Xinjian Li

PKM2 Phosphorylates Histone H3 and Promotes Gene Transcription and Tumorigenesis

Tumor-specific pyruvate kinase M2 (PKM2) is essential for the Warburg effect. In addition to its well-established role in aerobic glycolysis, PKM2 directly regulates gene transcription. However, the mechanism underlying this nonmetabolic function of PKM2 remains elusive. We show here that PKM2 directly binds to histone H3 and phosphorylates histone H3 at T11 upon EGF receptor activation. This phosphorylation is required for the dissociation of HDAC3 from the CCND1 and MYC promoter regions and subsequent acetylation of histone H3 at K9. PKM2-dependent histone H3 modifications are instrumental in EGF-induced expression of cyclin D1 and c-Myc, tumor cell proliferation, cell-cycle progression, and brain tumorigenesis. In addition, levels of histone H3 T11 phosphorylation correlate with nuclear PKM2 expression levels, glioma malignancy grades, and prognosis. These findings highlight the role of PKM2 as a protein kinase in its nonmetabolic functions of histone modification, which is essential for its epigenetic regulation of gene expression and tumorigenesis.

PKM2 Regulates Chromosome Segregation and Mitosis Progression of Tumor Cells

Tumor-specific pyruvate kinase M2 (PKM2) is instrumental in both aerobic glycolysis and gene transcription. PKM2 regulates G1-S phase transition by controlling cyclin D1 expression. However, it is not known whether PKM2 directly controls cell-cycle progression. We show here that PKM2, but not PKM1, binds to the spindle checkpoint protein Bub3 during mitosis and phosphorylates Bub3 at Y207. This phosphorylation is required for Bub3-Bub1 complex recruitment to kinetochores, where it interacts with Blinkin and is essential for correct kinetochore-microtubule attachment, mitotic/spindle-assembly checkpoint, accurate chromosome segregation, cell survival and proliferation, and active EGF receptor-induced brain tumorigenesis. In addition, the level of Bub3 Y207 phosphorylation correlated with histone H3-S10 phosphorylation in human glioblastoma specimens and with glioblastoma prognosis. These findings highlight the role of PKM2 as a protein kinase controlling the fidelity of chromosome segregation, cell-cycle progression, and tumorigenesis.

Mitochondria-Translocated PGK1 Functions as a Protein Kinase to Coordinate Glycolysis and the TCA Cycle in Tumorigenesis

It is unclear how the Warburg effect that exemplifies enhanced glycolysis in the cytosol is coordinated with suppressed mitochondrial pyruvate metabolism. We demonstrate here that hypoxia, EGFR activation, and expression of K-Ras G12V and B-Raf V600E induce mitochondrial translocation of phosphoglycerate kinase 1 (PGK1); this is mediated by ERK-dependent PGK1 S203 phosphorylation and subsequent PIN1-mediated cis-trans isomerization. Mitochondrial PGK1 acts as a protein kinase to phosphorylate pyruvate dehydrogenase kinase 1 (PDHK1) at T338, which activates PDHK1 to phosphorylate and inhibit the pyruvate dehydrogenase (PDH) complex. This reduces mitochondrial pyruvate utilization, suppresses reactive oxygen species production, increases lactate production, and promotes brain tumorigenesis. Furthermore, PGK1 S203 and PDHK1 T338 phosphorylation levels correlate with PDH S293 inactivating phosphorylation levels and poor prognosis in glioblastoma patients. This work highlights that PGK1 acts as a protein kinase in coordinating glycolysis and the tricarboxylic acid (TCA) cycle, which is instrumental in cancer metabolism and tumorigenesis.

