Cancer-Causing Mutations in a Novel Transcription-Dependent Nuclear Export Motif of VHL Abrogate Oxygen-Dependent Degradation of Hypoxia-Inducible Factor

Abstract

It is thought that degradation of nuclear proteins by the ubiquitylation system requires nuclear-cytoplasmic trafficking of E3 ubiquitin ligases. The von Hippel-Lindau (VHL) tumor suppressor protein is the substrate recognition component of a Cullin-2-containing E3 ubiquitin ligase that recruits hypoxia-inducible factor (HIF) for oxygen-dependent degradation. We demonstrated that VHL engages in nuclear-cytoplasmic trafficking that requires ongoing transcription to promote efficient HIF degradation. Here, we report the identification of a discreet motif, DXGX₂DX₂L, that directs transcription-dependent nuclear export of VHL and which is targeted by naturally occurring mutations associated with renal carcinoma and polycythemia in humans. The DXGX₂DX₂L motif is also found in other proteins, including poly(A)-binding protein 1, to direct its transcription-dependent nuclear export. We define DXGX₂DX₂L as TD-NEM (transcription-dependent nuclear export motif), since inhibition of transcription by actinomycin D or 5,6-dichlorobenzimidazole abrogates its nuclear export activity. Disease-causing mutations of key residues of TD-NEM restrain the ability of VHL to efficiently mediate oxygen-dependent degradation of HIF by altering its nuclear export dynamics without affecting interaction with its substrate. These results identify a novel nuclear export motif, further highlight the role of nuclear-cytoplasmic shuttling of E3 ligases in degradation of nuclear substrates, and provide evidence that disease-causing mutations can target subcellular trafficking.

SUPPLEMENTAL MATERIAL

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ACKNOWLEDGMENTS

This work is supported by a grant from the Canadian Institutes of Health Research to S.L. S.L. is the recipient of the National Cancer Institute of Canada Harold E. Johns Award. K.M. is a recipient of a Canadian Institutes of Health Research fellowship.