Pyruvate Kinase M2 Is a PHD3-Stimulated Coactivator for Hypoxia-Inducible Factor 1

PKM2-V5 expression significantly enhances p2.1 transcriptional activity in HeLa cells cotransfected with HIF-2α expression vector.

“PKM2-V5 expression significantly enhanced p2.1 transcriptional activity in HeLa cells cotransfected with HIF-2α expression vector (Figure S2E).”

Mass spectrometry analysis revealed hydroxylation of Pro-403 as well as Pro-408 in PKM2.

“Mass spectrometry analysis revealed hydroxylation of Pro-403 as well as Pro-408 (Figure 4A).”

The PKM2 HRE significantly increased FLuc activity in hypoxic HeLa cells.

“The PKM2 HRE significantly increased Fluc activity in hypoxic HeLa cells (Figure 1E).”

PKM2-V5 mainly binds to GST-HIF-1α (575-786), which encodes an inhibitory domain associated with regulation of the HIF-1α transactivation domain.

“We further mapped PKM2 binding within the transactivation domain of HIF-1α and found that PKM2-V5 mainly bound to GST-HIF-1α (575–786) (Figure 2E), which encodes an inhibitory domain associated with regulation of the HIF-1α transactivation domain (Jiang et al., 1997).”

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PKM2 occupancy by HIF-2α was not above background levels in ChIP assays.

“In contrast, PKM2 occupancy by HIF-2α was not above background levels (Figure 1D).”

PKM2-V5 is prolyl hydroxylated in HeLa cells cultured at 20% O2.

“Analysis of immunoprecipitated PKM2-V5 protein revealed that PKM2-V5 was prolyl hydroxylated in HeLa cells cultured at 20% O2 (Figure 4C).”

PKM2-V5 significantly increases GalA transcriptional activity in HeLa cells exposed to 20% or 1% O2 for 24 h.

“PKM2-V5 significantly increased GalA transcriptional activity in HeLa cells exposed to 20% or 1% O2 for 24 h (Figure 3E).”

Mutation of 5′-CGT-3′ to 5′-AAA-3′ at the core binding site significantly decreased hypoxia-induced FLuc activity.

“Mutation of 5′-CGT-3′ to 5′-AAA-3′ at the core binding site, which eliminates HIF-1 binding (Semenza et al., 1996), significantly decreased hypoxia-induced FLuc activity (Figure 1E).”

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PKM2-V5 expression does not affect HIF-1α or HIF-1β protein levels in HeLa cells.

“PKM2-V5 expression did not affect HIF-1α or HIF-1β protein levels (Figure 3C).”

The human PKM2 gene contains a candidate HRE within the first intron with the antisense HIF-1 binding site 5′-ACGTG-3′ followed by a 5′-CACA-3′ sequence.

“Analysis of the human PKM2 gene sequence revealed a candidate HRE within the first intron containing (on the antisense strand) the HIF-1 binding site 5′-ACGTG-3′ followed by a 5′-CACA-3′ sequence (Figure 1C), which is found in many HREs (Fukuda et al., 2007).”

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This prolyl hydroxylation motif is not present in PKM1.

“This prolyl hydroxylation motif is not present in PKM1 (Figure S3A).”

Prolyl hydroxylation of PKM2-V5 is also detected in HeLa cells cultured at 1% O2.

“Remarkably, prolyl hydroxylation of PKM2-V5 was also detected in HeLa cells cultured at 1% O2.”

PKM2 knockdown fails to alter protein levels of HIF-1α or HIF-1β in HeLa cells.

“PKM2 knockdown also failed to alter protein levels of HIF-1α or HIF-1β in HeLa cells (Figure 3D).”

HIF-2α interacts with PKM2-V5 in hypoxic cells.

“Co-IP assays using anti-HIF-2α antibody demonstrated that HIF-2α interacted with PKM2-V5 in hypoxic cells (Figure S2F).”

PKM2-V5 did not induce FLuc activity when cotransfected with GalO.

“PKM2-V5 did not induce FLuc activity when cotransfected with GalO (Figure 3E), indicating that PKM2 specifically stimulates HIF-1α transactivation domain function.”

Mutation of 5′-CAC-3′ to 5′-AAA-3′ abolished PKM2 HRE-driven FLuc activity.

“Mutation of 5′-CAC-3′ to 5′-AAA-3′ also abolished PKM2 HRE-driven FLuc activity (Figure 1F).”

GalA activity is stimulated by catalytically inactive PKM2(K270M).

“GalA activity was also stimulated by catalytically inactive PKM2(K270M) (Figure 3H).”