PKM2 phosphorylates MLC2 and regulates cytokinesis of tumour cells

Pyruvate kinase M2 (PKM2) is expressed at high levels during embryonic development and tumor progression and is important for cell growth. However, it is not known whether it directly controls cell division. Here, we found that Aurora B phosphorylates PKM2, but not PKM1, at T45; this phosphorylation is required for PKM2s localization and interaction with myosin light chain 2 (MLC2) in the contractile ring region of mitotic cells during cytokinesis. PKM2 phosphorylates MLC2 at Y118, which primes the binding of ROCK2 to MLC2 and subsequent ROCK2-dependent MLC2 S15 phosphorylation. PKM2-regulated MLC2 phosphorylation, which is greatly enhanced by EGF stimulation or EGFRvIII, K-Ras G12V, and B-Raf V600E mutant expression, plays a pivotal role in cytokinesis, cell proliferation, and brain tumor development. These findings underscore the instrumental function of PKM2 in oncogenic EGFR-, K-Ras-, and B-Raf-regulated cytokinesis and tumorigenesis.

A splicing switch from ketohexokinase-C to ketohexokinase-A drives hepatocellular carcinoma formation

Dietary fructose is primarily metabolized in the liver. Here we demonstrate that, compared with normal hepatocytes, hepatocellular carcinoma (HCC) cells markedly reduce the rate of fructose metabolism and the level of reactive oxygen species, as a result of a c-Myc-dependent and heterogeneous nuclear ribonucleoprotein (hnRNP) H1- and H2-mediated switch from expression of the high-activity fructokinase (KHK)-C to the low-activity KHK-A isoform. Importantly, KHK-A acts as a protein kinase, phosphorylating and activating phosphoribosyl pyrophosphate synthetase 1 (PRPS1) to promote pentose phosphate pathway-dependent de novo nucleic acid synthesis and HCC formation. Furthermore, c-Myc, hnRNPH1/2 and KHK-A expression levels and PRPS1 Thr225 phosphorylation levels correlate with each other in HCC specimens and are associated with poor prognosis for HCC. These findings reveal a pivotal mechanism underlying the distinct fructose metabolism between HCC cells and normal hepatocytes and highlight the instrumental role of KHK-A protein kinase activity in promoting de novo nucleic acid synthesis and HCC development.

Nucleus-Translocated ACSS2 Promotes Gene Transcription for Lysosomal Biogenesis and Autophagy

Overcoming metabolic stress is a critical step in tumor growth. Acetyl coenzyme A (acetyl-CoA) generated from glucose and acetate uptake is important for histone acetylation and gene expression. However, how acetyl-CoA is produced under nutritional stress is unclear. We demonstrate here that glucose deprivation results in AMP-activated protein kinase (AMPK)-mediated acetyl-CoA synthetase 2 (ACSS2) phosphorylation at S659, which exposed the nuclear localization signal of ACSS2 for importin α5 binding and nuclear translocation. In the nucleus, ACSS2 binds to transcription factor EB and translocates to lysosomal and autophagy gene promoter regions, where ACSS2 incorporates acetate generated from histone acetylation turnover to locally produce acetyl-CoA for histone H3 acetylation in these regions and promote lysosomal biogenesis, autophagy, cell survival, and brain tumorigenesis. In addition, ACSS2 S659 phosphorylation positively correlates with AMPK activity in glioma specimens and grades of glioma malignancy. These results underscore the significance of nuclear ACSS2-mediated histone acetylation in maintaining cell homeostasis and tumor development.

PKM2 dephosphorylation by Cdc25A promotes the Warburg effect and tumorigenesis

Many types of human tumour cells overexpress the dual-specificity phosphatase Cdc25A. Cdc25A dephosphorylates cyclin-dependent kinase and regulates the cell cycle, but other substrates of Cdc25A and their relevant cellular functions have yet to be identified. We demonstrate here that EGFR activation results in c-Src-mediated Cdc25A phosphorylation at Y59, which interacts with nuclear pyruvate kinase M2 (PKM2). Cdc25A dephosphorylates PKM2 at S37, and promotes PKM2-dependent β-catenin transactivation and c-Myc-upregulated expression of the glycolytic genes GLUT1, PKM2 and LDHA, and of CDC25A; thus, Cdc25A upregulates itself in a positive feedback loop. Cdc25A-mediated PKM2 dephosphorylation promotes the Warburg effect, cell proliferation and brain tumorigenesis. In addition, we identify positive correlations among Cdc25A Y59 phosphorylation, Cdc25A and PKM2 in human glioblastoma specimens. Furthermore, levels of Cdc25A Y59 phosphorylation correlate with grades of glioma malignancy and prognosis. These findings reveal an instrumental function of Cdc25A in controlling cell metabolism, which is essential for EGFR-promoted tumorigenesis.

