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Activating AMP-Activated Protein Kinase without AMP
Molecular Cell, Vol. 19, 289–296, August 5, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.07.012
Previews
Activating AMP-Activated Protein Kinase without AMP Though once believed to be regulated exclusively by the cellular energy state, AMPK has now been shown to be activated by a calcium-dependent signaling pathway.
The cellular mechanisms for dealing with nutritional stress are remarkably conserved, with the same signaling network used in organisms from yeast to mammals. In one sense, this is not terribly surprising since the most pressing response to such potential injury is always the same: suppress those biochemical reactions that consume energy while at the same time triggering those metabolic pathways most effective at generating ATP. But an interesting problem to contemplate is how this fundamental process has been adapted to the complexities of a multicellular organism in which recognition of the nutritional state is frequently provided by more subtle cues such as hormones or impulses generated within the central nervous system. Moreover, although the cellular responses to nutrient deprivation are conserved phylogenetically, organismal reactions in metazoans are more multifaceted in that one organ often sacrifices for the good of the whole. The major phylogenetically conserved pathway for signaling nutritional stress is now well established as centering on the protein kinase Snf1 in budding yeast, which is orthologous to AMPK-activated protein kinase (AMPK) in higher organisms. As its name suggests, mammalian AMPK is regulated by changes in the ratio of AMP to ATP, a sensitive indicator of the energy state of the cell. Now, three papers report the intriguing observation that an alternative pathway mediated by a Ca2+-dependent protein kinase is also capable of regulating AMPK (Hawley et al., 2005; Hurley et al., 2005; Woods et al., 2005). AMP binds directly to AMPK, stimulating it allosterically as well as making it a better substrate for the upstream activating enzyme AMPK kinase (AMPKK) (Kahn et al., 2005) (Figure 1). The molecular identity of AMPKK had long been a mystery until recent experiments demonstrated that three enzymes that phosphorylate Snf1 (Pak1p, Tos3p, and Elm1p) share homology with several kinases in the vertebrate genome (Hong et al., 2003; Sutherland et al., 2003). Most closely related is Ca2+/calmodulin-dependent kinase kinase (CaMKK), initially excluded as the physiological activating enzyme based on lack of copurification of CaMKK with AMPKK activity in liver and the prevalent idea that stimulation of AMPK is Ca2+ independent. Instead, another homologous kinase, LKB1, was demonstrated to be AMPKK. This discovery has broad implications, as LKB1 is a well-studied tumor suppressor mutated in Peutz-Jeghers syndrome, a hereditary disease charac-
terized by hamartomatous intestinal polyps (Spicer and Ashworth, 2004). The importance of AMPK to the control of growth and survival is incontestable, though its precise role in carcinogenesis remains uncertain, because LKB1 is capable of regulating a number of other targets including those involved in establishing epithelial polarity (Lizcano et al., 2004). Interestingly, this kinase cascade is distinguished by an unusual mode of regulation whereby LKB1, the upstream kinase, remains constitutively active while modification of the downstream substrate kinase dictates its phosphorylation. Though direct regulation of LKB1 has not been excluded definitively, it looked until recently as if the only way of activating AMPK was by modulating AMP levels. Nonetheless, a number of observations seemed incompatible with this hypothesis. For example, some conditions that activate AMPK do not produce a measurable increase in intracellular AMP, and several cell lines deficient in LKB1 display residual AMPK activity and phosphorylation at the critical activating amino acid residue. The physiological argument was just as compelling. As the AMPK system evolved to regulate metabolism in metazoans, it learned to respond not only to cell-autonomous signs of nutrient deprivation, but also to circulating or neuronal factors that provide information regarding the nutritional state of the organism. The adipocyte hormones adiponectin and leptin promote catabolism in muscle and liver, exerting their effects by activation of AMPK; there also appears to be a signal conveyed by the peripheral nervous system that has much the same consequence in muscle (Kahn et al., 2005). One problem is how these agonists can maintain long-term stimulation of AMPK if glucose and lipid oxidation were to rapidly replete energy stores and if AMP were the only signal capable of regulating AMPK. Both leptin and adiponectin are associated with chronic metabolic control, so a transient increase in AMPK activity alone would not be effective. The intracellular energy potential represents an ideal measure of nutritional stress in a unicellular organism, but in metazoans the cellular status does not always reflect the state of the organism. Moreover, since unlike mammals, yeast does not appear to use AMP as the sensor of glucose deprivation, there is likely another primitive signal that may have evolved into a vertebrate pathway that works without AMP. Consistent with these ideas, studies have now been published describing another “AMPKK” that functions independently of AMP but phosphorylates the same critical activating residue in AMPK (Hawley et al., 2005; Hurley et al., 2005; Woods et al., 2005) (Figure 1). For all three groups of investigators, the homology of yeast Pak1p to CaMKK and, in particular, the ability of the latter kinase to rescue much of the abnormal phenotype in budding yeast deficient in all three Snf1 kinases motivated the scientists to revisit the idea that CaMKK is a physiological activator of AMPK (Hong et al., 2005). The essential observation is consistent among these papers: in cells lacking LKB1, the activation of AMPK by elevations in intracellular calcium is dependent on the
Molecular Cell 290
leptin using AMP as a signaling intermediate in muscle but Ca2+ to modulate AMPK in neurons. The issue of which signaling pathways are responsible for long-term stimulation of AMPK in muscle and liver is more mysterious. LKB1 is required for activation of AMPK in muscle in response to contraction, but we still do not know if this is true for hormones and neurotransmitters (Sakamoto et al., 2005). As noted above, CaMKK levels are low in muscle and liver, at least raising the possibility that other activators of AMPK may yet be discovered. Identification of such enzymes would be of potential clinical importance, as pharmacological activation of AMPK produces profound insulin-like effects and one of the major drugs currently used in the treatment of diabetes mellitus appears to work through activation of AMPK. Though the importance of AMPK to the control of growth and metabolism is just beginning to be understood, the identification of a novel activating mechanism represents a significant step forward.