PKM1-V5 protein levels are comparable to PKM2-V5 protein levels in HeLa cells.

“PKM1-V5 protein levels were comparable to PKM2-V5 protein levels in HeLa cells (Figure S2G).”

PKM2 also regulates GalA activity in Hep3B cells.

“PKM2 also regulated GalA activity in Hep3B cells (Figures S2C and S2D).”

Endogenous PKM2 is specifically precipitated by anti-HIF-1α antibody, but not control IgG, in HeLa cells exposed to 1% O2.

“Endogenous PKM2 was specifically precipitated by anti-HIF-1α antibody, but not by control IgG (Figure 2B).”

The anti-Pro-OH antibody preferentially bound to WT FLAG-HIF-1α compared to double mutant (DM) FLAG-HIF-1α (P402/564A).

“The anti-Pro-OH antibody preferentially bound to WT FLAG-HIF-1α as compared to double mutant (DM) FLAG-HIF-1α (P402/564A), in which both hydroxylatable Pro residues have been changed to alanine (Figure 4B).”

PKM2 preferentially bound to GST-HIF-1α (531-826) fusion protein compared to GST alone in a SILAC-based proteomic screen.

“A stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic screen (Luo et al., 2010) revealed that PKM2 preferentially bound to a GST-HIF-1α (531–826) fusion protein containing amino acid residues 531–826 of HIF-1α as compared to GST alone (Figure 2A), suggesting that PKM2 is a novel HIF-1α-interacting protein.”

• cites Luo et al., 2010 sourceNot checked yet

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PKM2-V5 significantly increases HIF-1 transcriptional activity in a concentration-dependent manner in HeLa cells.

“PKM2-V5 significantly increased HIF-1 transcriptional activity in a concentration-dependent manner (Figure 3A).”

PKM2(P403/408A)-V5 is detected in the nucleus at levels similar to WT PKM2-V5.

“despite the fact that PKM2(P403/408A)-V5 was detected in the nucleus at levels similar to WT PKM2-V5 (Figure S3C).”

ChIP with HIF-1α antibody enriched the putative HRE sequence >70-fold compared to IgG in HeLa cells treated with DMOG.

“ChIP with HIF-1α antibody enriched the putative HRE sequence >70-fold compared to IP with IgG (Figure 1D).”

PKM2-V5 is efficiently co-immunoprecipitated with HIF-1α in nuclear extracts of hypoxic HeLa cells.

“PKM2-V5 was efficiently co-immunoprecipitated with HIF-1α (Figure 2C).”

Knockdown of endogenous PKM2 significantly decreases GalA activity in HeLa cells.

“Knockdown of endogenous PKM2 significantly decreased GalA activity in HeLa cells (Figure 3F).”

Similar results were observed in Hep3B hepatoblastoma cells.

“Similar results were observed in Hep3B hepatoblastoma cells (Figures S2A and S2B).”

The alternatively spliced domain of PKM2 contains a match with the prolyl hydroxylation motif LXXLAP403, which is present in all vertebrate HIF-1α and HIF-2α isoforms.

“Analysis of the amino acid sequence of the alternatively spliced domain of PKM2 (Figure S3A) revealed a match with the prolyl hydroxylation motif LXXLAP403, which is present in all vertebrate HIF-1α and HIF-2α isoforms (Epstein et al., 2001).”

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Double mutation of Pro-403/408 significantly reduces PKM2-mediated HIF-1 transactivation in HeLa cells.

“The p2.1 and GalA assays revealed that double mutation of Pro-403/408 significantly reduced PKM2-mediated HIF-1 transactivation in HeLa cells (Figures 4F and 4G), despite the fact that PKM2(P403/408A)-V5 was detected in the nucleus at levels similar to WT PKM2-V5 (Figure S3C).”

PKM2(K270M) increases HIF-1 transcriptional activity in HeLa cells, similar to WT PKM2.

“p2.1 reporter assays demonstrated that PKM2(K270M) increased HIF-1 transcriptional activity in HeLa cells, similar to WT PKM2 (Figure 3G).”

Knockdown of endogenous PKM2 by shRNA significantly reduces HIF-1 transcriptional activity in hypoxic HeLa cells.

“Conversely, knockdown of endogenous PKM2 expression by shRNA (shPKM2) significantly reduced HIF-1 transcriptional activity in hypoxic HeLa cells (Figure 3B).”

Double mutation of Pro-403/408 to alanine significantly reduces Pro-OH antibody binding by 50%.

“Double mutation of Pro-403/408 to alanine significantly reduced Pro-OH antibody binding by 50% (Figures 4E and S3B).”

