Structural basis for allosteric regulation of pyruvate kinase M2 by phosphorylation and acetylation

Nandi, Suparno; Razzaghi, Mortezaali; Srivastava, Dhiraj; Dey, Mishtu

doi:10.1074/jbc.ra120.015800

Structural basis for allosteric regulation of pyruvate kinase M2 by phosphorylation and acetylation

Journal Article · Mon Nov 30 23:00:00 EST 2020 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.ra120.015800· OSTI ID:1765218

^[1]; ^[1]; ^[1]; ^[1]

Univ. of Iowa, Iowa City, IA (United States)

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme and transcriptional coactivator and is critical for tumor metabolism. In cancer cells, native tetrameric PKM2 is phosphorylated or acetylated, which initiates a switch to a dimeric/monomeric form that translocates into the nucleus, causing oncogene transcription. However, it is not known how these post-translational modifications (PTMs) disrupt the oligomeric state of PKM2. We explored this question via crystallographic and biophysical analyses of PKM2 mutants containing residues that mimic phosphorylation and acetylation. We find that the PTMs elicit major structural reorganization of the fructose 1,6-bisphosphate (FBP), an allosteric activator, binding site, impacting the interaction with FBP and causing a disruption in oligomerization. To gain insight into how these modifications might cause unique outcomes in cancer cells, we examined the impact of increasing the intracellular pH (pHi) from ~7.1 (in normal cells) to ~7.5 (in cancer cells). Biochemical studies of WT PKM2 (wtPKM2) and the two mimetic variants demonstrated that the activity decreases as the pH is increased from 7.0 to 8.0, and wtPKM2 is optimally active and amenable to FBP-mediated allosteric regulation at pHi 7.5. However, the PTM mimetics exist as a mixture of tetramer and dimer, indicating that physiologically dimeric fraction is important and might be necessary for the modified PKM2 to translocate into the nucleus. Thus, our findings provide insight into how PTMs and pH regulate PKM2 and offer a broader understanding of its intricate allosteric regulation mechanism by phosphorylation or acetylation.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC); NSF

Grant/Contract Number:: AC02-06CH11357; AC02-05CH11231

OSTI ID:: 1765218

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 51 Vol. 295; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

References (54)

