Metabolic kinases moonlighting as protein kina.pdf

Metabolic kinases moonlighting as protein kinasesZhimin Lu1,2,3 and Tony Hunter41Brain Tumor Center and Department of Neuro-Oncology,The University of Texas MD Anderson Cancer Center,Houston,TX 77030,USA2Department of Molecular and Cellular Oncology,The University of Texas MD Anderson Cancer Center,Houston,TX 77030,USA3Cancer Biology Program,The University of Texas Graduate School of Biomedical Sciences at Houston,Houston,TX 77030,USA4Cancer Biology Program,MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences,The University of Texas,Houston,Texas 77030,USAAbstractProtein kinases regulate every aspect of cellular activity,whereas metabolic enzymes are responsible for energy production and catabolic and anabolic processes.Emerging evidence demonstrates that some metabolic enzymes,such as pyruvate kinase M2,phosphoglycerate kinase 1,ketohexokinase isoform A,hexokinase(HK),and nucleoside diphosphate kinase 1 and 2(NME1/2),that phosphorylate soluble metabolites can also function as protein kinases and phosphorylate a variety of protein substrates to regulate the Warburg effect,gene expression,cell cycle progression and proliferation,apoptosis,autophagy,exosome secretion,T-cell activation,iron transport,ion channel opening,and many other fundamental cellular functions.The elevated protein kinase functions of these moonlighting metabolic enzymes in tumor development make them promising therapeutic targets for cancer.Keywordsmetabolic enzymes;protein kinase;phosphorylationProtein kinases are important regulators of intracellular signal transduction pathways that mediate the development and regulation of both unicellular and multicellular organisms.They play critical roles in cell growth,division,differentiation,adhesion,motility,and death(Brognard and Hunter,2011;Lu and Hunter,2009).These protein kinases,more than 500 of which exist in the human genome,can be primarily subdivided into tyrosine(Y)-and serine(S)/threonine(T)-specific kinases based on their catalytic specificity(Manning et al.,2002).Correspondence:Zhimin Lua and Tony Hunterb,aTel:713-834-6231;Fax:713-834-6230,zhiminlumdanderson.org.bTel:858-453-4100;Fax:858-457-4765,huntersalk.edu.Publishers Disclaimer:This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting,typesetting,and review of the resulting proof before it is published in its final citable form.Please note that during the production process errors may be discovered which could affect the content,and all legal disclaimers that apply to the journal pertain.HHS Public AccessAuthor manuscriptTrends Biochem Sci.Author manuscript;available in PMC 2019 April 01.Published in final edited form as:Trends Biochem Sci.2018 April;43(4):301310.doi:10.1016/j.tibs.2018.01.006.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptDisruption of normal protein kinase functions by mutations,altered expression,or dysregulation can cause many human diseases,including cancer and diabetes(Lu and Hunter,2009).Cell metabolism comprises the life-sustaining chemical reactions ultimately responsible for all cellular processes;production of ATP and building blocks for proteins,lipids,nucleic acids,and some carbohydrates;and elimination of nitrogenous wastes.Metabolic enzymes catalyze these reactions,facilitating cell growth and proliferation and the response to all intracellular and extracellular signaling and stimuli.Each metabolic enzyme is known to catalyze a unidirectional and/or bidirectional reaction in a specific metabolic pathway.However,recent studies have demonstrated that several metabolic enzymes can also moonlight as protein kinases and phosphorylate multiple protein substrates.These phosphorylations are critical for key cellular functions,including the Warburg effect,a feature of which is a high rate of glycolysis and lactic acid fermentation in the cytosol of cancer cells regardless of oxygen level(Li et al.,2016b;Li et al.,2016d;Yang and Lu,2015).This review highlights the roles of the unexpected protein kinase activity of the metabolic enzymes pyruvate kinase M2(PKM2),phosphoglycerate kinase 1(PGK1),ketohexokinase isoform A(KHK-A,or fructokinase A),hexokinase(HK)and the NME1/2 histidine(H)kinases in regulation of a variety of cellular functions and the impact of this regulation on tumorigenesis.PKM2Pyruvate kinase regulates the final rate-limiting step of glycolysis by catalyzing the transfer of a phosphate group from phosphoenolpyruvate(PEP)to adenosine diphosphate(ADP)to produce pyruvate and adenosine triphosphate(ATP)(Yang and Lu,2015).This kinase has four isoforms:PKL,PKR,PKM1,and PKM2.PKL and PKR,which are encoded by the PKLR gene,are expressed in the liver and erythrocytes,respectively,whereas PKM1 and PKM2,which are encoded by the PKM gene,are expressed in different types of cells and tissues.The heterogeneous nuclear ribonucleoproteins A1(hnRNPH1)and A2(hnRNPH2)and polypyrimidine-tract(PPT)binding