- Email: [email protected]
Please Keep Me 2uned to PKM2
Molecular Cell
Previews Please Keep Me 2uned to PKM2 Steven L. McKnight1,* 1Department of Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9152, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2014.02.022
In this issue of Molecular Cell, Keller et al. (2014) found that binding of the metabolite SAICAR to PKM2 induces the protein kinase activity of an enzyme normally designed to terminate the glycolytic pathway. I gave a talk at the Cold Spring Harbor Symposium on Quantitative Biology several years ago showing that mouse embryonic stem cells rely on threonine as a metabolic fuel. At the end of my talk, I was asked whether mouse ES cells express the M2 isoform of pyruvate kinase (PKM2), even though there was no ostensible relationship between our findings on ES cells and PKM2. Soon after, I realized that in the trendy field of cancer metabolism, PKM2 had ‘‘gone viral.’’ It was claimed to be at the heart of aerobic glycolysis, perhaps alone explaining the vaunted ‘‘Warburg effect’’ (Christofk et al., 2008). This is white-hot stuff—since 2008, over 200 papers have been published referring to PKM2. In order to begin to consider the relationship between PKM2 and cancer, let’s start with some background. Pyruvate kinase is the glycolytic enzyme that converts phosphoenolpyruvate (PEP) into pyruvate, generating ATP and pyruvate. The enzyme comes in four flavors, two encoded by the PKLR gene, and another two encoded by the PKM gene. The PKM1 and PKM2 isoforms differ via the alternative inclusion of one of two mutually exclusive exons (Noguchi et al., 1986). PKM1 is expressed in heart, brain, and muscle—tissues with a high ATP demand. PKM2, by contrast, is expressed in developing tissues and many cancer cell lines. The PKM1 and PKM2 isoforms differ primarily with respect to their differential access to regulatory input. PKM1 forms stable, enzymatically active tetramers. The oligomeric state and enzymatic activity of PKM2 are malleable by virtue of regulatory input from glycolytic metabolites (Anastasiou et al., 2012; Christofk et al., 2008), tyrosine-phosphorylated proteins (Christofk et al., 2008), posttranslational modification (Anastasiou et al., 2012;
Luo et al., 2011; Lv et al., 2011; Yang et al., 2011), and intracellular metabolites unrelated to glycolysis (Keller et al., 2012). When cancer cells are programmed to exclusively express the PKM1 isoform instead of PKM2, the forced switch was reported to reverse the Warburg effect and attenuate the ability of the resultant cells to grow as tumor xenografts in living mice (Christofk et al., 2008). The mystery of why cancer cells or tumors favor expression of the M2 isoform of pyruvate kinase relative to normal tissues now thickens upon analysis of mice tailored to exclusively express the M1 isoform. Quite by surprise, mice missing the exon necessary to create PKM2 exhibit an accelerated rate of tumor formation (Israelsen et al., 2013). If cancer cells strongly prefer expression of PKM2, why are cancer rates not diminished in mice that are unable to produce the M2 isoform? Indeed, it is now reported that immunohistochemical (IHC) staining of PKM2 in human breast cancer tumors is anticorrelative with the most aggressive HER2-positive and triple-negative breast tumor subtypes. Has the PKM2 story now been turned on its head, such that instead of being cancer-abetting, we are now asked to believe that the M2 isoform of pyruvate kinase is protective? Somewhere within this array of confusion comes some cool science that may or may not be relevant to cancer. The story began a year or two ago, when two groups reported the surprising ability of PKM2 to function as a protein kinase (Gao et al., 2012; Yang et al., 2012). Given that ADP inhibits this reaction, it was concluded that the same active site normally used to convert PEP to pyruvate and ATP is also used for PKM2 to phosphorylate protein substrates using PEP instead of ATP as the phosphoryl donor.
