Structural insight into mechanisms for dynamic regulation of PKM2

Abstract

Pyruvate kinase isoform M2 (PKM2) converts phosphoenolpyruvate (PEP) to pyruvate and plays an important role in cancer metabolism. Here, we show that posttranslational modifications and a patient-derived mutation regulate pyruvate kinase activity of PKM2 through modulating the conformation of the PKM2 tetramer. We determined crystal structures of human PKM2 mutants and proposed a “seesaw” model to illustrate conformational changes between an inactive T-state and an active R-state tetramers of PKM2. Biochemical and structural analyses demonstrate that PKM2^Y105E (phosphorylation mimic of Y105) decreases pyruvate kinase activity by inhibiting FBP (fructose 1,6-bisphosphate)-induced R-state formation, and PKM2^K305Q (acetylation mimic of K305) abolishes the activity by hindering tetramer formation. K422R, a patient-derived mutation of PKM2, favors a stable, inactive T-state tetramer because of strong intermolecular interactions. Our study reveals the mechanism for dynamic regulation of PKM2 by posttranslational modifications and a patient-derived mutation and provides a structural basis for further investigation of other modifications and mutations of PKM2 yet to be discovered.

Key words： pyruvate kinase M2; crystal structure; allosteric regulation; Warburg effect; post-translational modifications

References

1 Akhtar K, Gupta V, Koul A, Alam N, Bhat R, Bamezai RN (2009) Differential behavior of missense mutations in the intersubunit contact domain of the human pyruvate kinase M2 isozyme. J Biol Chem284: 11971-11981

2 Anastasiou D, Poulogiannis G, Asara JM, Boxer MB, Jiang JK, Shen M, Bellinger G, Sasaki AT, Locasale JW, Auld DS (2011) Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science334: 1278-1283

3 Anastasiou D, Yu Y, Israelsen WJ, Jiang JK, Boxer MB, Hong BS, Tempel W, Dimov S, Shen M, Jha A (2012) Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis. Nat Chem Biol8: 839-847

4 Anitha M, Kaur G, Baquer NZ, Bamezai R (2004) Dominant negative effect of novel mutations in pyruvate kinase-M2. DNA Cell Biol23: 442-449

5 Boxer MB, Jiang J, Vander Heiden MG, Shen M, Veith H, Cantley LC, Thomas CJ (2010) Identification of activators for the M2 isoform of human pyruvate kinase version 3. In: Probe reports from the NIH molecular libraries program. National Center for Biotechnology Information, Bethesda

6 Chaneton B, Gottlieb E (2012) Rocking cell metabolism: revised functions of the key glycolytic regulator PKM2 in cancer. Trends Biochem Sci37: 309-316

7 Chaneton B, Hillmann P, Zheng L, Martin AC, Maddocks OD, Chokkathukalam A, Coyle JE, Jankevics A, Holding FP, Vousden KH (2012) Serine is a natural ligand and allosteric activator of pyruvate kinase M2. Nature491: 458-462

8 Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A, Gerszten RE, Wei R, Fleming MD, Schreiber SL, Cantley LC (2008a) The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature452: 230-233

9 Christofk HR, Vander Heiden MG, Wu N, Asara JM, Cantley LC (2008b) Pyruvate kinase M2 is a phosphotyrosine-binding protein. Nature452: 181-186

10 Dombrauckas JD, Santarsiero BD, Mesecar AD (2005) Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis. Biochemistry44: 9417-9429

11 Gao X, Wang H, Yang JJ, Liu X, Liu ZR (2012) Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Mol Cell45: 598-609

12 Gao X, Wang H, Yang JJ, Chen J, Jie J, Li L, Zhang Y, Liu ZR (2013) Reciprocal regulation of protein kinase and pyruvate kinase activities of pyruvate kinase M2 by growth signals. J Biol Chem288: 15971-15979

13 Gupta V, Kalaiarasan P, Faheem M, Singh N, Iqbal MA, Bamezai RN (2010) Dominant negative mutations affect oligomerization of human pyruvate kinase M2 isozyme and promote cellular growth and polyploidy. J Biol Chem285: 16864-16873

14 Hitosugi T, Kang S, Vander Heiden MG, Chung TW, Elf S, Lythgoe K, Dong S, Lonial S, Wang X, Chen GZ (2009) Tyrosine phosphorylation inhibits PKM2 to promote the Warburg effect and tumor growth. Sci Signal2: ra73