PKM1-V5 is not hydroxylated.

“In contrast, PKM1-V5 was not hydroxylated (Figure 4C), again demonstrating the selectivity of the antibody.”

PKM2 interacts with HIF-1α within multiple domains.

“Remarkably, PKM2 interacts with HIF-1α within multiple domains.”

PHD3 knockdown inhibits PKM2 coactivator function.

“PHD3 knockdown inhibits PKM2 coactivator function, reduces glucose uptake and lactate production, and increases O2 consumption in cancer cells.”

PKM2-V5 physically interacted with p300 in HeLa cells.

“Co-IP assays demonstrated that PKM2-V5 physically interacted with p300 in HeLa cells (Figure 6E).”

Expression of PKM1 and PKM2, the alternative isoenzymes of the PKM2 gene, is controlled by HIF-1.

“In the present study, we demonstrate that PKM2 is a direct HIF-1 target gene and that expression of PKM1 and PKM2, the alternative isoenzymes of the PKM2 gene, is controlled by HIF-1.”

HIF-1α levels were not altered in PHD3 knockdown HeLa cells.

“HIF-1α levels were not altered in PHD3 knockdown HeLa cells (Figure S6C), indicating that reduced HIF-1 transactivation accounts for the inhibition of HIF-1 target gene expression in PHD3 knockdown cells.”

PHD3 overexpression in RCC4 cells enhances HIF-1α transactivation mediated by WT PKM2 but not PKM2(P403/408A).

“PHD3 overexpression in RCC4 cells enhanced HIF-1α transactivation mediated by WT PKM2 but not PKM2(P403/408A) (mutPKM2, Figure 5D).”

Similar results on glucose uptake, lactate production and OCR were observed in RCC4 cells transduced with lentivirus encoding a different shRNA, which completely knocked down PHD3 protein levels.

“Similar results on glucose uptake, lactate production and OCR were also observed in RCC4 cells transduced with lentivirus encoding a different shRNA (Figures S6F–H), which completely knocked down PHD3 protein levels (Figure S6I).”

PKM2 stimulates HIF-1/HIF-2-dependent VEGF gene expression in hypoxic HeLa cells.

“PKM2 also stimulates HIF-1/HIF-2-dependent VEGF gene expression in hypoxic HeLa cells, suggesting that through its function as a HIF-1 coactivator PKM2 may play a far broader role in promoting cancer progression than has been appreciated heretofore.”

PHD3 knockdown increases O2 consumption in cancer cells.

“PHD3 knockdown inhibits PKM2 coactivator function, reduces glucose uptake and lactate production, and increases O2 consumption in cancer cells.”

PKM2 knockdown had no effect on HIF-1α or HIF-1β protein levels in HeLa cells.

“PKM2 knockdown had no effect on HIF-1α or HIF-1β protein levels in HeLa cells (Figures 3D and S5D).”

shPKM2 significantly decreased expression of the glycolytic genes LDHA, PDK1, SLC2A1, HK1 and the angiogenesis gene VEGF in HeLa cells.

“qRT-PCR assays revealed that shPKM2 significantly decreased expression of the glycolytic genes LDHA, PDK1, SLC2A1, HK1 and the angiogenesis gene VEGF in HeLa cells, whereas expression of the non-HIF-1 target genes COX4I1, LDHB, and RPL13A was not affected (Figures 7A and S6A).”

Levels of H3K9-ac at the LDHA HRE were significantly decreased in hypoxic HeLa cells transfected with shPKM2 vector.

“Levels of histone H3 acetylated at lysine-9 (H3K9-ac) at the LDHA HRE were significantly decreased in hypoxic HeLa cells transfected with shPKM2 vector (Figure 6G), whereas PKM2 knockdown did not affect H3K9-ac levels at the RPL13A gene (Figure 6G) or total histone H3 occupancy at the LDHA or RPL13A gene (Figures S5H and S5I).”

PKM2 localizes in the nucleus.

“PKM2 localizes in the nucleus, is recruited with HIF-1 to HREs, and enhances HIF-1 occupancy, p300 recruitment, and H3K9 acetylation, thereby promoting transactivation of genes encoding glucose transporters and glycolytic enzymes in cancer cells (Figure 7G).”

PHD3 overexpression did not stimulate the interaction between PKM2(P403/408A)-V5 and FLAG-HIF-1α(DM).

“PHD3 overexpression enhanced the interaction of PKM2-V5 with FLAG-HIF-1α(DM) in transfected HeLa cells (Figure 5F), but did not stimulate the interaction between PKM2(P403/408A)-V5 and FLAG-HIF-1α(DM) (Figure S4D).”