Whereas PKM2 purified from mammalian cells has this protein kinase activity, recombinant PKM2 does not. The latter enigma has begun to clarify upon reports showing that the succinylaminoimidazolecarboxamide ribose-50 -phosphate (SAICAR) intermediate in purine biosynthesis binds to PKM2 (Keller et al., 2012) and, as reported in this issue by Lee and colleagues (Keller et al., 2014), stimulates its ability to act as a protein kinase instead of the terminal enzyme in glycolysis. Why are we to believe that SAICAR binding to PKM2 is of biological significance? First and foremost, this discovery was made in an unbiased manner. Recombinant PKM2 was mixed with metabolites extracted from cells grown in either the presence or absence of glucose. The latter extracts yielded a prominent metabolite that copurified with PKM2. Following analytical sleuthing, the PKM2-bound metabolite was identified as SAICAR (Keller et al., 2012). Second, SAICAR only affected the enzymatic activity of PKM2, not PKM1, and did so at physiologically relevant concentrations. Third, mutation of a single glutamine residue within the PKM2-specific exon to lysine yielded a SAICAR-resistant variant that maintained full basal enzymatic activity. Finally, perturbations of the purine biosynthetic pathway leading to either elevation or reduction of intracellular levels of SAICAR correspondingly influenced cellular PKM2 enzyme activity. The most recent studies from the Lee group add significantly to the evolving SAICAR:PKM2 story (Keller et al., 2014). By probing a protein microarray containing thousands of recombinant mammalian proteins with a mixture of PKM2, PEP, and SAICAR, Lee and colleagues discovered that scores of protein kinase enzymes are PKM2 substrates. This list includes many mitogen-activated protein
Molecular Cell 53, March 6, 2014 ª2014 Elsevier Inc. 683
Molecular Cell
Previews Epidermal growth factor MEK
ERK
SAICAR
PKM2
Sustained Proliferation Signal Figure 1. Reciprocal, Feed-Forward Regulation of Erk and PKM2 Enzymes Erk phosphorylation of PKM2 favors the ability of SAICAR to stimulate the protein kinase active of PKM2, instead of its role as the terminal enzyme in glycolysis wherein it converts phosphoenolpyruvate (PEP) into pyruvate and ATP. The Erkphosphorylated isoform of PKM2, in turn, favors PEP-dependent phosphorylation of the activation loop of Erk. These reciprocal activation events yield a feed-forward pathway regulation, helping to allow cells to adapt to their ambient metabolic state.
kinases (MAP kinases), as well as dozens of receptor tyrosine kinases, including EGFR, FGFR, and PDGFR. Singling out the Erk1 protein kinase as a substrate for phosphorylation by PKM2, Keller et al. confirmed that the reaction is PEP dependent. Moreover, incubation of PKM2, PEP, and SAICAR with Erk1 produced from bacteria led to phosphorylation of the activation loop threonine (residue 202 in the human Erk1 enzyme). Thus, not only can one conclude that PKM2 is able to phosphorylate Erk1, but that the resultant modification of Erk1 can be anticipated to activate the latter enzyme. Recall that Cantley, Lu, and colleagues discovered Erk1/2-dependent phosphor-
ylation of PKM2 (Yang et al., 2012). In this case, Erk1/2-mediated modification of PKM2 was shown to move the enzyme from cytoplasm to nucleus, where it was reported to work collaboratively with b-catenin to induce c-Myc expression. Here the plot thickens. If PKM2 can phosphorylate Erk1/2 in a manner that activates its protein kinase activity, and if Erk1/2 can phosphorylate PKM2 causing reciprocal activation, are we seeing evidence of a feed-forward collaborative network among the two enzymes (Figure 1)? Lee and colleagues, as reported in this issue, come to this very conclusion. Erk1/2-mediated phosphorylation of PKM2 causes the enzyme to be sensitized to the stimulatory effects of SAICAR, thereby causing PKM2 to have enhanced activity as a protein kinase (relative to its activity as a glycolytic enzyme). The SAICAR-activated form of PKM2, in turn, can phosphorylate many canonical protein kinase enzymes— including the Erk1/2 kinases that themselves phosphorylate PKM2 leading to its activation. The beauty of this web of crossfertilizing reactions is in its demonstration of the means by which cells sense their metabolic state. The key discovery enabling this new conceptualization came from the biochemical purification and identification of SAICAR as a direct activator of PKM2 (Keller et al., 2012). When cells are deprived of glucose, SAICAR levels rise and are hypothesized to trigger adaptation fanning out from PKM2 to a multitude of protein kinase enzymes well understood to be of regulatory significance (Keller et al., 2014). Liaisons such as the partnership between SAICAR and PKM2 are hard to come by—they require tough biochemistry and analytical chemistry. One can’t simply