Cancer‐associated mutations in human pyruvate kinase M2 impair enzyme activity Liu, Vivian M.; Howell, Andrea J.; Hosios, Aaron M. FEBS Letters, Vol. 594, Issue 4 https://doi.org/10.1002/1873-3468.13648	journal	November 2019
Isotype-specific inhibitors of the glycolytic key regulator pyruvate kinase subtype M2 moderately decelerate tumor cell proliferation: Isotype-Specific Inhibitors of M2-PK Spoden, Gilles A.; Mazurek, Sybille; Morandell, Dieter International Journal of Cancer, Vol. 123, Issue 2 https://doi.org/10.1002/ijc.23512	journal	April 2008
MRI of the tumor microenvironment Gillies, Robert J.; Raghunand, Natarajan; Karczmar, Gregory S. Journal of Magnetic Resonance Imaging, Vol. 16, Issue 4 https://doi.org/10.1002/jmri.10181	journal	September 2002
Regulation of Pyruvate Kinase M Gene Expression Yamada, Kazuya; Noguchi, Tamio Biochemical and Biophysical Research Communications, Vol. 256, Issue 2 https://doi.org/10.1006/bbrc.1999.0228	journal	March 1999
Structural insight into mechanisms for dynamic regulation of PKM2 Wang, Ping; Sun, Chang; Zhu, Tingting Protein & Cell, Vol. 6, Issue 4 https://doi.org/10.1007/s13238-015-0132-x	journal	February 2015
Identification of small molecule inhibitors of pyruvate kinase M2 Vander Heiden, Matthew G.; Christofk, Heather R.; Schuman, Eli Biochemical Pharmacology, Vol. 79, Issue 8 https://doi.org/10.1016/j.bcp.2009.12.003	journal	April 2010
PKM2 Phosphorylates Histone H3 and Promotes Gene Transcription and Tumorigenesis Yang, Weiwei; Xia, Yan; Hawke, David Cell, Vol. 150, Issue 4 https://doi.org/10.1016/j.cell.2012.07.018	journal	August 2012
Inference of Macromolecular Assemblies from Crystalline State Krissinel, Evgeny; Henrick, Kim Journal of Molecular Biology, Vol. 372, Issue 3 https://doi.org/10.1016/j.jmb.2007.05.022	journal	September 2007
Acetylation Targets the M2 Isoform of Pyruvate Kinase for Degradation through Chaperone-Mediated Autophagy and Promotes Tumor Growth Lv, Lei; Li, Dong; Zhao, Di Molecular Cell, Vol. 42, Issue 6 https://doi.org/10.1016/j.molcel.2011.04.025	journal	June 2011
Mitogenic and Oncogenic Stimulation of K433 Acetylation Promotes PKM2 Protein Kinase Activity and Nuclear Localization Lv, Lei; Xu, Yan-Ping; Zhao, Di Molecular Cell, Vol. 52, Issue 3 https://doi.org/10.1016/j.molcel.2013.09.004	journal	November 2013
SAICAR Induces Protein Kinase Activity of PKM2 that Is Necessary for Sustained Proliferative Signaling of Cancer Cells Keller, Kirstie E.; Doctor, Zainab M.; Dwyer, Zachary W. Molecular Cell, Vol. 53, Issue 5 https://doi.org/10.1016/j.molcel.2014.02.015	journal	March 2014
Pyruvate kinase: Function, regulation and role in cancer Israelsen, William J.; Vander Heiden, Matthew G. Seminars in Cell & Developmental Biology, Vol. 43 https://doi.org/10.1016/j.semcdb.2015.08.004	journal	July 2015
Pyruvate kinase isoenzyme M2 is a glycolytic sensor differentially regulating cell proliferation, cell size and apoptotic cell death dependent on glucose supply Spoden, Gilles A.; Rostek, Ursula; Lechner, Stefan Experimental Cell Research, Vol. 315, Issue 16 https://doi.org/10.1016/j.yexcr.2009.06.024	journal	October 2009
Succinyl-5-aminoimidazole-4-carboxamide-1-ribose 5′-Phosphate (SAICAR) Activates Pyruvate Kinase Isoform M2 (PKM2) in Its Dimeric Form Yan, Ming; Chakravarthy, Srinivas; Tokuda, Joshua M. Biochemistry, Vol. 55, Issue 33 https://doi.org/10.1021/acs.biochem.6b00658	journal	August 2016
Structural Investigation of a Dimeric Variant of Pyruvate Kinase Muscle Isoform 2 Srivastava, Dhiraj; Razzaghi, Mortezaali; Henzl, Michael T. Biochemistry, Vol. 56, Issue 50 https://doi.org/10.1021/acs.biochem.7b01013	journal	December 2017
Mechanistic and Structural Insights into Cysteine-Mediated Inhibition of Pyruvate Kinase Muscle Isoform 2 Srivastava, Dhiraj; Nandi, Suparno; Dey, Mishtu Biochemistry, Vol. 58, Issue 35 https://doi.org/10.1021/acs.biochem.9b00349	journal	August 2019
Synergistic Allosteric Mechanism of Fructose-1,6-bisphosphate and Serine for Pyruvate Kinase M2 via Dynamics Fluctuation Network Analysis Yang, Jingxu; Liu, Hao; Liu, Xiaorui Journal of Chemical Information and Modeling, Vol. 56, Issue 6 https://doi.org/10.1021/acs.jcim.6b00115	journal	May 2016
