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. Remarkably, PKM2 interacts with HIF-1α within multiple domains. PKM2 binding to the transactivation domain of HIF-1α stimulates HIF-1 transcriptional activity. 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. 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 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 (). In contrast, PKM1 does not regulate HIF-1 transcriptional activity. Together, these findings delineate a molecular mechanism underlying the shift from oxidative to glycolytic metabolism that is associated with the expression of PKM2 in cancer 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.
HIF-1α and HIF-2α were the first PHD3 substrates identified (Epstein et al., 2001
), although PHD3 is less active in mediating HIF-1α hydroxylation than PHD2, which is the primary regulator of HIF-1α hydroxylation and proteasomal degradation in well-oxygenated cells (Berra et al., 2003
). PHD3 was subsequently shown to promote degradation of the β2
-adrenergic receptor (Xie et al., 2009
) and the transcription factor ATF-4 (Köditz et al., 2007
). Two Pro residues in the β2
-adrenergic receptor were hydroxylated by PHD3 (Xie et al., 2009
). Here we identified a putative PHD hydroxylation motif LRRLAP403
within the unique exon 10 domain of PKM2. Hydroxylation of Pro-403 and Pro-408 in PKM2 was demonstrated by mass spectrometry. 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
). 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. 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 (), which compensates for the reduction in hydroxylase activity. Quantitative mass spectrometric data comparing the prolyl hydroxylation of PKM2 in cells expressing or deficient in PHD3 cannot be obtained because the relevant PKM2 tryptic peptide is subject to multiple modifications in addition to prolyl hydroxylation (data not shown). Nevertheless, experimental evidence presented above indicates that PKM2 is hydroxylated by PHD3.
Intratumoral hypoxia is commonly found in aggressive solid cancers, leading to HIF-1α accumulation (Harris, 2002
; Semenza, 2003
). 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
). Although HIF-1α accumulates in VHL-null cells under aerobic conditions, FIH-1-mediated hydroxylation of the HIF-1α transactivation domain should block recruitment of p300 and inhibit transactivation. The interaction of PKM2 with HIF-1α and p300 may provide a mechanism to bypass negative regulation by FIH-1. 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
). 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 (). Our data on PKM2
and previous studies of the EGLN3
which encodes PHD3 (Pescador et al., 2005
), indicate that PKM2
are both direct HIF-1 target genes, resulting in a positive feedback loop that amplifies HIF-1 activity and may accelerate metabolic reprogramming and other critical aspects of cancer progression that are mediated by HIF-1 ().