Growth suppression induced by the TRC8 hereditary kidney cancer gene is dependent upon JAB1/CSN5.
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.
Gemmill
Robert M
RM
Division of Medical Oncology, University of Colorado Health Sciences Center, 12801 E 17th Avenue, Aurora, CO 80010, USA. robert.gemmill@uchsc.edu
Lee
Jason P
JP
Chamovitz
Daniel A
DA
Segal
Daniel
D
Hooper
Joan E
JE
Drabkin
Harry A
HA
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D000075686:COP9 Signalosome Complex; D002292:Carcinoma, Renal Cell / Q000235:genetics* / Q000503:physiopathology; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics*; D004330:Drosophila; D029721:Drosophila Proteins / Q000096:biosynthesis* / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000503:physiopathology; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D010641:Phenotype; D017354:Point Mutation; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D015398:Signal Transduction; D015854:Up-Regulation; D015003:Yeasts
D048868:Adaptor Proteins, Signal Transducing; C000593220:CSN7 protein, Drosophila; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; C456159:TRC8 protein, Drosophila; D000075686:COP9 Signalosome Complex; D008666:Metalloendopeptidases; C468276:Rri1 protein, S cerevisiae