15 Iqbal MA, Siddiqui FA, Chaman N, Gupta V, Kumar B, Gopinath P, Bamezai RN (2014) Missense mutations in pyruvate kinase M2 promote cancer metabolism, oxidative endurance, anchorage independence and tumor growth in a dominant negative manner. J Biol Chem289: 8098-8105

16 Jiang JK, Boxer MB, Vander Heiden MG, Shen M, Skoumbourdis AP, Southall N, Veith H, Leister W, Austin CP, Park HW (2010) Evaluation of thieno[3,2-b]pyrrole[3,2-d]pyridazinones as activators of the tumor cell specific M2 isoform of pyruvate kinase. Bioorg Med Chem Lett20: 3387-3393

17 Keller KE, Tan IS, Lee YS (2012) SAICAR stimulates pyruvate kinase isoform M2 and promotes cancer cell survival in glucoselimited conditions. Science338: 1069-1072

18 Keller KE, Doctor ZM, Dwyer ZW, Lee Y-S (2014) SAICAR induces protein kinase activity of PKM2 that is necessary for sustained proliferative signaling of cancer cells. Mol Cell53: 700-709

19 Koppenol WH, Bounds PL, Dang CV (2011) Otto Warburg’s contributions to current concepts of cancer metabolism. Nat Rev Cancer11: 325-337

20 Kung C, Hixon J, Choe S, Marks K, Gross S, Murphy E, DeLaBarre B, Cianchetta G, Sethumadhavan S, Wang X (2012) Small molecule activation of PKM2 in cancer cells induces serine auxotrophy. Chem Biol19: 1187-1198

21 Liebermeister W (2005) Predicting physiological concentrations of metabolites from their molecular structure. J Comput Biol12: 1307-1315

22 Luo W, Hu H, Chang R, Zhong J, Knabel M, O’Meally R, Cole RN, Pandey A, Semenza GL (2011) Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell145: 732-744

23 Lv L, Li D, Zhao D, Lin R, Chu Y, Zhang H, Zha Z, Liu Y, Li Z, Xu Y (2011) Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperone-mediated autophagy and promotes tumor growth. Mol Cell42: 719-730

24 Lv L, Xu YP, Zhao D, Li FL, Wang W, Sasaki N, Jiang Y, Zhou X, Li TT, Guan KL (2013) Mitogenic and oncogenic stimulation of K433 acetylation promotes PKM2 protein kinase activity and nuclear localization. Mol Cell52: 340-352

25 Morgan HP, McNae IW, Nowicki MW, Hannaert V, Michels PA, Fothergill-Gilmore LA, Walkinshaw MD (2010) Allosteric mechanism of pyruvate kinase from Leishmania mexicana uses a rock and lock model. J Biol Chem285: 12892-12898

26 Morgan HP, O’Reilly FJ, Wear MA, O’Neill JR, Fothergill-Gilmore LA, Hupp T, Walkinshaw MD (2013) M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation. Proc Natl Acad Sci U S A110: 5881-5886

27 Noguchi T, Inoue H, Tanaka T (1986) The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. J Biol Chem261: 13807-13812

28 Noguchi T, Yamada K, Inoue H, Matsuda T, Tanaka T (1987) The Land R-type isozymes of rat pyruvate kinase are produced from a single gene by use of different promoters. J Biol Chem262: 14366-14371

29 Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol276: 307-326

30 Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science324: 1029-1033

31 Warburg O (1956) On the origin of cancer cells. Science123: 309-323

32 Wong N, Ojo D, Yan J, Tang D (2015) PKM2 contributes to cancer metabolism. Cancer Lett356: 184-191

33 Yang W, Lu Z (2013) Regulation and function of pyruvate kinase M2 in cancer. Cancer Lett339: 153-158

34 Yang W, Xia Y, Hawke D, Li X, Liang J, Xing D, Aldape K, Hunter T, Alfred Yung WK, Lu Z (2012a) PKM2 phosphorylates histone H3 and promotes gene transcription and tumorigenesis. Cell150: 685-696

35 Yang W, Zheng Y, Xia Y, Ji H, Chen X, Guo F, Lyssiotis CA, Aldape K, Cantley LC, Lu Z (2012b) ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect. Nat Cell Biol14: 1295-1304