The Ca2+/Calmodulin-dependent Protein Kinase Kinases Are AMP-activated Protein Kinase Kinases

The AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism in response to metabolic stress and to other regulatory signals. AMPK activity is absolutely dependent upon phosphorylation of AMPKαThr-172 in its activation loop by one or more AMPK kinases (AMPKKs). The tumor suppressor kinase, LKB1, is a major AMPKK present in a variety of tissues and cells, but several lines of evidence point to the existence of other AMPKKs. We have employed three cell lines deficient in LKB1 to study AMPK regulation and phosphorylation, HeLa, A549, and murine embryo fibroblasts derived from LKB-/- mice. In HeLa and A549 cells, mannitol, 2-deoxyglucose, and ionomycin, but not 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), treatment activates AMPK by αThr-172 phosphorylation. These responses, as well as the downstream effects of AMPK on the phosphorylation of acetyl-CoA carboxylase, arelargelyinhibitedbytheCa2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, STO-609. AMPKK activity in HeLa cell lysates measured in vitro is totally inhibited by STO-609 with an IC50 comparable with that of the known CaMKK isoforms, CaMKKα and CaMKKβ. Furthermore, 2-deoxyglucose- and ionomycin-stimulated AMPK activity, αThr-172 phosphorylation, and acetyl-CoA carboxylase phosphorylation are substantially reduced in HeLa cells transfected with small interfering RNAs specific for CaMKKα and CaMKKβ. Lastly, the activation of AMPK in response to ionomycin and 2-deoxyglucose is not impaired in LKB1-/- murine embryo fibroblasts. These data indicate that the CaMKKs function in intact cells as AMPKKs, predicting wider roles for these kinases in regulating AMPK activity in vivo. The AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism in response to metabolic stress and to other regulatory signals. AMPK activity is absolutely dependent upon phosphorylation of AMPKαThr-172 in its activation loop by one or more AMPK kinases (AMPKKs). The tumor suppressor kinase, LKB1, is a major AMPKK present in a variety of tissues and cells, but several lines of evidence point to the existence of other AMPKKs. We have employed three cell lines deficient in LKB1 to study AMPK regulation and phosphorylation, HeLa, A549, and murine embryo fibroblasts derived from LKB-/- mice. In HeLa and A549 cells, mannitol, 2-deoxyglucose, and ionomycin, but not 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), treatment activates AMPK by αThr-172 phosphorylation. These responses, as well as the downstream effects of AMPK on the phosphorylation of acetyl-CoA carboxylase, arelargelyinhibitedbytheCa2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, STO-609. AMPKK activity in HeLa cell lysates measured in vitro is totally inhibited by STO-609 with an IC50 comparable with that of the known CaMKK isoforms, CaMKKα and CaMKKβ. Furthermore, 2-deoxyglucose- and ionomycin-stimulated AMPK activity, αThr-172 phosphorylation, and acetyl-CoA carboxylase phosphorylation are substantially reduced in HeLa cells transfected with small interfering RNAs specific for CaMKKα and CaMKKβ. Lastly, the activation of AMPK in response to ionomycin and 2-deoxyglucose is not impaired in LKB1-/- murine embryo fibroblasts. These data indicate that the CaMKKs function in intact cells as AMPKKs, predicting wider roles for these kinases in regulating AMPK activity in vivo. The AMP-activated protein kinase (AMPK) 1The abbreviations used are: AMPK, AMP-activated protein kinase; AMPKK, AMPK kinase; MEF, mouse embryo fibroblast; ACC, acetyl-CoA carboxylase; CaM, calmodulin; CaMKK, CaM-dependent protein kinase kinase; siRNA, small interfering RNA; HRP, horseradish peroxidase; ANOVA, analysis of variance; 2-DG, 2-deoxyglucose; MAP, mitogen-activated protein; AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside. regulates many aspects of cellular metabolism, especially in response to metabolic stress (1Hardie D. G. Scott J. W. Pan D. A. Hudson E. R. FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (729) Google Scholar). AMPK is a serine/threonine protein kinase and a member of the Snf1/AMPK protein kinase family (1Hardie D. G. Scott J. W. Pan D. A. Hudson E. R. FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (729) Google Scholar). It is an αβγ heterotrimeric protein, consisting of