protein splicing factors regulate alternative splicing of PKM pre-mRNA and generate PKM2 via the inclusion of the PKM2-specific exon 10 and exclusion of the PKM1-specific exon 9(David et al.,2010;Noguchi et al.,1987).An isoform switch from PKM1 to PKM2 and enhanced PKM2 expression have been found in many cancer types(Bluemlein et al.,2011;Desai et al.,2014;Yang et al.,2012a).Genetic replacement of PKM2 with PKM1 inhibits aerobic glycolysis and tumor growth in mice,although PKM1 has higher glycolytic activity than PKM2 does(Christofk et al.,2008;Guminska et al.,1988;Mellati et al.,1992).One of the fundamental functional differences between PKM1 and PKM2 is that the latter has unique nuclear functions.Specifically,PKM2 contains a nuclear localization signal(NLS)encoded by exon 10,whereas PKM1 lacks an NLS.Activated extracellular signal-regulated kinase(ERK)1/2 mitogen-activated protein kinases bind to an ERK docking groove encoded by PKM2 exon 10 thus allowing ERK1/2 to phosphorylate PKM2 but not PKM1 at Ser37.Once phosphorylated,the pSer37.Pro38 bond in PKM2 is subject to cis-trans isomerization by the phospho-specific peptidyl-proline isomerase protein interacting with never in mitosis A-1(PIN1),causing a Lu and HunterPage 2Trends Biochem Sci.Author manuscript;available in PMC 2019 April 01.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptconformational change in PKM2,conversion of PKM2 from a tetramer to a monomer,and exposure of the PKM2 NLS for importin 5 binding and subsequent nuclear translocation(Yang and Lu,2013;Yang et al.,2012c).Interaction of PKM2 with JMJD5,a Jumonji C domain-containing dioxygenase,hinders PKM2 tetrameric assembly and facilitates PKM2s nuclear translocation(Wang et al.,2014a).In addition,enhanced nuclear translocation of PKM2 is induced by sumoylation of PKM2 mediated by the SUMO-E3 ligase PIAS3 and by acetylation of PKM2 at Lys433 mediated by the p300 acetyltransferase that prevents the binding of fructose-1,6-bisphosphate to PKM2 and formation of a PKM2 tetramer(Lv et al.,2013;Spoden et al.,2009).This evidence supports the possibility that PKM2 translocates into the nucleus as a monomer.In the nucleus,PKM2 phosphorylated at Ser37 in the cytosol in response to growth factor receptor(EGFR)activation,is dephosphorylated by the dual-specificity Cdc25A phosphatase,thereby forming a complex with-catenin that has been phosphorylated by c-Src at Tyr333,which then binds to-cateninregulated target promoter regions of genes,such as MYC and CCND1(Liang et al.,2016b;Yang et al.,2011).Using PEP as a phosphate donor,nuclear PKM2 phosphorylates histone H3 at Thr11 in nucleosomes associated with gene promoter regions.This phosphorylation is required for the dissociation of histone deacetylase 3(HDAC3)from the gene promoter regions and the subsequent acetylation of histone H3 at Lys9.PKM2-dependent histone H3 phosphorylation is essential for EGFR-promoted cell proliferation and tumorigenesis as well as expression of specific genes,including c-Mycregulated genes such as GLUT1 and lactate dehydrogenase A(LDHA),and polypyrimidine-tract binding(PTB)protein,which increases PKM2 mRNA expression levels via alternative splicing of PKM pre-mRNA.Thus,nuclear PKM2 enhances the Warburg effect by upregulating expression of glycolytic genes,including itself(Yang et al.,2012b;Yang et al.,2012c).PKM2 also binds to other transcription factors,such as hypoxia-inducible factor(HIF)1,signal transducer and activator of transcription(STAT)3,and Oct4,and enhances their target gene expression(Gao et al.,2012;Luo et al.,2011;Yang and Lu,2015).Notably,PKM2 phosphorylates STAT3 at Tyr705 and promotes STAT3 transcriptional activity(Gao et al.,2012).Although one study of the ability of recombinant PKM2 to phosphorylate proteins in PKM2-deficient cell lysates failed to detect PEP-dependent protein kinase activity,possibly due to insufficient levels of key target substrate proteins in the cell lysates and the low concentration of 32P-PEP used in the reactions(Hosios et al.,2015),a later study using similar phosphoproteomic analyses of the proteome of renal cell carcinoma cells demonstrated that recombinant PKM2 in the presence of PEP phosphorylated 974 protein substrates(He et al.,2016).In addition,multiple research groups have demonstrated and validated PKM2s protein kinase activity using both yeast and mammalian cells(Keller et al.,2014;Li et al.,2015).Succinyl-5-aminoimidazole-4-carboxamide-1-ribose-5-phosphate(SAICA)binds to PKM2 and enhances PKM2-mediated histone H3 phosphorylation at Thr11,as well as phosphorylation of ERK1/2 and more than 100 other proteins(Keller et al.,2014).Another report demonstrated that the yeast PKM2 homolog PYK1 directly phosphorylates histone H3 at Thr11 and regulates the cross-talk between H3 Thr11 phosphorylation and H3 Lys4 methylation in autoregulation of PYK1 expression(Li et al.,2015).Transgenic mouse studies demonstrated that PKM2 is not required for breast cancer formation promoted by BRCA1 tumor suppressor deficiency but Lu and HunterPage 3Trends Biochem Sci.Author manuscript;available in PMC 2019 April 01.