PKM2 knockdown did not affect total histone H3 occupancy at the LDHA or RPL13A gene.

“Levels of histone H3 acetylated at lysine-9 (H3K9-ac) at the LDHA HRE were significantly decreased in hypoxic HeLa cells transfected with shPKM2 vector (Figure 6G), whereas PKM2 knockdown did not affect H3K9-ac levels at the RPL13A gene (Figure 6G) or total histone H3 occupancy at the LDHA or RPL13A gene (Figures S5H and S5I).”

Expression of COX4I1 and RPL13A was not affected by PHD3 knockdown.

“However, expression of COX4I1 and RPL13A, which are not regulated by HIF-1, was not affected by PHD3 knockdown (Figure 7C).”

p300 bound to the LDHA HRE in HeLa cells transfected with shSC vector.

“ChIP assays revealed that p300 bound to the LDHA HRE, and hypoxia increased p300 occupancy in HeLa cells transfected with shSC vector (Figure 6F).”

PKM2 enhances p300 recruitment.

“PKM2 localizes in the nucleus, is recruited with HIF-1 to HREs, and enhances HIF-1 occupancy, p300 recruitment, and H3K9 acetylation, thereby promoting transactivation of genes encoding glucose transporters and glycolytic enzymes in cancer cells (Figure 7G).”

Intracellular glucose and extracellular lactate levels were significantly decreased in RCC4 cells transduced with shPHD3 retrovirus, compared to cells transduced with shSC retrovirus.

“Consistent with altered expression of glucose transporters and glycolytic enzymes, intracellular glucose and extracellular lactate levels were significantly decreased in RCC4 cells transduced with shPHD3 retrovirus, compared to cells transduced with shSC retrovirus (Figures 7D and 7E).”

Double mutation of Pro-403/408 markedly reduces binding of PKM2-V5 to endogenous HIF-1α in hypoxic cells.

“Co-IP assays revealed that double mutation of Pro-403/408 markedly reduced the binding of PKM2-V5 to endogenous HIF-1α in hypoxic cells (Figure 4H).”

PKM2 is recruited with HIF-1 to HREs.

“PKM2 localizes in the nucleus, is recruited with HIF-1 to HREs, and enhances HIF-1 occupancy, p300 recruitment, and H3K9 acetylation, thereby promoting transactivation of genes encoding glucose transporters and glycolytic enzymes in cancer cells (Figure 7G).”

Prolyl hydroxylation of PKM2-V5 is inhibited by 50% in HeLa cells transduced with retrovirus encoding PHD3 shRNA.

“Immunoblot assays indicated that prolyl hydroxylation of PKM2-V5 was inhibited by 50% in HeLa cells transduced with retrovirus encoding PHD3 shRNA (shPHD3) (Figure 5C), which was similar to the effect of exposing cells to near-anoxic conditions (Figure 4D) or mutation of P403/408A (Figure 4E).”

PKM2 knockdown did not affect H3K9-ac levels at the RPL13A gene.

“Levels of histone H3 acetylated at lysine-9 (H3K9-ac) at the LDHA HRE were significantly decreased in hypoxic HeLa cells transfected with shPKM2 vector (Figure 6G), whereas PKM2 knockdown did not affect H3K9-ac levels at the RPL13A gene (Figure 6G) or total histone H3 occupancy at the LDHA or RPL13A gene (Figures S5H and S5I).”

PKM2-V5 binding to FLAG-HIF-1α(DM) is reduced by DMOG treatment of HeLa cells.

“PKM2-V5 binding to FLAG-HIF-1α(DM) was also reduced by DMOG treatment of HeLa cells (Figure 4J), thereby eliminating any potential effects of DMOG on prolyl hydroxylation of HIF-1α.”

PHD3 knockdown reduces lactate production in cancer cells.

“PHD3 knockdown inhibits PKM2 coactivator function, reduces glucose uptake and lactate production, and increases O2 consumption in cancer cells.”

PHD2 knockdown failed to affect prolyl hydroxylation of PKM2 in HeLa cells transduced with lentivirus encoding an shRNA that effectively reduced PHD2 protein levels.

“Although PHD2 co-immunoprecipitated with PKM2 (Figure S4F), PHD2 knockdown failed to affect prolyl hydroxylation of PKM2 in HeLa cells transduced with lentivirus encoding an shRNA that effectively reduced PHD2 protein levels (Figure 5C).”

PKM2 also binds strongly to the PAS domain of HIF-1α.

“Although PKM2 also binds strongly to the PAS domain of HIF-1α, it does not affect the two known biological functions of the PAS domain, HIF-1α/HIF-1β dimerization and HIF-1α protein stability.”