an α catalytic subunit, a β subunit important both for enzyme activity and for targeting, and a γ regulatory subunit, which binds the allosteric activator, AMP. The activity of AMPK absolutely requires phosphorylation of the α subunit on Thr-172 in its activation loop by one or more upstream kinases (AMPKK) (1Hardie D. G. Scott J. W. Pan D. A. Hudson E. R. FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (729) Google Scholar). The major breakthrough in identifying AMPK upstream kinases came from the study of the regulation of the AMPK ortholog, Snf1, in Saccharomyces cerevisiae, in which Pak1 was shown to act as a Snf1p kinase kinase (2Nath N. McCartney R. R. Schmidt M. C. Mol. Cell. Biol. 2003; 23: 3909-3917Crossref PubMed Scopus (126) Google Scholar). Subsequently, it was shown that three closely related kinases, Pak1p, Tos3p, and Elm1p, needed to be deleted to generate the Snf1- phenotype (3Hong S. P. Leiper F. C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (487) Google Scholar, 4Sutherland C. M. Hawley S. A. McCartney R. R. Leech A. Stark M. J. Schmidt M. C. Hardie D. G. Curr. Biol. 2003; 13: 1299-1305Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Sequence comparison revealed that the human LKB1 tumor suppressor kinase was the most closely related mammalian kinase. LKB1 was subsequently identified by several groups as being an important upstream kinase active on AMPK (5Hawley S. A. Boudeau J. Reid J. L. Mustard K. J. Udd L. Makela T. P. Alessi D. R. Hardie D. G. J. Biol. 2003; 2: 28-37Crossref PubMed Google Scholar, 6Woods A. Johnstone S. R. Dickerson K. Leiper F. C. Fryer L. G. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar, 7Shaw R. J. Kosmatka M. Bardeesy N. Hurley R. L. Witters L. A. DePinho R. A. Cantley L. C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1480) Google Scholar). Several lines of evidence point to the presence of non-LKB1 AMPKKs. Multiple AMPKK activities are separable during chromatography of extracts from rodent heart (8Altarejos J. Y. Taniguchi M. Clanachan A. S. Lopaschuk G. D. J. Biol. Chem. 2005; 280: 183-190Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 9Baron S. J. Li J. Russell III, R. R. Neumann D. Miller E. J. Tuerk R. Wallimann T. Hurley R. L. Witters L. A. Young L. H. Circ. Res. 2005; 96: 337-345Crossref PubMed Scopus (90) Google Scholar). Murine fibroblasts obtained from LKB1-/- embryos by two different groups still demonstrate residual AMPKT172α phosphorylation and AMPK activity, albeit not as responsive to the usual activators of AMPK, such as the nucleoside AICAR (5Hawley S. A. Boudeau J. Reid J. L. Mustard K. J. Udd L. Makela T. P. Alessi D. R. Hardie D. G. J. Biol. 2003; 2: 28-37Crossref PubMed Google Scholar, 7Shaw R. J. Kosmatka M. Bardeesy N. Hurley R. L. Witters L. A. DePinho R. A. Cantley L. C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1480) Google Scholar). Partially purified Ca2+/CaM-dependent protein kinase kinase (CaMKK) from pig brain has been shown to be active in vitro on AMPK, but it was concluded that the kinetics of phosphorylation by CaMKK were weaker than those for a partially purified AMPKK and that CaMKKs were unlikely to function as AMPKKs in intact cells and tissues (10Hawley 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 (371) Google Scholar). Although this view has been widely accepted (28Carling D. Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (970) Google Scholar), Nath et al. (2Nath N. McCartney R. R. Schmidt M. C. Mol. Cell. Biol. 2003; 23: 3909-3917Crossref PubMed Scopus (126) Google Scholar) suggested that CaMKKβ may be an AMPKK based on homology with yeast PAK1. Recently, CaMKKα has been shown directly to function as a Snf1-activating kinase in yeast cells lacking the three Snf-activating kinases, Pak1, Tos3, and Elm1 (29Hong S. -P. Momcilovic M. Carlson M. J. Biol. Chem. 2005; 280: 21804-21809Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The protein products of the CaMKK gene family, CaMKKα and CaMKKβ, show significant homology to LKB1 and to the three aforementioned yeast kinases (3Hong S. P. Leiper F. C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (487) Google Scholar, 4Sutherland C. M. Hawley S. A. McCartney R. R. Leech A. Stark M. J. Schmidt M. C. Hardie D. G. Curr. Biol. 2003; 13: 1299-1305Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 