Figure 1. Activation of AMPK by Two Distinct Upstream Pathways AMPK is made up of three distinct subunits. As shown in the righthand part of the figure, the canonical activation of AMPK is initiated by the binding of AMP to the γ subunit. This results in an increase in AMPK activity but also alters the conformation of the kinase to make it a better substrate for LKB1. LKB1 activates AMPK further by phosphorylating it on the α subunit. The left-hand part of the figure described the recently described Ca2+-dependent pathway. Ca2+/calmodulin binds to CaMKK, altering its conformation such that the catalytic domain is exposed. This permits CaMKK to phosphorylate and activate AMPK. The sites of interaction of either LKB1 or CaMKK with AMPK are not known, so the figure has been drawn only for illustrative purposes in this regard.
presence and activity of CaMKK. Most importantly, Hawley et al. convincingly demonstrate the physiological relevance of this signaling pathway by showing that depolarization-induced activation of AMPK in brain slices is dependent on CaMKK (Hawley et al., 2005). CaMKK is expressed at highest abundance in the central nervous system, though much lower levels are detected in a variety of other tissues. The suggestion that CaMKK’s greatest importance in AMPK activation resides in neurons may well solve a paradox raised recently. Barbara Kahn and her colleagues have shown that leptin stimulates AMPK activity in muscle whereas it inhibits the enzyme in the hypothalamus (Kahn et al., 2005). It is likely that this difference is accounted for by
Morris J. Birnbaum Howard Hughes Medical Institute and The Department of Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104
Selected Reading Hawley, S.A., Pan, D.A., Mustard, K.J., Ross, L., Bain, J., Edelman, A.M., Frenguelli, B.G., and Hardie, D.G. (2005). Cell Met. 2, 9–19. Hong, S.P., Leiper, F.C., Woods, A., Carling, D., and Carlson, M. (2003). Proc. Natl. Acad. Sci. USA 100, 8839–8843. Hong, S.P., Momcilovic, M., and Carlson, M. (2005). J. Biol. Chem. 280, 21804–21809. Hurley, R.L., Anderson, K.A., Franzone, J.M., Kemp, B.E., Means, A.R., and Witters, L.A. (2005). J. Biol. Chem. in press. Published online June 24, 2005. 10.1074/jbc.M503824200. Kahn, B.B., Alquier, T., Carling, D., and Hardie, D.G. (2005). Cell Met. 1, 15–25. Lizcano, J.M., Goransson, O., Toth, R., Deak, M., Morrice, N.A., Boudeau, J., Hawley, S.A., Udd, L., Makela, T.P., Hardie, D.G., and Alessi, D.R. (2004). EMBO J. 23, 833–843. Sakamoto, K., McCarthy, A., Smith, D., Green, K.A., Grahame Hardie, D., Ashworth, A., and Alessi, D.R. (2005). EMBO J. 24, 1810– 1820. Spicer, J., and Ashworth, A. (2004). Curr. Biol. 14, R383–R385. Sutherland, C.M., Hawley, S.A., McCartney, R.R., Leech, A., Stark, M.J., Schmidt, M.C., and Hardie, D.G. (2003). Curr. Biol. 13, 1299– 1305. Woods, A., Dickerson, K., Heath, R., Hong, S.-P., Momcilovic, M., Johnstone, S.R., Carlson, M., and Carling, D. (2005). Cell Met. 2, 21–33.