684 Molecular Cell 53, March 6, 2014 ª2014 Elsevier Inc.
do a yeast two-hybrid screen and clone out the SAICAR metabolite! In closing, one wonders whether SAICAR:PKM2like interactions are incredibly rare, or whether there might be much more extensive shoulder rubbing between our metabalome and proteome? ACKNOWLEDGMENTS I thank Kosaku Uyeda, Benjamin Tu, and Deepak Nijhawan for valuable editorial comments. REFERENCES Anastasiou, D., Yu, Y., Israelsen, W.J., Jiang, J.K., Boxer, M.B., Hong, B.S., Tempel, W., Dimov, S., Shen, M., Jha, A., et al. (2012). Nat. Chem. Biol. 8, 839–847. Christofk, H.R., Vander Heiden, M.G., Harris, M.H., Ramanathan, A., Gerszten, R.E., Wei, R., Fleming, M.D., Schreiber, S.L., and Cantley, L.C. (2008). Nature 452, 230–233. Gao, X., Wang, H., Yang, J.J., Liu, X., and Liu, Z.R. (2012). Mol. Cell 45, 598–609. Israelsen, W.J., Dayton, T.L., Davidson, S.M., Fiske, B.P., Hosios, A.M., Bellinger, G., Li, J., Yu, Y., Sasaki, M., Horner, J.W., et al. (2013). Cell 155, 397–409. Keller, K.E., Tan, I.S., and Lee, Y.S. (2012). Science 338, 1069–1072. Keller, K.E., Doctor, Z.M., Dwyer, Z.W., and Lee, Y.-S. (2014). Mol. Cell 53, this issue, 700–709. Luo, W., Hu, H., Chang, R., Zhong, J., Knabel, M., O’Meally, R., Cole, R.N., Pandey, A., and Semenza, G.L. (2011). Cell 145, 732–744. Lv, L., Li, D., Zhao, D., Lin, R., Chu, Y., Zhang, H., Zha, Z., Liu, Y., Li, Z., Xu, Y., et al. (2011). Mol. Cell 42, 719–730. Noguchi, T., Inoue, H., and Tanaka, T. (1986). J. Biol. Chem. 261, 13807–13812. Yang, W., Zheng, Y., Xia, Y., Ji, H., Chen, X., Guo, F., Lyssiotis, C.A., Aldape, K., Cantley, L.C., and Lu, Z. (2011). Nat. Cell Biol. 14, 1295–1304. Yang, W., Xia, Y., Hawke, D., Li, X., Liang, J., Xing, D., Aldape, K., Hunter, T., Alfred Yung, W.K., and Lu, Z. (2012). Cell 150, 685–696.
Previews Please Keep Me 2uned to PKM2 Steven L. McKnight1,* 1Department of Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9152, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2014.02.022
In this issue of Molecular Cell, Keller et al. (2014) found that binding of the metabolite SAICAR to PKM2 induces the protein kinase activity of an enzyme normally designed to terminate the glycolytic pathway. I gave a talk at the Cold Spring Harbor Symposium on Quantitative Biology several years ago showing that mouse embryonic stem cells rely on threonine as a metabolic fuel. At the end of my talk, I was asked whether mouse ES cells express the M2 isoform of pyruvate kinase (PKM2), even though there was no ostensible relationship between our findings on ES cells and PKM2. Soon after, I realized that in the trendy field of cancer metabolism, PKM2 had ‘‘gone viral.’’ It was claimed to be at the heart of aerobic glycolysis, perhaps alone explaining the vaunted ‘‘Warburg effect’’ (Christofk et al., 2008). This is white-hot stuff—since 2008, over 200 papers have been published referring to PKM2. In order to begin to consider the relationship between PKM2 and cancer, let’s start with some background. Pyruvate kinase is the glycolytic enzyme that converts phosphoenolpyruvate (PEP) into pyruvate, generating ATP and pyruvate. The enzyme comes in four flavors, two encoded by the PKLR gene, and another two encoded by the PKM gene. The PKM1 and PKM2 isoforms differ via the alternative inclusion of one of two mutually exclusive exons (Noguchi et al., 1986). PKM1 is expressed in heart, brain, and muscle—tissues with a high ATP demand. PKM2, by contrast, is expressed in developing tissues and many cancer cell lines. The PKM1 and PKM2 isoforms differ primarily with respect to their differential access to regulatory input. PKM1 forms stable, enzymatically active tetramers. The oligomeric state and enzymatic activity of PKM2 are malleable by virtue of regulatory input from glycolytic metabolites (Anastasiou et al., 2012; Christofk et al., 2008), tyrosine-phosphorylated proteins (Christofk et al., 2008), posttranslational modification (Anastasiou et al., 2012;
