Growth suppression induced by the TRC8 hereditary kidney cancer gene is dependent upon JAB1/CSN5
Robert M Gemmill1, Jason P Lee, Daniel A Chamovitz
1Division of Medical Oncology, University of Colorado Health Sciences Center, 12801 E 17th Avenue, Aurora, CO 80010, USA. robert.gemmill@uchsc.edu
Abstract:
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 (CSN5(3)), relieved growth suppression by DTrc8, whereas CSN5(1) (E160V) and CSN5(2) (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.
Insights
TRC8 overexpression inhibits growth by interacting with the COP9 signalosome subunit CSN5. Genetic analysis reveals CSN5 and CSN6 are crucial for this growth suppression, impacting signalosome levels.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- TRC8, an E3-ubiquitin ligase, is implicated in hereditary renal cell carcinoma (RCC).
- Previous studies showed Drosophila Trc8 (DTrc8) overexpression inhibits growth and interacts with CSN5.
- The precise mechanism linking DTrc8 growth suppression to CSN5 remained unclear.
Purpose of the Study:
- To elucidate the mechanistic link between DTrc8 overexpression and growth suppression via the COP9 signalosome (CSN).
- To investigate the role of specific CSN subunits and mutations in mediating DTrc8's effects.
- To explore genetic interactions between DTrc8 and other components of the CSN and proteasome.
Main Methods:
- Utilized Drosophila genetics to assess the impact of CSN5 mutations and haploinsufficiency on DTrc8-induced growth suppression.
- Employed yeast two-hybrid assays to quantify DTrc8-CSN5 interaction strength.
- Analyzed protein levels of CSN subunits and proteasome components using Western blotting and digitonin-based complex extraction.
Main Results:
- CSN5 haploinsufficiency and a specific CSN5 mutation (T100I) relieved DTrc8-mediated growth suppression.
- Interaction strength between DTrc8 and CSN5 correlated with observed phenotypes.
- DTrc8 overexpression increased CSN5 and CSN7 levels and the formation of the CSN holocomplex, but did not affect Cul-1 neddylation.
- CSN6 haploinsufficiency also restored growth, while proteasome subunit mutations had no effect.
Conclusions:
- DTrc8-induced growth suppression is dependent on CSN5 and CSN6.
- TRC8 appears to modulate CSN levels or compartmentalization rather than deneddylation activity.
- These findings provide mechanistic insights into TRC8 function and its link to hereditary RCC.
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