Hsp70 and CHIP Selectively Mediate Ubiquitination and Degradation of Hypoxia-inducible Factor (HIF)-1 alpha but Not HIF-2 alpha
ABSTRACT Hypoxia-inducible factors (HIFs) are transcription factors that mediate adaptive responses to reduced oxygen availability. HIF-alpha subunits are stabilized under conditions of acute hypoxia. However, prolonged hypoxia leads to decay of HIF-1alpha but not HIF-2alpha protein levels by unknown mechanisms. Here, we identify Hsp70 and CHIP (carboxyl terminus of Hsc70-interacting protein) as HIF-1alpha-interacting proteins. Hsp70, through recruiting the ubiquitin ligase CHIP, promotes the ubiquitination and proteasomal degradation of HIF-1alpha but not HIF-2alpha, thereby inhibiting HIF-1-dependent gene expression. Disruption of Hsp70-CHIP interaction blocks HIF-1alpha degradation mediated by Hsp70 and CHIP. Inhibition of Hsp70 or CHIP synthesis by RNA interference increases protein levels of HIF-1alpha but not HIF-2alpha and attenuates the decay of HIF-1alpha levels during prolonged hypoxia. Thus, Hsp70- and CHIP-dependent ubiquitination represents a molecular mechanism by which prolonged hypoxia selectively reduces the levels of HIF-1alpha but not HIF-2alpha protein.
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ABSTRACT: HIF-1α is degraded by oxygen-dependent mechanisms but stabilized in hypoxia to form transcriptional complex HIF-1, which transactivates genes promoting cancer hallmarks. However, how HIF-1α is specifically regulated in hypoxia is poorly understood. Here, we report that the histone methyltransferase SET9 promotes HIF-1α protein stability in hypoxia and enhances HIF-1 mediated glycolytic gene transcription, thereby playing an important role in mediating cancer cell adaptation and survival to hypoxic stress. Specifically, SET9 interacts with HIF-1α and promotes HIF-1α protein stability in hypoxia. Silencing SET9 by siRNA reduces HIF-1α protein stability in hypoxia, and attenuates the hypoxic induction of HIF-1 target genes mediating hypoxic glycolysis. Mechanistically, we find that SET9 is enriched at the hypoxia response elements (HRE) within promoters of the HIF-1-responsive glycolytic genes. Silencing SET9 reduces HIF-1α levels at these HREs in hypoxia, thereby attenuating HIF-1-mediated gene transcription. Further, silencing SET9 by siRNA reduces hypoxia-induced glycolysis and inhibits cell viability of hypoxic cancer cells. Our findings suggest that SET9 enriches at HRE sites of HIF-1 responsive glycolytic genes and stabilizes HIF-1α at these sites in hypoxia, thus establishes an epigenetic mechanism of the metabolic adaptation in hypoxic cancer cells.Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 01/2015; 1853(5). DOI:10.1016/j.bbamcr.2015.01.011 · 5.30 Impact Factor
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ABSTRACT: MCM proteins are components of a DNA helicase that plays an essential role in DNA replication and cell proliferation. However, MCM proteins are present in excess relative to origins of replication, suggesting they may serve other functions. Decreased proliferation is a fundamental physiological response to hypoxia in many cell types, and hypoxia-inducible factor 1 (HIF-1) has been implicated in this process. Here, we demonstrate that multiple MCM proteins bind directly to the HIF-1α subunit and synergistically inhibit HIF-1 transcriptional activity via distinct O(2)-dependent mechanisms. MCM3 inhibits transactivation domain function, whereas MCM7 enhances HIF-1α ubiquitination and proteasomal degradation. HIF-1 activity decreases when quiescent cells re-enter the cell cycle, and this effect is MCM dependent. Exposure to hypoxia leads to MCM2-7 downregulation in diverse cell types. These studies reveal a function of MCM proteins apart from their DNA helicase activity and establish a direct link between HIF-1 and the cell-cycle machinery.Molecular cell 06/2011; 42(5):700-12. DOI:10.1016/j.molcel.2011.03.029 · 14.46 Impact Factor
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ABSTRACT: The pyruvate kinase isoforms PKM1 and PKM2 are alternatively spliced products of the PKM2 gene. PKM2, but not PKM1, alters glucose metabolism in cancer cells and contributes to tumorigenesis by mechanisms that are not explained by its known biochemical activity. We show that PKM2 gene transcription is activated by hypoxia-inducible factor 1 (HIF-1). 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. Interaction of PKM2 with prolyl hydroxylase 3 (PHD3) enhances PKM2 binding to HIF-1α and PKM2 coactivator function. Mass spectrometry and anti-hydroxyproline antibody assays demonstrate PKM2 hydroxylation on proline-403/408. PHD3 knockdown inhibits PKM2 coactivator function, reduces glucose uptake and lactate production, and increases O(2) consumption in cancer cells. Thus, PKM2 participates in a positive feedback loop that promotes HIF-1 transactivation and reprograms glucose metabolism in cancer cells.Cell 05/2011; 145(5):732-44. DOI:10.1016/j.cell.2011.03.054 · 33.12 Impact Factor