Determinants of Allosteric Activation of Yeast Pyruvate Kinase and Identification of Novel Effectors Using Computational Screening ^† Bond, Christopher J.; Jurica, Melissa S.; Mesecar, Andrew Biochemistry, Vol. 39, Issue 50 https://doi.org/10.1021/bi001443i	journal	December 2000
Structural Basis for Tumor Pyruvate Kinase M2 Allosteric Regulation and Catalysis ^† ^, ^‡ Dombrauckas, Jill D.; Santarsiero, Bernard D.; Mesecar, Andrew D. Biochemistry, Vol. 44, Issue 27 https://doi.org/10.1021/bi0474923	journal	July 2005
Role of Lysine 240 in the Mechanism of Yeast Pyruvate Kinase Catalysis ^† Bollenbach, Thomas J.; Mesecar, Andrew D.; Nowak, Thomas Biochemistry, Vol. 38, Issue 28 https://doi.org/10.1021/bi990690n	journal	July 1999
Pyruvate kinase M2 is a phosphotyrosine-binding protein Christofk, Heather R.; Vander Heiden, Matthew G.; Wu, Ning Nature, Vol. 452, Issue 7184 https://doi.org/10.1038/nature06667	journal	March 2008
Nuclear PKM2 regulates β-catenin transactivation upon EGFR activation Yang, Weiwei; Xia, Yan; Ji, Haitao Nature, Vol. 480, Issue 7375 https://doi.org/10.1038/nature10598	journal	December 2011
Serine is a natural ligand and allosteric activator of pyruvate kinase M2 Chaneton, Barbara; Hillmann, Petra; Zheng, Liang Nature, Vol. 491, Issue 7424 https://doi.org/10.1038/nature11540	journal	October 2012
The metabolic function of cyclin D3–CDK6 kinase in cancer cell survival Wang, Haizhen; Nicolay, Brandon N.; Chick, Joel M. Nature, Vol. 546, Issue 7658 https://doi.org/10.1038/nature22797	journal	June 2017
Therapeutic targets in cancer cell metabolism and autophagy Cheong, Heesun; Lu, Chao; Lindsten, Tullia Nature Biotechnology, Vol. 30, Issue 7 https://doi.org/10.1038/nbt.2285	journal	July 2012
ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect Yang, Weiwei; Zheng, Yanhua; Xia, Yan Nature Cell Biology, Vol. 14, Issue 12 https://doi.org/10.1038/ncb2629	journal	November 2012
PKM2 dephosphorylation by Cdc25A promotes the Warburg effect and tumorigenesis Liang, Ji; Cao, Ruixiu; Zhang, Yajuan Nature Communications, Vol. 7, Issue 1 https://doi.org/10.1038/ncomms12431	journal	August 2016
The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis Cardone, Rosa A.; Casavola, Valeria; Reshkin, Stephan J. Nature Reviews Cancer, Vol. 5, Issue 10 https://doi.org/10.1038/nrc1713	journal	September 2005
Announcing the worldwide Protein Data Bank Berman, Helen; Henrick, Kim; Nakamura, Haruki Nature Structural & Molecular Biology, Vol. 10, Issue 12 https://doi.org/10.1038/nsb1203-980	journal	December 2003
Mutations in the PKM2 exon-10 region are associated with reduced allostery and increased nuclear translocation Chen, Tsan-Jan; Wang, Hung-Jung; Liu, Jai-Shin Communications Biology, Vol. 2, Issue 1 https://doi.org/10.1038/s42003-019-0343-4	journal	March 2019
The regulatory properties of rabbit muscle pyruvate kinase. The influence of effector concentrations Gregory, R. B.; Ainsworth, S.; Kinderlerer, J. Biochemical Journal, Vol. 209, Issue 2 https://doi.org/10.1042/bj2090413	journal	February 1983
M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation Morgan, H. P.; O'Reilly, F. J.; Wear, M. A. Proceedings of the National Academy of Sciences, Vol. 110, Issue 15 https://doi.org/10.1073/pnas.1217157110	journal	March 2013
Modulation of type M2 pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein Zwerschke, W.; Mazurek, S.; Massimi, P. Proceedings of the National Academy of Sciences, Vol. 96, Issue 4 https://doi.org/10.1073/pnas.96.4.1291	journal	February 1999
Bacillus anthracis Prolyl 4-Hydroxylase Modifies Collagen-like Substrates in Asymmetric Patterns Schnicker, Nicholas J.; Dey, Mishtu Journal of Biological Chemistry, Vol. 291, Issue 25 https://doi.org/10.1074/jbc.M116.725432	journal	June 2016
Biochemical and structural insights into how amino acids regulate pyruvate kinase muscle isoform 2 Nandi, Suparno; Dey, Mishtu Journal of Biological Chemistry, Vol. 295, Issue 16 https://doi.org/10.1074/jbc.RA120.013030	journal	April 2020
Dual roles of PKM2 in cancer metabolism Wu, S.; Le, H. Acta Biochimica et Biophysica Sinica, Vol. 45, Issue 1 https://doi.org/10.1093/abbs/gms106	journal	December 2012
