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The LIMD1 protein bridges an association between the prolyl hydroxylases and VHL to repress HIF-1 activity

Abstract

There are three prolyl hydroxylases (PHD1, 2 and 3) that regulate the hypoxia-inducible factors (HIFs), the master transcriptional regulators that respond to changes in intracellular O2 tension1,2. In high O2 tension (normoxia) the PHDs hydroxylate two conserved proline residues on HIF-1α, which leads to binding of the von Hippel–Lindau (VHL) tumour suppressor, the recognition component of a ubiquitin–ligase complex, initiating HIF-1α ubiquitylation and degradation3,4,5,6. However, it is not known whether PHDs and VHL act separately to exert their enzymatic activities on HIF-1α or as a multiprotein complex. Here we show that the tumour suppressor protein LIMD1 (LIM domain-containing protein) acts as a molecular scaffold, simultaneously binding the PHDs and VHL, thereby assembling a PHD–LIMD1–VHL protein complex and creating an enzymatic niche that enables efficient degradation of HIF-1α. Depletion of endogenous LIMD1 increases HIF-1α levels and transcriptional activity in both normoxia and hypoxia. Conversely, LIMD1 expression downregulates HIF-1 transcriptional activity in a manner depending on PHD and 26S proteasome activities. LIMD1 family member proteins Ajuba and WTIP also bind to VHL and PHDs 1 and 3, indicating that these LIM domain-containing proteins represent a previously unrecognized group of hypoxic regulators.

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Figure 1: The Ajuba/zyxin family members interact differentially with PHD1–3 and VHL.
Figure 2: LIMD1 is a negative regulator of HIF-1α levels and transcription activity.
Figure 3: LIMD1-induced ODD degradation is dependent on PHD, proteasomal activities and PHD2/VHL binding.
Figure 4: Depletion of endogenous LIMD1 induces expression of endogenous HIF-1-targeted genes.
Figure 5: Proposed model of LIMD1-mediated/dependent degradation of HIF-1α.

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Acknowledgements

We thank R. Layfield, T. Hagen, M. Cockman and S. Kristjansdottir for reagents and technical advice/assistance. K.S.B. is supported by a Biotechnology and Biological Sciences Research Council Doctorate Training Award. V.J. and D.E.F. were supported by funding from the Biotechnology and Biological Sciences Research Council (BB/F006470/1 and BB/I007571/1) awarded to T.V.S.

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D.E.F., K.S.B., V.J., T.M.W. and T.V.S. designed experiments and wrote the paper. M.M., S.C.K.W., D.C-T. and T.E.P. carried out experiments. Y.F., P.J.R., S.I., J.B. and G.D.L. provided reagents. P.J.R. and G.D.L. contributed to experimental design and writing and editing the paper. T.V.S. initiated and managed this investigation.

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Correspondence to Tyson V. Sharp.

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Foxler, D., Bridge, K., James, V. et al. The LIMD1 protein bridges an association between the prolyl hydroxylases and VHL to repress HIF-1 activity. Nat Cell Biol 14, 201–208 (2012). https://doi.org/10.1038/ncb2424

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