TRC8 encodes an E3-ubiquitin ligase disrupted in a family with hereditary renal cell carcinoma (RCC). We previously reported that Drosophila Trc8 (DTrc8) overexpression inhibits growth and that human and fly proteins interact with with the COP9 signalosome (CSN) subunit JAB1/CSN5. However, further mechanistic evidence linking DTrc8 growth suppression to CSN5 was lacking. Here, we show that haploinsufficiency of CSN5, or a T100I point mutation (CSN53), relieved growth suppression by DTrc8, whereas CSN51 (E160V) and CSN52 (G147D) mutations had no effect. The strength of yeast two-hybrid interactions between DTrc8 and CSN5 were in complete agreement with the observed phenotypes. DTrc8 overexpression resulted in elevated levels of CSN5 and CSN7, but had no effect on NEDD8-modified Cul-1. In contrast to CSN5, heterozygosity for CSN4null had no effect on the DTrc8 phenotype. We also looked for genetic interactions between DTrc8 and other MPN domain proteins in the CSN and 26S proteasome lid. CSN6 haploinsufficiency restored growth, whereas reduction of proteasome subunits RPN8 or RPN11 had no effect. DTrc8 expression increased the level of digitonin-extractable CSN complex, consistent with elevated levels of CSN5 and 7. Our genetic results confirm that DTrc8-induced growth suppression is CSN5 (and CSN6) dependent. While there was no obvious influence on CSN deneddylation activity, the increase in CSN subunits and holocomplex suggests that TRC8 modulates signalosome levels or compartmentalization.
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We thank Drs Steven Beckendorf and CT Chien for providing multiple fly strains, including the CSN52 missense mutation. Statistical analysis was performed by Drs Anna Baron and Chan Zeng of the University of Colorado Cancer Center Biostatistics Core. We also thank D Kent and C Korch for helpful discussions during the course of this work. These studies were supported by an NIH grant (CA76035) to HAD and RMG, and from the Israel Science Foundation to DAC and DS.
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Gemmill, R., Lee, J., Chamovitz, D. et al. Growth suppression induced by the TRC8 hereditary kidney cancer gene is dependent upon JAB1/CSN5. Oncogene 24, 3503–3511 (2005). https://doi.org/10.1038/sj.onc.1208509
- COP9 signalosome