PHD3 knockdown reduces glucose uptake in cancer cells.

“PHD3 knockdown inhibits PKM2 coactivator function, reduces glucose uptake and lactate production, and increases O2 consumption in cancer cells.”

PKM2 knockdown significantly reduced p300 occupancy at the LDHA HRE in non-hypoxic and hypoxic HeLa cells.

“However, PKM2 knockdown significantly reduced p300 occupancy at the LDHA HRE in non-hypoxic and hypoxic HeLa cells (Figure 6F).”

LDHA, PDK1, and SLC2A1 gene expression was significantly decreased in HeLa cells transduced with shPHD3 retrovirus, compared to cells transduced with shSC retrovirus.

“To determine whether PHD3 activity stimulates expression of HIF-1 target genes, we performed qRT-PCR assays, which demonstrated that LDHA, PDK1, and SLC2A1 gene expression was significantly decreased in HeLa cells transduced with shPHD3 retrovirus, compared to cells transduced with shSC retrovirus (Figure 7C).”

FLAG-PHD2 overexpression had no effect on HIF-1α transactivation domain function.

“FLAG-PHD2 overexpression also had no effect on HIF-1α transactivation domain function (Figure S4G).”

DMOG largely inhibits prolyl hydroxylation of PKM2-V5.

“DMOG largely inhibited prolyl hydroxylation of PKM2-V5 (Figure 4I).”

PHD3 knockdown decreased PKM2-V5 interaction with endogenous HIF-1α in hypoxic HeLa cells.

“Conversely, PHD3 knockdown decreased PKM2-V5 interaction with endogenous HIF-1α in hypoxic HeLa cells (Figure 5G).”

HIF-1β occupancy at the PDK1 and LDHA genes, but not at the RPL13A gene, was significantly reduced by PKM2 knockdown in hypoxic HeLa cells.

“HIF-1β occupancy at the PDK1 and LDHA genes, but not at the RPL13A gene, was also significantly reduced by PKM2 knockdown in hypoxic HeLa cells (Figures 6B and S5B).”

PHD3, but not PHD3(H135A/D137A), also potentiates PKM2-mediated HIF-1α transactivation in HeLa cells.

“PHD3, but not PHD3(H135A/D137A), also potentiated PKM2-mediated HIF-1α transactivation in HeLa cells (Figure S4C).”

PKM2 occupancy at the HRE of HIF-1 target genes LDHA and PDK1, but not at the RPL13A gene, was detected in hypoxic HeLa cells.

“ChIP assays demonstrated PKM2 occupancy at the HRE of HIF-1 target genes LDHA and PDK1, but not at the RPL13A gene in hypoxic HeLa cells (Figures 6C and S5F).”

PKM2 is a direct HIF-1 target gene.

“In the present study, we demonstrate that PKM2 is a direct HIF-1 target gene and that expression of PKM1 and PKM2, the alternative isoenzymes of the PKM2 gene, is controlled by HIF-1.”

Hypoxia significantly increased HIF-1α occupancy at the LDHA and PDK1 HREs.

“Hypoxia significantly increased HIF-1α occupancy at the LDHA and PDK1 HREs.”

LDHA, PDK1, and GLUT1 mRNA and protein levels were reduced by PHD3 knockdown in RCC4 cells.

“LDHA, PDK1, and GLUT1 mRNA and protein levels were also reduced by PHD3 knockdown in RCC4 cells (Figures S6D and S6E).”

FLAG-PHD1 failed to interact with PKM2-V5.

“FLAG-PHD1 failed to interact with PKM2-V5 (Figure S4E).”

In non-hypoxic HeLa cells transfected with shSC vector, HIF-1α occupancy was detected at the HRE of HIF-1 target genes LDHA and PDK1, but not at the non-HIF-1 target gene RPL13A.

“ChIP assays demonstrated that, relative to IgG background signals, HIF-1α occupancy was detected at the HRE of HIF-1 target genes, including LDHA and PDK1, but not at the non-HIF-1 target gene RPL13A, in non-hypoxic HeLa cells transfected with shSC vector (Figures 6A and S5A).”

PHD3 overexpression enhanced the interaction of PKM2-V5 with FLAG-HIF-1α(DM) in transfected HeLa cells.

“PHD3 overexpression enhanced the interaction of PKM2-V5 with FLAG-HIF-1α(DM) in transfected HeLa cells (Figure 5F), but did not stimulate the interaction between PKM2(P403/408A)-V5 and FLAG-HIF-1α(DM) (Figure S4D).”