Previews
Activating AMP-Activated Protein Kinase without AMP Though once believed to be regulated exclusively by the cellular energy state, AMPK has now been shown to be activated by a calcium-dependent signaling pathway.
The cellular mechanisms for dealing with nutritional stress are remarkably conserved, with the same signaling network used in organisms from yeast to mammals. In one sense, this is not terribly surprising since the most pressing response to such potential injury is always the same: suppress those biochemical reactions that consume energy while at the same time triggering those metabolic pathways most effective at generating ATP. But an interesting problem to contemplate is how this fundamental process has been adapted to the complexities of a multicellular organism in which recognition of the nutritional state is frequently provided by more subtle cues such as hormones or impulses generated within the central nervous system. Moreover, although the cellular responses to nutrient deprivation are conserved phylogenetically, organismal reactions in metazoans are more multifaceted in that one organ often sacrifices for the good of the whole. The major phylogenetically conserved pathway for signaling nutritional stress is now well established as centering on the protein kinase Snf1 in budding yeast, which is orthologous to AMPK-activated protein kinase (AMPK) in higher organisms. As its name suggests, mammalian AMPK is regulated by changes in the ratio of AMP to ATP, a sensitive indicator of the energy state of the cell. Now, three papers report the intriguing observation that an alternative pathway mediated by a Ca2+-dependent protein kinase is also capable of regulating AMPK (Hawley et al., 2005; Hurley et al., 2005; Woods et al., 2005). AMP binds directly to AMPK, stimulating it allosterically as well as making it a better substrate for the upstream activating enzyme AMPK kinase (AMPKK) (Kahn et al., 2005) (Figure 1). The molecular identity of AMPKK had long been a mystery until recent experiments demonstrated that three enzymes that phosphorylate Snf1 (Pak1p, Tos3p, and Elm1p) share homology with several kinases in the vertebrate genome (Hong et al., 2003; Sutherland et al., 2003). Most closely related is Ca2+/calmodulin-dependent kinase kinase (CaMKK), initially excluded as the physiological activating enzyme based on lack of copurification of CaMKK with AMPKK activity in liver and the prevalent idea that stimulation of AMPK is Ca2+ independent. Instead, another homologous kinase, LKB1, was demonstrated to be AMPKK. This discovery has broad implications, as LKB1 is a well-studied tumor suppressor mutated in Peutz-Jeghers syndrome, a hereditary disease charac-
terized by hamartomatous intestinal polyps (Spicer and Ashworth, 2004). The importance of AMPK to the control of growth and survival is incontestable, though its precise role in carcinogenesis remains uncertain, because LKB1 is capable of regulating a number of other targets including those involved in establishing epithelial polarity (Lizcano et al., 2004). Interestingly, this kinase cascade is distinguished by an unusual mode of regulation whereby LKB1, the upstream kinase, remains constitutively active while modification of the downstream substrate kinase dictates its phosphorylation. Though direct regulation of LKB1 has not been excluded definitively, it looked until recently as if the only way of activating AMPK was by modulating AMP levels. Nonetheless, a number of observations seemed incompatible with this hypothesis. For example, some conditions that activate AMPK do not produce a measurable increase in intracellular AMP, and several cell lines deficient in LKB1 display residual AMPK activity and phosphorylation at the critical activating amino acid residue. The physiological argument was just as compelling. As the AMPK system evolved to regulate metabolism in metazoans, it learned to respond not only to cell-autonomous signs of nutrient deprivation, but also to circulating or neuronal factors that provide information regarding the nutritional state of the organism. The adipocyte hormones adiponectin and leptin promote catabolism in muscle and liver, exerting their effects by activation of AMPK; there also appears to be a signal conveyed by the peripheral nervous system that has much the same consequence in muscle (Kahn et al., 2005). One problem is how these agonists can maintain long-term stimulation of AMPK if glucose and lipid oxidation were to rapidly replete energy stores and if AMP were the only signal capable of regulating AMPK. Both leptin and adiponectin are associated with chronic metabolic control, so a transient increase in AMPK activity alone would not be effective. The intracellular energy potential represents an ideal measure of nutritional stress in a unicellular organism, but in metazoans the cellular status does not always reflect the state of the organism. Moreover, since unlike mammals, yeast does not appear to use AMP as the sensor of glucose deprivation, there is likely another primitive signal that may have evolved into a vertebrate pathway that works without AMP. Consistent with these ideas, studies have now been published describing another “AMPKK” that functions independently of AMP but phosphorylates the same critical activating residue in AMPK (Hawley et al., 2005; Hurley et al., 2005; Woods et al., 2005) (Figure 1). For all three groups of investigators, the homology of yeast Pak1p to CaMKK and, in particular, the ability of the latter kinase to rescue much of the abnormal phenotype in budding yeast deficient in all three Snf1 kinases motivated the scientists to revisit the idea that CaMKK is a physiological activator of AMPK (Hong et al., 2005). The essential observation is consistent among these papers: in cells lacking LKB1, the activation of AMPK by elevations in intracellular calcium is dependent on the
Molecular Cell 290
leptin using AMP as a signaling intermediate in muscle but Ca2+ to modulate AMPK in neurons. The issue of which signaling pathways are responsible for long-term stimulation of AMPK in muscle and liver is more mysterious. LKB1 is required for activation of AMPK in muscle in response to contraction, but we still do not know if this is true for hormones and neurotransmitters (Sakamoto et al., 2005). As noted above, CaMKK levels are low in muscle and liver, at least raising the possibility that other activators of AMPK may yet be discovered. Identification of such enzymes would be of potential clinical importance, as pharmacological activation of AMPK produces profound insulin-like effects and one of the major drugs currently used in the treatment of diabetes mellitus appears to work through activation of AMPK. Though the importance of AMPK to the control of growth and metabolism is just beginning to be understood, the identification of a novel activating mechanism represents a significant step forward.