KAT2A coupled with the α-KGDH complex acts as a histone H3 succinyltransferase

Histone modifications, such as the frequently occurring lysine succinylation, are central to the regulation of chromatin-based processes. However, the mechanism and functional consequences of histone succinylation are unknown. Here we show that the α-ketoglutarate dehydrogenase (α-KGDH) complex is localized in the nucleus in human cell lines and binds to lysine acetyltransferase 2A (KAT2A, also known as GCN5) in the promoter regions of genes. We show that succinyl-coenzyme A (succinyl-CoA) binds to KAT2A. The crystal structure of the catalytic domain of KAT2A in complex with succinyl-CoA at 2.3 Å resolution shows that succinyl-CoA binds to a deep cleft of KAT2A with the succinyl moiety pointing towards the end of a flexible loop 3, which adopts different structural conformations in succinyl-CoA-bound and acetyl-CoA-bound forms. Site-directed mutagenesis indicates that tyrosine 645 in this loop has an important role in the selective binding of succinyl-CoA over acetyl-CoA. KAT2A acts as a succinyltransferase and succinylates histone H3 on lysine 79, with a maximum frequency around the transcription start sites of genes. Preventing the α-KGDH complex from entering the nucleus, or expression of KAT2A(Tyr645Ala), reduces gene expression and inhibits tumour cell proliferation and tumour growth. These findings reveal an important mechanism of histone modification and demonstrate that local generation of succinyl-CoA by the nuclear α-KGDH complex coupled with the succinyltransferase activity of KAT2A is instrumental in histone succinylation, tumour cell proliferation, and tumour development.

Protein kinase activity of the glycolytic enzyme PGK1 regulates autophagy to promote tumorigenesis

ABSTRACT Macroautophagy/autophagy is a cellular defense response to stress conditions and is crucial for cell homeostasis maintenance. However, the precise mechanism underlying autophagy initiation, especially in response to glutamine deprivation and hypoxia, is yet to be explored. We recently discovered that PGK1 (phosphoglycerate kinase 1), a glycolytic enzyme, functions as a protein kinase, phosphorylating BECN1/Beclin 1 to initiate autophagy. Under glutamine deprivation or hypoxia stimulation, PGK1 is acetylated at K388 by NAA10/ARD1 in an MTOR-inhibition-dependent manner, leading to the interaction between PGK1 and BECN1 and the subsequent phosphorylation of BECN1 at S30 by PGK1. This phosphorylation enhances ATG14-associated PIK3C3/VPS34-BECN1-PIK3R4/VPS15 complex activity, thereby increasing phosphatidylinositol-3-phosphate (PtdIns3P) generation in the initiation stage of autophagy. Furthermore, NAA10-dependent PGK1 acetylation and PGK1-dependent BECN1 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumor formation. Our work reveals the important dual roles of PGK1 as a glycolytic enzyme and a protein kinase in the mutual regulation of cell metabolism and autophagy in maintaining cell homeostasis.