PHD3 occupancy at the HRE of LDHA and PDK1 genes was detected in hypoxic HeLa cells.

“PHD3 occupancy at the HRE of LDHA and PDK1 genes was also detected in hypoxic HeLa cells (Figures 6D and S5G).”

PKM2-V5 protein levels are not altered by shPHD3.

“PKM2-V5 protein levels were not altered by shPHD3 (Figure 5C).”

Hypoxia dramatically increases PHD3 levels.

“Hypoxia dramatically increased PHD3 levels, which compensated for the decreased hydroxylase activity at 1% O2 (Epstein et al., 2001), resulting in similar levels of prolyl hydroxylation of PKM2 in cells incubated at 20% and 1% O2 for 24 h (Figure 5C).”

• cites Epstein et al., 2001 sourceNot checked yet

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Hypoxia increased p300 occupancy at the LDHA HRE in HeLa cells transfected with shSC vector.

“ChIP assays revealed that p300 bound to the LDHA HRE, and hypoxia increased p300 occupancy in HeLa cells transfected with shSC vector (Figure 6F).”

PKM2 enhances HIF-1 occupancy.

“PKM2 localizes in the nucleus, is recruited with HIF-1 to HREs, and enhances HIF-1 occupancy, p300 recruitment, and H3K9 acetylation, thereby promoting transactivation of genes encoding glucose transporters and glycolytic enzymes in cancer cells (Figure 7G).”

PKM2 enhances H3K9 acetylation.

“PKM2 localizes in the nucleus, is recruited with HIF-1 to HREs, and enhances HIF-1 occupancy, p300 recruitment, and H3K9 acetylation, thereby promoting transactivation of genes encoding glucose transporters and glycolytic enzymes in cancer cells (Figure 7G).”

LDHA, PDK1, and GLUT1 protein levels were significantly reduced in HeLa cells transduced with shPKM2 retrovirus.

“LDHA, PDK1, and GLUT1 protein levels were also significantly reduced in HeLa cells transduced with shPKM2 retrovirus, which was comparable to the effect of treatment with digoxin (Figures 7B and S6B), a drug that inhibits HIF-1α synthesis (Zhang et al., 2008).”

PHD3 knockdown significantly reduced HIF-1α transactivation in hypoxic HeLa cells.

“PHD3 knockdown significantly reduced HIF-1α transactivation in hypoxic HeLa cells (Figure 5E).”

shPKM2 did not affect expression of the non-HIF-1 target genes COX4I1, LDHB, and RPL13A in HeLa cells.

“qRT-PCR assays revealed that shPKM2 significantly decreased expression of the glycolytic genes LDHA, PDK1, SLC2A1, HK1 and the angiogenesis gene VEGF in HeLa cells, whereas expression of the non-HIF-1 target genes COX4I1, LDHB, and RPL13A was not affected (Figures 7A and S6A).”

PKM1 does not regulate HIF-1 transcriptional activity.

“In contrast, PKM1 does not regulate HIF-1 transcriptional activity.”

In VHL-null RCC4 cells, PKM2 promotes HIF-1 transactivation of target genes that mediate increased glucose uptake (GLUT1), increased lactate production (LDHA), and decreased oxidative metabolism (PDK1).

“We have shown that in VHL-null RCC4 cells, PKM2 promotes HIF-1 transactivation of target genes that mediate increased glucose uptake (GLUT1), increased lactate production (LDHA), and decreased oxidative metabolism (PDK1).”

Hydroxylation of Pro-403 and Pro-408 in PKM2 was demonstrated by mass spectrometry.

“Hydroxylation of Pro-403 and Pro-408 in PKM2 was demonstrated by mass spectrometry.”

A putative PHD hydroxylation motif LRRLAP403 is within the unique exon 10 domain of PKM2.

“Here we identified a putative PHD hydroxylation motif LRRLAP403 within the unique exon 10 domain of PKM2.”

Double mutation of Pro-403/408 reduced PKM2 hydroxylation to a degree similar to knockdown of PHD3 or exposure to near-anoxic conditions.

“Double mutation of Pro-403/408 reduced PKM2 hydroxylation to a degree that was similar to knockdown of PHD3 or exposure to near-anoxic conditions.”

Several acidic residues are present on either side of Pro-403/408.

“The presence of acidic (Asp/Glu) residues near the Pro residue appears to be important for hydroxylation by PHD3 (Li et al., 2004; Xie et al., 2009) and several acidic residues are present on either side of Pro-403/408 (Figure S3A).”

PKM2 physically interacts with HIF-1α in the nuclei of hypoxic human cancer cells.

