hardie D.G. J. Biol. Chem. 1995; 270: 27186-27191Abstract Full Text Full Text PDF PubMed Scopus (365) Google of phosphorylation sites in the AMPK and in a new of the phosphorylation sites to acidic residues to mimic phosphorylation in the bacterially expressed proteins indicated that Thr172 phosphorylation was for AMPK activation, but that Thr258 and Ser485 phosphorylation was not for in phosphorylation at the sites no on the AMP of the on the that a at these sites with AMPKK, the of the wild-type complex was that of of the constitutively active This could be for mutation of Thr172 to phosphorylation at site. AMPK activation could on phosphorylation at sites other or on between AMPK and of Thr258 and Ser485 to a non-phosphorylatable alanine and in CCL13 cells did not AMPK activation by and activation of the T258A to be which be with an role of Thr258 The role of phosphorylation is at Ser485 phosphorylation to be in bacterially expressed and to by protein could play a role or be involved in that the new phosphorylation sites not involved in activation or AMP of AMPK, to in the complex AMPK e.g. via subcellular localization or substrate recognition. L. Hue for of the and for Hawley and Hardie for
Woods et al. (Fri,) studied this question.