Figure 1. Activation of AMPK by Two Distinct Upstream Pathways AMPK is made up of three distinct subunits. As shown in the righthand part of the figure, the canonical activation of AMPK is initiated by the binding of AMP to the γ subunit. This results in an increase in AMPK activity but also alters the conformation of the kinase to make it a better substrate for LKB1. LKB1 activates AMPK further by phosphorylating it on the α subunit. The left-hand part of the figure described the recently described Ca2+-dependent pathway. Ca2+/calmodulin binds to CaMKK, altering its conformation such that the catalytic domain is exposed. This permits CaMKK to phosphorylate and activate AMPK. The sites of interaction of either LKB1 or CaMKK with AMPK are not known, so the figure has been drawn only for illustrative purposes in this regard.
presence and activity of CaMKK. Most importantly, Hawley et al. convincingly demonstrate the physiological relevance of this signaling pathway by showing that depolarization-induced activation of AMPK in brain slices is dependent on CaMKK (Hawley et al., 2005). CaMKK is expressed at highest abundance in the central nervous system, though much lower levels are detected in a variety of other tissues. The suggestion that CaMKK’s greatest importance in AMPK activation resides in neurons may well solve a paradox raised recently. Barbara Kahn and her colleagues have shown that leptin stimulates AMPK activity in muscle whereas it inhibits the enzyme in the hypothalamus (Kahn et al., 2005). It is likely that this difference is accounted for by
Morris J. Birnbaum Howard Hughes Medical Institute and The Department of Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104
Selected Reading Hawley, S.A., Pan, D.A., Mustard, K.J., Ross, L., Bain, J., Edelman, A.M., Frenguelli, B.G., and Hardie, D.G. (2005). Cell Met. 2, 9–19. Hong, S.P., Leiper, F.C., Woods, A., Carling, D., and Carlson, M. (2003). Proc. Natl. Acad. Sci. USA 100, 8839–8843. Hong, S.P., Momcilovic, M., and Carlson, M. (2005). J. Biol. Chem. 280, 21804–21809. Hurley, R.L., Anderson, K.A., Franzone, J.M., Kemp, B.E., Means, A.R., and Witters, L.A. (2005). J. Biol. Chem. in press. Published online June 24, 2005. 10.1074/jbc.M503824200. Kahn, B.B., Alquier, T., Carling, D., and Hardie, D.G. (2005). Cell Met. 1, 15–25. Lizcano, J.M., Goransson, O., Toth, R., Deak, M., Morrice, N.A., Boudeau, J., Hawley, S.A., Udd, L., Makela, T.P., Hardie, D.G., and Alessi, D.R. (2004). EMBO J. 23, 833–843. Sakamoto, K., McCarthy, A., Smith, D., Green, K.A., Grahame Hardie, D., Ashworth, A., and Alessi, D.R. (2005). EMBO J. 24, 1810– 1820. Spicer, J., and Ashworth, A. (2004). Curr. Biol. 14, R383–R385. Sutherland, C.M., Hawley, S.A., McCartney, R.R., Leech, A., Stark, M.J., Schmidt, M.C., and Hardie, D.G. (2003). Curr. Biol. 13, 1299– 1305. Woods, A., Dickerson, K., Heath, R., Hong, S.-P., Momcilovic, M., Johnstone, S.R., Carlson, M., and Carling, D. (2005). Cell Met. 2, 21–33.