5Hawley S. A. Boudeau J. Reid J. L. Mustard K. J. Udd L. Makela T. P. Alessi D. R. Hardie D. G. J. Biol. 2003; 2: 28-37Crossref PubMed Google Scholar). In the present study, we have investigated the possibility that one or both CaMKKs might serve as AMPKKs to regulate AMPK in cell lines lacking expression of LKB1. Cell Culture and Incubations—Panc-1, AsPC-1, and COS cells were purchased from ATCC. Mouse embryo fibroblasts (MEFs) from LKB1+/+ and LKB1-/- mice and HeLa cells were kindly provided by Reuben Shaw (Harvard University). These cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. A549 cells (ATCC) were grown in Ham's F-12 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. All cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cells were incubated in 6-well plates after various additions (see the figure legends) prior to extraction for immunoblotting and analysis of AMPK activity. Preparation of Cell Extracts—Cell extracts were prepared by three different methods. For analysis of AMPK activity, digitonin lysis followed by ammonium sulfate precipitation was employed, as in Ref. 11Witters L. A. Kemp B. E. J. Biol. Chem. 1992; 267: 2864-2867Abstract Full Text PDF PubMed Google Scholar. For immunoblotting of total cellular protein, cells were lysed either in a Triton X-100-containing buffer, as in Ref. 12Hamilton 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: 892-898Crossref PubMed Scopus (55) Google Scholar, or in an SDS-containing buffer. For the latter, cells were rinsed with phosphate-buffered saline (2×) and lysed directly on the plate with boiling SDS buffer (1% SDS, 100 mm NaCl, 10 mm Tris-HCl, pH 7. 5). These extracts were then sheared with a 25-gauge needle and boiled for 5 min. Protein concentration in all extracts was determined with a BCA assay (Pierce), according to the manufacturer's protocol. Enzyme Activities and Immunoblotting—AMPK activity against the SAMS peptide was determined at a saturating concentration of AMP, as in Ref. 11Witters L. A. Kemp B. E. J. Biol. Chem. 1992; 267: 2864-2867Abstract Full Text PDF PubMed Google Scholar. AMPKK activity in Triton X-100 cell lysates was determined by phosphorylation of a recombinant AMPKα protein, as in Ref. 12Hamilton 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: 892-898Crossref PubMed Scopus (55) Google Scholar. Cell extracts were examined by immunoblotting, as in Ref. 12Hamilton 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: 892-898Crossref PubMed Scopus (55) Google Scholar, employing a panel of different antibodies/reagents. These included anti-AMPK total α (reactive against α and α2), anti-AMPKαT172p, anti-ACCS79p, streptavidin-HRP (13Hamilton S. R. Stapleton D. O'Donnell Jr. , J. B. Kung J. T. Dalal S. R. Kemp B. E. Witters L. A. FEBS Lett. 2001; 500: 163-168Crossref PubMed Scopus (96) Google Scholar), anti-CaMKKα/β (C-terminal; BD Transduction Laboratories catalog number 610544), and anti-LKB1 (a kind gift from Reuben Shaw and Ronald DePhino (Harvard University) ) (7Shaw R. J. Kosmatka M. Bardeesy N. Hurley R. L. Witters L. A. DePinho R. A. Cantley L. C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1480) Google Scholar). RNA Interference—siRNA oligonucleotides against a scrambled, non-targeting sequence as a negative control (D-001206-13-20) and CaMKKβ (human CaMKK2, SiGENOME SMARTpool reagent M-004842-00-0050, accession number NM₀06549) were obtained from Dharmacon Research (Lafeyette, CO). siRNA oligonucleotides designed against CaMKKα (siRNA Gene Silencers, human) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). HeLa cells were plated at a density of 5 × 105 cells/well in a 6-well plate and allowed to adhere overnight. Cells were transfected with a non-targeting control siRNA pool (200 nm), CaMKKβ siRNA pool (100 nm), or CaMKKα siRNA (200 nm). Preliminary studies established that the latter two were employed at their maximally effective concentrations (data not shown). Transfection was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. At 48 h after transfection, cells were either harvested or treated with AMPK activators and then harvested. Preparation of LKB1/STRAD/Mo25 Complex—COS cells were triply transfected with cDNAs expressing glutathione S-transferase-tagged LKB1 and FLAG-tagged STRAD and Mo25 (generous gifts from Reuben Shaw (Harvard University) and Dario Alessi (University of Dundee) ) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Cells were lysed in buffer containing 1% Triton X-100, as described above, 48 h after transfection. Cleared supernatants were aliquoted into microcentrifuge tubes (1 ml/tube) and incubated with 100 μl of 50% glutathione bead slurry (Sigma) for 2 h at 4 °C. Lysates were spun down briefly (20 to The was and were with buffer containing 1% Triton X-100 and then subsequently two with assay buffer were then in assay buffer containing mm glutathione (Sigma) to the enzyme a on are spun down for at to and the containing the purified LKB1/STRAD/Mo25 was and used in the in vitro kinase kinase analysis of data were by a with using the significant by the In an to AMPKKs from LKB1, two human cell lines that are deficient in LKB1 were for study, the HeLa and the A549 LKB1 is not in these two lines in LKB1-/- mouse embryo fibroblasts by immunoblotting We expression of LKB1 in other human lines and AsPC-1, the latter to be S. S. A. 2003; PubMed Google Scholar). 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Goldstein E. G. Means A. R. Edelman A. M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. Selbert M. A. Stapleton D. Goldstein E. G. Means A. R. Kemp B. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Means R. L. Kemp B. E. Goldstein E. G. Selbert M. A. Edelman A. M. Means A. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Trends Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar, J. T. Chem. 2001; 101: PubMed Scopus Google Scholar, Means A. R. Edelman A. M. Selbert M. A. Stapleton D. Goldstein E. G. Kemp B. E. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). CaMKK from pig brain prior to the that were two of the has been shown to and AMPK in this phosphorylation is by the of to the AMPK (10Hawley S. A. Selbert M. A. Goldstein E. G. Edelman A. M. Carling D. Hardie D. G. J. Biol. 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At a concentration of STO-609 AMPK activation and phosphorylation in response to all three in HeLa cells The of of STO-609 was in the ionomycin-stimulated cells STO-609 totally the activation and phosphorylation of AMPK in response to and 2-DG, this might that these cells residual LKB1 by or STO-609 inhibited AMPK activation in response to and in A549 cells activation by of AMPK phosphorylation and phosphorylation of a downstream of AMPK, acetyl-CoA carboxylase were inhibited by STO-609 in HeLa cells at concentrations of mm The IC50 for STO-609 of phosphorylation in HeLa cells was μg/ml (data not shown). In other (data not we have that STO-609 the in vitro activity of purified AMPK against the SAMS the IC50 is than that for the we the possibility that of the STO-609 of phosphorylation in intact cells is to the of AMPK, the concentrations we employed in these intact cells studies was is not an AMPK effects of STO-609 be for by of CaMKKα activity is dependent upon and CaM, CaMKKβ has activity in its it be by a in M. A. Goldstein E. G. Means A. R. Edelman A. M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. Selbert M. A. Stapleton D. Goldstein E. G. Means A. R. Kemp B. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Means R. L. Kemp B. E. Goldstein E. G. Selbert M. A. Edelman A. M. Means A. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). an of and a R. Biochem. J. PubMed Scopus Google Scholar). In HeLa cells, the in AMPK activity and phosphorylation in response to ionomycin might be to a of either of the In of this STO-609 the of ionomycin to these in AMPK AMPKK activity, measured in HeLa cell lysates against a recombinant AMPKα in was inhibited by STO-609 The IC50 for this in vitro STO-609 was μg/ml (data not shown). IC50 is comparable with that for the of recombinant CaMKKα and CaMKKβ phosphorylation of a kinase protein in vitro M. R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The of STO-609 against several other protein kinases kinases and kinase, protein kinase protein kinase, and 10 its and as a CaMKK M. R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). we have that at these STO-609 not the activity of a LKB1/STRAD/Mo25 against the recombinant AMPKα in vitro the of this in the roles of the CaMKKs and LKB1 as AMPKKs in various tissues or cell specific for the the two CaMKK isoforms, CaMKKα and HeLa cells have been to both CaMKKα and CaMKKβ M. R. FEBS Lett. 2003; PubMed Scopus Google Scholar). The CaMKKβ gene several of as a of of the M. R. FEBS Lett. 2003; PubMed Scopus Google Scholar, G. D. J. Y. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), the of the protein products kinase activity, and have not been In HeLa cells, the and are two CaMKK and have been M. R. FEBS Lett. 2003; PubMed Scopus Google Scholar). with a that a sequence to CaMKKα and CaMKKβ, HeLa cell extracts two to a CaMKKα and at the of and M. R. FEBS Lett. 2003; PubMed Scopus Google Scholar). the possibility that the effects of STO-609 to AMPK activity were not by specific of the we employed RNA as an to their activity. with transfected non-targeting siRNA, siRNA substantially and AMPK activity and phosphorylation, siRNA but still effects on these AMPK significant effects of the on phosphorylation are and this is by the residual AMPK activity We have that the phosphorylation of in other is to small in AMPK activity S. M. Witters L. A. Kemp B. E. 2003; PubMed Scopus Google Scholar). The of both CaMKK both and AMPK activity and and phosphorylation and reduced ionomycin-stimulated AMPK activity siRNA the and the on immunoblotting, siRNA the that the in HeLa extracts is a of and CaMKKα and that the is We have been to the of a CaMKKα (Santa Cruz Biotechnology, to more the of the The have effects on either or total total AMPKα is in the presence of both The all of the CaMKK with a of AMPK activity and phosphorylation of CaMKKs AMPK activation by HeLa cells were transfected with against a non-targeting CaMKKα as or CaMKKβ as and incubated for 48 Cells were then incubated in the presence or of ionomycin for 5 and harvested by digitonin were against the SAMS peptide as described to AMPK kinase activity. These 4 lysates at are as of AMPK activity as of into the SAMS peptide of activity, and ionomycin-stimulated activity. determined by the of AMPK activity by both is significant at and that of the of the by ionomycin by siRNA is significant at For the data for the effects of the roles of LKB1 and the CaMKKs in a cell in which all three kinases are LKB1+/+ were with ionomycin and in the presence and of STO-609. and AMPK activity was then with that in LKB1-/- cells We and have that AICAR, and to AMPK in LKB1-/- (5Hawley S. A. Boudeau J. Reid J. L. Mustard K. J. Udd L. Makela T. P. Alessi D. R. Hardie D. G. J. Biol. 2003; 2: 28-37Crossref PubMed Google Scholar, 7Shaw R. J. Kosmatka M. Bardeesy N. Hurley R. L. Witters L. A. DePinho R. A. Cantley L. C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1480) Google Scholar). of AMPK and phosphorylation in response to ionomycin and is not impaired in LKB1-/- as with LKB1+/+ the response to ionomycin to be in the of STO-609 partially the response to ionomycin in both cell its to AMPK activation is in the LKB1-/- These data revealed that AMPK activation is one or both the response may be to by either LKB1 or the In the cells, of CaMKKs might be to that of LKB1. We of the of AMPKKs in either cell The data evidence that both CaMKKs function as AMPKKs in the cell lines for non-LKB1 AMPKKs in other tissues and cell Although and from both CaMKKα and CaMKKβ have a expression in rodent tissues A. M. Selbert M. A. Stapleton D. Goldstein E. G. Means A. R. Kemp B. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Means R. L. Kemp B. E. Goldstein E. G. Selbert M. A. Edelman A. M. Means A. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. M. N. N. Res. Mol. Res. 2003; PubMed Scopus Google Scholar), important roles for in the regulation of AMPK activity in vivo. The data indicate that both cellular and may or roles in the regulation of AMPK activity. the of three mammalian AMPKKs, be to in their to AMPK regulation in other tissues and cell Although LKB1 may be the AMPKK regulating AMPK to and in murine K. and D. R. Scholar), it expression of these AMPKKs, that may be in the regulation of AMPK both and dependent on the of the In data from yeast indicate that the different β of the kinase for Pak1, Tos3, and Elm1 upstream kinases R. R. Schmidt M. C. Curr. 2005; PubMed Scopus Google Scholar). it might be that different AMPK of of different catalytic α and and and might be by the three mammalian AMPKKs, the of regulation of AMPK activity in vivo. We the and the of from Reuben Shaw and Ronald DePinho (Harvard and Dario Alessi (University of with the with