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptis essential for BCR-ABL or MLL-AF9induced leukemia development(Israelsen et al.,2013;Wang et al.,2014b),suggesting that the roles of PKM2 in tumor suppressor-and oncogene-induced tumorigenesis can differ.Besides regulating gene expression,PKM2s protein kinase activity is instrumental for many critical cellular activities.PKM2 binds to the spindle checkpoint protein Bub3 during mitosis and phosphorylates Bub3 at Tyr207(Jiang et al.,2014a).This phosphorylation leads to recruitment of the Bub3-Bub1 complex to the outer kinetochore protein Blinkin and governs kinetochore-spindle attachment and the mitotic checkpoint,thereby promoting accurate chromosome segregation and proliferation of tumor cells(Jiang et al.,2014a).PKM2 is also involved in cytokinesis.When PKM2 Thr45 is phosphorylated by Aurora B,PKM2 interacts with myosin light chain 2(MLC2)in the contractile ring regions of mitotic cells and phosphorylates it at Tyr118,and MLC2 Tyr118 phosphorylation is greatly enhanced by EGFR variant III,K-Ras G12V,and B-Raf V600E oncogenic mutant expression(Jiang et al.,2014a).This phosphorylation primes MLC2 phosphorylation at Ser15 mediated by Rho-associated protein kinase 2(ROCK2)and is required for contraction of the actomyosin complex at the cleavage furrow,completion of cytokinesis,and tumor cell proliferation(Jiang et al.,2014b).In addition,PKM2,whose expression is transcriptionally enhanced by EGFR-dependent activation of nuclear factor(NF)-B,promotes hormonal and nutrient signal-independent activation of mammalian target of rapamycin complex 1(mTORC1)via phosphorylation of mTORC1 inhibitor AKT1 substrate 1(AKT1S1)at Ser202/203 and its release from Raptor(He et al.,2016;Yang et al.,2012a).In renal cell carcinomas and breast cancers,PKM2 overexpression has been correlated with elevated AKT1S1 phosphorylation at Ser202/203,activated mTORC1,and reduced autophagy(He et al.,2016).The protein kinase activity of PKM2 is also involved in cell apoptosis.In response to oxidative stress,PKM2 translocates to the outer membrane of mitochondria,where heat shock protein(HSP)901-mediates a conformational change in PKM2,which then interacts with and phosphorylates Bcl-2 at Thr69.This phosphorylation prevents the binding of a Cul3-based BCR(BTB-CUL3-RBX1)E3 ligase to Bcl-2,thereby stabilizing Bcl-2 and enhancing the resistance of tumor cells to oxidative stress(Liang et al.,2016a).PKM2 also participates in the remodeling of tumor microenvironments by regulating tumor cell exosome secretion.PKM2 acts as a protein kinase to phosphorylate synaptosome-associated protein 23(SNAP-23)at Ser95,which in turn enables the formation of the SNARE complex to facilitate exosome release(Wei et al.,2017).In summary,in addition to its originally characterized metabolic function in the glycolytic pathway,it has now been established that PKM2 also acts as a dual-specificity protein kinase that can phosphorylate protein substrates at both serine/threonine and tyrosine residues.PKM2 possesses nonmetabolic functions,acting as a protein kinase and phosphorylating a variety of protein substrates to regulate the Warburg effect,gene expression,mitosis and cytokinesis progression,cell proliferation,apoptosis,and exosome secretion(Fig.1).Lu and HunterPage 4Trends Biochem Sci.Author manuscript;available in PMC 2019 April 01.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptPGK1Pyruvate kinase and PGK1 are the only two ATP-generating enzymes in the glycolysis pathway.PGK1 is the first ATP-generating enzyme in this pathway and is highly expressed in many types of cancer(Li et al.,2016d).It catalyzes the reversible conversion of 1,3-diphosphoglycerate and ADP to 3-phosphoglycerate and ATP,respectively(Li et al.,2016d).Nuclear PKM2 enhances the Warburg effect by upregulating the expression of glycolytic enzymes,including PKM2 itself,to increase glucose uptake and lactate production(Yang et al.,2011),but how this enhanced aerobic glycolysis coordinates with suppression of mitochondrial pyruvate metabolism is a central question in understanding the Warburg effect.Our recent work demonstrated that EGFR activation,oncogenic K-Ras G12V and B-Raf V600E mutant expression,and hypoxic stress all result in ERK activation-dependent mitochondrial translocation of a small portion of cytosolic PGK1(Li et al.,2016a).Activated ERK1/2 phosphorylates PGK1 at Ser203.This phosphorylation recruits the PIN1 prolyl isomerase,leading to isomerization of the Ser203.Pro204 bond and subsequent exposure of the presequence of PGK1(38-QRIKAA-43)on its surface.The exposed PGK1 presequence is then recognized by the mitochondrial translocase of the outer membrane(TOM)complex,leading to tr