Luo et al., 2011; Lv et al., 2011; Yang et al., 2011), and intracellular metabolites unrelated to glycolysis (Keller et al., 2012). When cancer cells are programmed to exclusively express the PKM1 isoform instead of PKM2, the forced switch was reported to reverse the Warburg effect and attenuate the ability of the resultant cells to grow as tumor xenografts in living mice (Christofk et al., 2008). The mystery of why cancer cells or tumors favor expression of the M2 isoform of pyruvate kinase relative to normal tissues now thickens upon analysis of mice tailored to exclusively express the M1 isoform. Quite by surprise, mice missing the exon necessary to create PKM2 exhibit an accelerated rate of tumor formation (Israelsen et al., 2013). If cancer cells strongly prefer expression of PKM2, why are cancer rates not diminished in mice that are unable to produce the M2 isoform? Indeed, it is now reported that immunohistochemical (IHC) staining of PKM2 in human breast cancer tumors is anticorrelative with the most aggressive HER2-positive and triple-negative breast tumor subtypes. Has the PKM2 story now been turned on its head, such that instead of being cancer-abetting, we are now asked to believe that the M2 isoform of pyruvate kinase is protective? Somewhere within this array of confusion comes some cool science that may or may not be relevant to cancer. The story began a year or two ago, when two groups reported the surprising ability of PKM2 to function as a protein kinase (Gao et al., 2012; Yang et al., 2012). Given that ADP inhibits this reaction, it was concluded that the same active site normally used to convert PEP to pyruvate and ATP is also used for PKM2 to phosphorylate protein substrates using PEP instead of ATP as the phosphoryl donor.
Whereas PKM2 purified from mammalian cells has this protein kinase activity, recombinant PKM2 does not. The latter enigma has begun to clarify upon reports showing that the succinylaminoimidazolecarboxamide ribose-50 -phosphate (SAICAR) intermediate in purine biosynthesis binds to PKM2 (Keller et al., 2012) and, as reported in this issue by Lee and colleagues (Keller et al., 2014), stimulates its ability to act as a protein kinase instead of the terminal enzyme in glycolysis. Why are we to believe that SAICAR binding to PKM2 is of biological significance? First and foremost, this discovery was made in an unbiased manner. Recombinant PKM2 was mixed with metabolites extracted from cells grown in either the presence or absence of glucose. The latter extracts yielded a prominent metabolite that copurified with PKM2. Following analytical sleuthing, the PKM2-bound metabolite was identified as SAICAR (Keller et al., 2012). Second, SAICAR only affected the enzymatic activity of PKM2, not PKM1, and did so at physiologically relevant concentrations. Third, mutation of a single glutamine residue within the PKM2-specific exon to lysine yielded a SAICAR-resistant variant that maintained full basal enzymatic activity. Finally, perturbations of the purine biosynthetic pathway leading to either elevation or reduction of intracellular levels of SAICAR correspondingly influenced cellular PKM2 enzyme activity. The most recent studies from the Lee group add significantly to the evolving SAICAR:PKM2 story (Keller et al., 2014). By probing a protein microarray containing thousands of recombinant mammalian proteins with a mixture of PKM2, PEP, and SAICAR, Lee and colleagues discovered that scores of protein kinase enzymes are PKM2 substrates. This list includes many mitogen-activated protein
Molecular Cell 53, March 6, 2014 ª2014 Elsevier Inc. 683
Molecular Cell
Previews Epidermal growth factor MEK
ERK
SAICAR
PKM2
Sustained Proliferation Signal Figure 1. Reciprocal, Feed-Forward Regulation of Erk and PKM2 Enzymes Erk phosphorylation of PKM2 favors the ability of SAICAR to stimulate the protein kinase active of PKM2, instead of its role as the terminal enzyme in glycolysis wherein it converts phosphoenolpyruvate (PEP) into pyruvate and ATP. The Erkphosphorylated isoform of PKM2, in turn, favors PEP-dependent phosphorylation of the activation loop of Erk. These reciprocal activation events yield a feed-forward pathway regulation, helping to allow cells to adapt to their ambient metabolic state.