“These data demonstrate that PKM2 physically interacts with HIF-1α in the nuclei of hypoxic human cancer cells.”

• Also stated in: § Abstract · plain

  “PKM2 interacts directly with the HIF-1α subunit”

PKM2 also promotes HIF-2 transcriptional activity.

“Thus, PKM2 also promotes HIF-2 transcriptional activity.”

PKM2 directly stimulates HIF-1α transactivation domain function.

“Taken together, these data indicate that PKM2 directly stimulates HIF-1α transactivation domain function.”

• Also stated in: § PKM2 Regulates HIF-1 Transcriptional Activity · hedged

  “indicating that PKM2 specifically stimulates HIF-1α transactivation domain function.”

Stimulation of HIF-1α transactivation by PKM2 is independent of its catalytic activity as a glycolytic enzyme.

“We conclude that stimulation of HIF-1α transactivation by PKM2 is independent of its catalytic activity as a glycolytic enzyme.”

An effect on HIF-1α protein stability is ruled out as the cause of PKM2-stimulated HIF-1 transcriptional activity.

“These data rule out an effect on HIF-1α protein stability as the cause of PKM2-stimulated HIF-1 transcriptional activity.”

PKM2 promotes HIF-1 transcriptional activity in human cancer cells.

“These data indicate that PKM2 promotes HIF-1 transcriptional activity in human cancer cells.”

The anti-Pro-OH antibody is selective for prolyl hydroxylated proteins.

“These results validated the selectivity of the anti-Pro-OH antibody.”

Physical association between the HIF-1α transactivation domain and the alternatively spliced domain of PKM2 is critical for stimulation of HIF-1α transactivation.

“Taken together, these data indicate that PKM2, but not PKM1, stimulates HIF-1 transcriptional activity, and that physical association between the HIF-1α transactivation domain and the alternatively spliced domain of PKM2 is critical for stimulation of HIF-1α transactivation.”

PKM2 binds to multiple domains of HIF-1α in vitro.

“These data indicate that PKM2 binds to multiple domains of HIF-1α in vitro.”

PKM2 is hydroxylated at Pro-403/408 within the domain that is not present in PKM1.

“Together, these data indicate that PKM2 is hydroxylated at Pro-403/408 within the domain that is not present in PKM1.”

• Also stated in: § Abstract · boosted

  “Mass spectrometry and anti-hydroxyproline antibody assays demonstrate PKM2 hydroxylation on proline-403/408.”

PKM2 specifically enhances HIF-1α binding at the HRE of target genes.

“Knockdown of endogenous PKM2 significantly reduced HIF-1α occupancy at the LDHA and PDK1 HREs in hypoxic HeLa cells, but not at the RPL13A gene (Figures 6A and S5A), indicating that PKM2 specifically enhances HIF-1α binding at the HRE of target genes.”

PHD3 is a specific regulator of PKM2-stimulated HIF-1 transcriptional activity.

“Therefore, PHD3 is a specific regulator of PKM2-stimulated HIF-1 transcriptional activity.”

PKM2 promotes transactivation of genes encoding glucose transporters and glycolytic enzymes in cancer cells.

“PKM2 localizes in the nucleus, is recruited with HIF-1 to HREs, and enhances HIF-1 occupancy, p300 recruitment, and H3K9 acetylation, thereby promoting transactivation of genes encoding glucose transporters and glycolytic enzymes in cancer cells (Figure 7G).”

PKM2 specifically enhances p300 recruitment to the HRE of HIF-1 target genes.

“These data indicate that PKM2 specifically enhances p300 recruitment to the HRE of HIF-1 target genes and promotes H3K9 acetylation, which is an epigenetic modification that is associated with gene transcription.”

Inhibition of prolyl hydroxylase activity impairs HIF-1α transactivation.

“These results indicate that inhibition of prolyl hydroxylase activity impairs HIF-1α transactivation.”

Hydroxylation of PKM2 at Pro-403/408 potentiates PKM2-mediated HIF-1α transactivation.

“The data presented in Figure 5 strongly suggest that PHD3 induces hydroxylation of PKM2 at Pro-403/408, thereby increasing the interaction of PKM2 with HIF-1α and potentiating PKM2-mediated HIF-1α transactivation.”

Reduced HIF-1 transactivation accounts for the inhibition of HIF-1 target gene expression in PHD3 knockdown cells.

“HIF-1α levels were not altered in PHD3 knockdown HeLa cells (Figure S6C), indicating that reduced HIF-1 transactivation accounts for the inhibition of HIF-1 target gene expression in PHD3 knockdown cells.”