Na ⁺ /H ⁺ exchanger‐dependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformation‐associated phenotypes Reshkin, Stephan J.; Bellizzi, Antonia; Caldeira, Sandra The FASEB Journal, Vol. 14, Issue 14 https://doi.org/10.1096/fj.00-0029com	journal	November 2000
Phaser crystallographic software McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D. Journal of Applied Crystallography, Vol. 40, Issue 4 https://doi.org/10.1107/S0021889807021206	journal	July 2007
XDS Kabsch, Wolfgang Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2 https://doi.org/10.1107/S0907444909047337	journal	January 2010
PHENIX: a comprehensive Python-based system for macromolecular structure solution Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221 https://doi.org/10.1107/S0907444909052925	journal	January 2010
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
Overview of the CCP 4 suite and current developments Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D. Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4 https://doi.org/10.1107/S0907444910045749	journal	March 2011
SAICAR Stimulates Pyruvate Kinase Isoform M2 and Promotes Cancer Cell Survival in Glucose-Limited Conditions Keller, K. E.; Tan, I. S.; Lee, Y. -S. Science, Vol. 338, Issue 6110 https://doi.org/10.1126/science.1224409	journal	October 2012
Tyrosine Phosphorylation Inhibits PKM2 to Promote the Warburg Effect and Tumor Growth Hitosugi, T.; Kang, S.; Vander Heiden, M. G. Science Signaling, Vol. 2, Issue 97 https://doi.org/10.1126/scisignal.2000431	journal	November 2009
PKM2, a Central Point of Regulation in Cancer Metabolism Wong, Nicholas; De Melo, Jason; Tang, Damu International Journal of Cell Biology, Vol. 2013 https://doi.org/10.1155/2013/242513	journal	January 2013
Acidity Generated by the Tumor Microenvironment Drives Local Invasion Estrella, V.; Chen, T.; Lloyd, M. Cancer Research, Vol. 73, Issue 5 https://doi.org/10.1158/0008-5472.CAN-12-2796	journal	January 2013
Immunohistological demonstration of pyruvate kinase isoenzyme type L in rat with monoclonal antibodies. Domingo, M.; Einig, C.; Eigenbrodt, E. Journal of Histochemistry & Cytochemistry, Vol. 40, Issue 5 https://doi.org/10.1177/40.5.1374093	journal	May 1992
Pyruvate kinase M2 at a glance Yang, W.; Lu, Z. Journal of Cell Science, Vol. 128, Issue 9 https://doi.org/10.1242/jcs.166629	journal	March 2015
In Silico Screening, Genotyping, Molecular Dynamics Simulation and Activity Studies of SNPs in Pyruvate Kinase M2 Kalaiarasan, Ponnusamy; Kumar, Bhupender; Chopra, Rupali PLOS ONE, Vol. 10, Issue 3 https://doi.org/10.1371/journal.pone.0120469	journal	March 2015
Biochemical, Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW—A Mononuclear Iron-Dependent DMSP Lyase Brummett, Adam E.; Schnicker, Nicholas J.; Crider, Alexander PLOS ONE, Vol. 10, Issue 5 https://doi.org/10.1371/journal.pone.0127288	journal	May 2015
Glycolysis, tumor metabolism, cancer growth and dissemination. A new pH-based etiopathogenic perspective and therapeutic approach to an old cancer question Alfarouk, Khalid O.; Verduzco, Daniel; Rauch, Cyril Oncoscience, Vol. 1, Issue 12 https://doi.org/10.18632/oncoscience.109	journal	November 2014
pH sensing and regulation in cancer Damaghi, Mehdi; Wojtkowiak, Jonathan W.; Gillies, Robert J. Frontiers in Physiology, Vol. 4 https://doi.org/10.3389/fphys.2013.00370	journal	January 2013
Nuclear PKM2 regulates the Warburg effect Yang, Weiwei; Lu, Zhimin Cell Cycle, Vol. 12, Issue 19 https://doi.org/10.4161/cc.26182	journal	October 2013
Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation Macpherson, Jamie A.; Theisen, Alina; Masino, Laura eLife, Vol. 8 https://doi.org/10.7554/eLife.45068	journal	July 2019

Similar Records

Structural Investigation of a Dimeric Variant of Pyruvate Kinase Muscle Isoform 2

Journal Article · Mon Nov 27 23:00:00 EST 2017 · Biochemistry · OSTI ID:1423366

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

Journal Article · Sun Aug 26 00:00:00 EDT 2012 · Nature Chemical Biology · OSTI ID:1054015

Pyruvate kinase M2 is a phosphotyrosine-binding protein

Journal Article · Tue Jun 03 00:00:00 EDT 2008 · Nature · OSTI ID:1006614

Related Subjects

59 BASIC BIOLOGICAL SCIENCES

Structural basis for allosteric regulation of pyruvate kinase M2 by phosphorylation and acetylation

Citation Formats

References (54)

Similar Records

Related Subjects