PGK1 is a new member of the protein kinome

In the glycolysis pathway, pyruvate kinase (PK) and phosphoglycerate kinase 1 (PGK1) are the only 2 ATP-generating enzymes. PK is a rate-limiting glycolytic enzyme that catalyzes the conversion of phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP. Splicing inclusion of exon 9 and exon 10 from PKM pre-mRNA results in expression of the PKM1 and PKM2 isoforms, respectively. Our previous studies demonstrated that PKM2 but not PKM1 has dual enzymatic activities as both a glycolytic enzyme and protein kinase. PKM2 uses PEP as a phosphate donor to phosphorylate its protein substrates, which include histone H3 T11, STAT3 Y705, Bub3 Y207, and MLC2 Y118. In addition, more than 100 protein substrates of PKM2 were identified via protein array screening analysis. Of note, a recent report debated that PKM2 lacks evidence of being a protein kinase. However, these results may owe to the drawbacks of using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cannot distinguish differences in the phosphorylation spectrum between cells wild-type for PKM2 and PKM2-null cells. Importantly, a more recent publication confirmed that the yeast PKM2 homolog, acting as a protein kinase, directly phosphorylated histone H3 T11 in the presence of PEP but not ATP. Additionally, it was demonstrated that AKT1 substrate 1 is a new protein substrate of PKM2. Taken together, these reports support that PKM2 possesses protein kinase activity (Fig. 1). PGK1 is the first ATP-generating enzyme in the glycolysis pathway. It catalyzes the reversible conversion of 1,3-diphosphoglycerate and ADP to 3-phosphoglycerate and ATP, respectively. Our recent studies demonstrated that PGK1 translocates into mitochondria under hypoxic stress, epidermal growth factor receptor (EGFR) activation, or expression of oncogenic KRas G12V or the B-Raf V600E mutation. Mechanistically, mitochondrial translocation of PGK1 is mediated by extracellular signal-regulated kinase (ERK) 1/2-dependent S203 phosphorylation, which results in PGK1 isomerization by Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1), and subsequent exposure of the presequence of PGK1 (38-QRIKAA-43) to recognition by the mitochondrial translocase of the outer membrane (TOM) complex. In mitochondria, PGK1 directly phosphorylated pyruvate dehydrogenase kinase isozyme 1 (PDHK1) at T338 using ATP as a phosphate donor (Fig. 1). PGK1-dependent phosphorylation of PDHK1 activated PDHK1 and enhanced PDHK1-mediated pyruvate dehydrogenase E1a S293 phosphorylation, which inactivated pyruvate dehydrogenase complex, an enzyme complex that converts pyruvate and coenzyme A to acetyl-coenzyme A and CO2. Thus, mitochondrial translocation of PGK1 resulted in inhibition of pyruvate oxidation in mitochondria and enhancement of lactate production from pyruvate in cytosol. Deficiency in mitochondrial translocation of PGK1 via CRISPR/Cas9-mediated knock-in of PGK1 S203A or reconstituted expression of a kinase-dead PGK1 T378P mutant in PGK1-depleted cells blocked hypoxiaand EGFR activation-induced lactate production and attenuation of mitochondrial pyruvate oxidation. These results indicated that the protein kinase activity of mitochondrial PGK1 is a gatekeeper for the tricarboxylic acid (TCA) cycle by shunting pyruvate from the mitochondria into the cytosol for lactate production. In addition, we revealed that hypoxia-enhanced expression of PDHK1 had a limited effect on pyruvate dehydrogenase E1a S293 phosphorylation in PGK1 mitochondrial translocation-deficient cells, which indicated that PDHK1 relied on mitochondrial PGK1-dependent phosphorylation of PDHK1 more than PDHK1 protein expression for its activity. Functional studies demonstrated that replacement of endogenous PGK1 with mitochondrial translocation-deficient mutant PGK1 S203A dramatically reduced the growth of tumors orthotopically transplanted in the brains of mice, which resulted from a low proliferation rate and enhanced apoptosis of tumor cells. Immunohistochemical staining of human glioblastoma tissues demonstrated that PGK1 pS203 and PDHK1 pT338 are positively correlated with each other, and the levels of PGK1 pS203 and PDHK1 pT338 staining are inversely correlated with survival duration in glioblastoma patients, supporting a pivotal role for PGK1-dependent PDHK1 phosphorylation in glioblastoma progression. In summary, we expanded the protein kinome via characterization and demonstration of the protein kinase activities of the