TRC8 encodes an ER-resident E3-ubiquitin ligase disrupted in a family with the (3;8)(p14.2;q24.1) translocation and hereditary kidney and thyroid tumors. TRC8 encodes a 664 amino acid protein with 10 transmembrane segments and a C-terminal RING-H2 domain, a feature common to many E3 ubiquitin ligases. The (3;8) translocation, which was one of the first genetic abnormalities identified in a familial form of cancer, disrupted TRC8 in its second transmembrane segment. The Drosophila homolog of TRC8 (DTrc8) inhibited growth when over-expressed in selected fly tissues yet provided an essential function since universal under-expression by RNAi was lethal. We have now extended these findings into mammalian cells utilizing a TET-on system in HEK293 cells. In vitro cell growth was strongly inhibited when wild type TRC8 was expressed in this system. However, several independent mutations in the RING-H2 domain blocked growth inhibitory activity. Cells expressing wild type (but not RING-mutant) TRC8 accumulated in G2/M. Tumor formation by HEK293 cells in nude mice was completely blocked by wild type TRC8 induction but not when the RING domain was mutated. Wild type TRC8 catalyzed in vitro ubiquitination reactions while RING-mutant proteins lost this function. In vitro analysis of multiple ubiquitin conjugating enzymes (E2s) indicated that TRC8 effectively utilized Ube2g2 (Ubc7) but not the closely related Ube2g1. Both E2s are implicated in ER-associated protein degradation (ERAD). Genetic screens for modifiers of the DTrc8 growth-inhibition phenotype in Drosophila identified several MPN-domain subunits of the COP9 signalosome and related subunits of the translation initiation factor eIF3. In vitro GST-pulldowns demonstrated strong interactions between DTrc8 and these eIF3 subunits. Ectopic DTrc8 expression yielded cuticular phenotypes consistent with Minute mutations that impair protein translation. Induction of human TRC8 in HEK293 cells suppressed polysome loading in general and selectively reduced ribosome loading of specific mRNAs. In summary, TRC8 is an E3 ubiquitin ligase whose tumor suppressor activity resides, in part, by modulating translation initiation, possibly in conjunction with the COP9 signalosome.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]