PKM2 promotes transactivation of HIF-1 target genes by enhancing HIF-1 binding and p300 recruitment to hypoxia response elements.

“PKM2 interacts directly with the HIF-1α subunit and promotes transactivation of HIF-1 target genes by enhancing HIF-1 binding and p300 recruitment to hypoxia response elements, whereas PKM1 fails to regulate HIF-1 activity.”

PKM2 promotes H3K9 acetylation.

“These data indicate that PKM2 specifically enhances p300 recruitment to the HRE of HIF-1 target genes and promotes H3K9 acetylation, which is an epigenetic modification that is associated with gene transcription.”

• AssumptionRests on an assumption: H3K9 acetylation is associated with gene transcription.

PKM2 stabilizes binding of HIF-1 to DNA.

“ChIP data indicate that PKM2 stabilizes binding of HIF-1 to DNA and further studies are required to determine whether this property is dependent upon interaction of PKM2 with the PAS domain of HIF-1α.”

PKM2 and PHD3 both co-localize with HIF-1α at the HRE of HIF-1 target genes.

“These data indicate that PKM2 and PHD3 both co-localize with HIF-1α at the HRE of HIF-1 target genes.”

PHD3 may mediate prolyl hydroxylation of PKM2.

“suggesting that PHD3 may mediate prolyl hydroxylation of PKM2.”

The lack of HIF-2α occupancy on HIF-1-selective target genes LDHA and PDK1 demonstrated the specificity of the assay.

“The lack of HIF-2α occupancy on HIF-1-selective target genes LDHA and PDK1 demonstrated the specificity of the assay (Figures 6A and S5A).”

PHD3 induces hydroxylation of PKM2 at Pro-403/408.

“The data presented in Figure 5 strongly suggest that PHD3 induces hydroxylation of PKM2 at Pro-403/408, thereby increasing the interaction of PKM2 with HIF-1α and potentiating PKM2-mediated HIF-1α transactivation.”

Hydroxylation of PKM2 at Pro-403/408 increases the interaction of PKM2 with HIF-1α.

“The data presented in Figure 5 strongly suggest that PHD3 induces hydroxylation of PKM2 at Pro-403/408, thereby increasing the interaction of PKM2 with HIF-1α and potentiating PKM2-mediated HIF-1α transactivation.”

PKM2 coactivates expression of HIF-1 target genes encoding glucose transporter and glycolytic enzymes.

“Thus, PKM2 coactivates expression of HIF-1 target genes encoding glucose transporter and glycolytic enzymes.”

PKM2 binding to the transactivation domain of HIF-1α stimulates HIF-1 transcriptional activity.

“PKM2 binding to the transactivation domain of HIF-1α stimulates HIF-1 transcriptional activity.”

The novel coactivator function of PKM2 provides a molecular mechanism by which it can act with HIF-1 to reprogram glucose metabolism in cancer cells.

“This novel coactivator function of PKM2 provides a molecular mechanism by which it can act with HIF-1 to reprogram glucose metabolism in cancer cells, thus answering a major question regarding the role of PKM2 in cancer progression (Figure 7H).”

• AssumptionRests on an assumption: FIH-1-mediated hydroxylation of the HIF-1α transactivation domain blocks recruitment of p300 and inhibits transactivation.

PKM2 is hydroxylated by PHD3.

“Nevertheless, experimental evidence presented above indicates that PKM2 is hydroxylated by PHD3.”

• AssumptionRests on an assumption: Anti-Pro-OH antibody binding measures PKM2 prolyl hydroxylation.

HIF-1α is also highly expressed in well-oxygenated cancer cells with loss of function of certain tumor suppressors, most notably VHL.

“However, HIF-1α is also highly expressed in well-oxygenated cancer cells with loss of function of certain tumor suppressors, most notably VHL (Melillo, 2007; Semenza, 2010).”

• cites Melillo, 2007 sourceNot checked yet

  Not checked in the free run.

• cites Semenza, 2010 sourceNot checked yet

  Not checked in the free run.

The similar degree of PKM2 hydroxylation at 20% vs 1% O2 for 24 h is due to the dramatic increase in PHD3 protein levels under hypoxic conditions, which compensates for the reduction in hydroxylase activity.

“The similarity in the degree of PKM2 hydroxylation (as measured by anti-Pro-OH antibody binding) in cells incubated at 20% vs 1% O2 for 24 h is due to the dramatic increase in PHD3 protein levels under hypoxic conditions (Figure 5C), which compensates for the reduction in hydroxylase activity.”

• AssumptionRests on an assumption: Anti-Pro-OH antibody binding measures PKM2 prolyl hydroxylation.