kinases (MAP kinases), as well as dozens of receptor tyrosine kinases, including EGFR, FGFR, and PDGFR. Singling out the Erk1 protein kinase as a substrate for phosphorylation by PKM2, Keller et al. confirmed that the reaction is PEP dependent. Moreover, incubation of PKM2, PEP, and SAICAR with Erk1 produced from bacteria led to phosphorylation of the activation loop threonine (residue 202 in the human Erk1 enzyme). Thus, not only can one conclude that PKM2 is able to phosphorylate Erk1, but that the resultant modification of Erk1 can be anticipated to activate the latter enzyme. Recall that Cantley, Lu, and colleagues discovered Erk1/2-dependent phosphor-
ylation of PKM2 (Yang et al., 2012). In this case, Erk1/2-mediated modification of PKM2 was shown to move the enzyme from cytoplasm to nucleus, where it was reported to work collaboratively with b-catenin to induce c-Myc expression. Here the plot thickens. If PKM2 can phosphorylate Erk1/2 in a manner that activates its protein kinase activity, and if Erk1/2 can phosphorylate PKM2 causing reciprocal activation, are we seeing evidence of a feed-forward collaborative network among the two enzymes (Figure 1)? Lee and colleagues, as reported in this issue, come to this very conclusion. Erk1/2-mediated phosphorylation of PKM2 causes the enzyme to be sensitized to the stimulatory effects of SAICAR, thereby causing PKM2 to have enhanced activity as a protein kinase (relative to its activity as a glycolytic enzyme). The SAICAR-activated form of PKM2, in turn, can phosphorylate many canonical protein kinase enzymes— including the Erk1/2 kinases that themselves phosphorylate PKM2 leading to its activation. The beauty of this web of crossfertilizing reactions is in its demonstration of the means by which cells sense their metabolic state. The key discovery enabling this new conceptualization came from the biochemical purification and identification of SAICAR as a direct activator of PKM2 (Keller et al., 2012). When cells are deprived of glucose, SAICAR levels rise and are hypothesized to trigger adaptation fanning out from PKM2 to a multitude of protein kinase enzymes well understood to be of regulatory significance (Keller et al., 2014). Liaisons such as the partnership between SAICAR and PKM2 are hard to come by—they require tough biochemistry and analytical chemistry. One can’t simply
684 Molecular Cell 53, March 6, 2014 ª2014 Elsevier Inc.
do a yeast two-hybrid screen and clone out the SAICAR metabolite! In closing, one wonders whether SAICAR:PKM2like interactions are incredibly rare, or whether there might be much more extensive shoulder rubbing between our metabalome and proteome? ACKNOWLEDGMENTS I thank Kosaku Uyeda, Benjamin Tu, and Deepak Nijhawan for valuable editorial comments. REFERENCES Anastasiou, D., Yu, Y., Israelsen, W.J., Jiang, J.K., Boxer, M.B., Hong, B.S., Tempel, W., Dimov, S., Shen, M., Jha, A., et al. (2012). Nat. Chem. Biol. 8, 839–847. Christofk, H.R., Vander Heiden, M.G., Harris, M.H., Ramanathan, A., Gerszten, R.E., Wei, R., Fleming, M.D., Schreiber, S.L., and Cantley, L.C. (2008). Nature 452, 230–233. Gao, X., Wang, H., Yang, J.J., Liu, X., and Liu, Z.R. (2012). Mol. Cell 45, 598–609. Israelsen, W.J., Dayton, T.L., Davidson, S.M., Fiske, B.P., Hosios, A.M., Bellinger, G., Li, J., Yu, Y., Sasaki, M., Horner, J.W., et al. (2013). Cell 155, 397–409. Keller, K.E., Tan, I.S., and Lee, Y.S. (2012). Science 338, 1069–1072. Keller, K.E., Doctor, Z.M., Dwyer, Z.W., and Lee, Y.-S. (2014). Mol. Cell 53, this issue, 700–709. Luo, W., Hu, H., Chang, R., Zhong, J., Knabel, M., O’Meally, R., Cole, R.N., Pandey, A., and Semenza, G.L. (2011). Cell 145, 732–744. Lv, L., Li, D., Zhao, D., Lin, R., Chu, Y., Zhang, H., Zha, Z., Liu, Y., Li, Z., Xu, Y., et al. (2011). Mol. Cell 42, 719–730. Noguchi, T., Inoue, H., and Tanaka, T. (1986). J. Biol. Chem. 261, 13807–13812. Yang, W., Zheng, Y., Xia, Y., Ji, H., Chen, X., Guo, F., Lyssiotis, C.A., Aldape, K., Cantley, L.C., and Lu, Z. (2011). Nat. Cell Biol. 14, 1295–1304. Yang, W., Xia, Y., Hawke, D., Li, X., Liang, J., Xing, D., Aldape, K., Hunter, T., Alfred Yung, W.K., and Lu, Z. (2012). Cell 150, 685–696.