The Vhl E3 ubiquitin ligase complex regulates melanisation via sima, cnc and the copper import protein Ctr1A

Abstract

VHL encodes a tumour suppressor, which possesses E3 ubiquitin ligase activity in complex with EloC and Cul2. In tumour cells or in response to hypoxia, VHL activity is lost, causing accumulation of the transcription factor HIF-1alpha. In this study, we demonstrated that in Drosophila, Rpn9, a regulatory component of the 26 s proteasome, participates in the Vhl-induced proteasomal degradation of sima, the Drosophila orthologue of HIF-1alpha. Knockdown of Vhl induces increased melanisation in the adult fly thorax and concurrent decrease in pigmentation in the abdomen. Both these defects are rescued by knockdown of sima and partially by knockdown of cnc, which encodes the fly orthologue of the transcription factor Nrf2, the master regulator of oxidative stress response. We further show that sima overexpression and Rpn9 knockdown both result in post-translational down-regulation of the copper uptake transporter Ctr1A in the fly eye and that Ctr1A expression exacerbates Vhl knockdown defects in the thorax and rescues these defects in the abdomen. We conclude that Vhl negatively regulates both sima and cnc and that in the absence of Vhl, these transcription factors interact to regulate Ctr1A, copper uptake and consequently melanin formation. We propose a model whereby the co-regulatory relationship between sima and cnc flips between thorax and abdomen: in the thorax, sima is favoured leading to upregulation of Ctr1A; in the abdomen, cnc dominates, resulting in the post-translational downregulation of Ctr1A.

Keywords: Ctr1A copper transporter; HIF-1 / sima; Melanisation; Nrf2 / cnc; Rpn9; Vhl E3 ubiquitin ligase.