Control of Multicellular Development by the Physically Interacting Deneddylases DEN1/DenA and COP9 Signalosome

Abstract

Deneddylases remove the ubiquitin-like protein Nedd8 from modified proteins. An increased deneddylase activity has been associated with various human cancers. In contrast, we show here that a mutant strain of the model fungus Aspergillus nidulans deficient in two deneddylases is viable but can only grow as a filament and is highly impaired for multicellular development. The DEN1/DenA and the COP9 signalosome (CSN) deneddylases physically interact in A. nidulans as well as in human cells, and CSN targets DEN1/DenA for protein degradation. Fungal development responds to light and requires both deneddylases for an appropriate light reaction. In contrast to CSN, which is necessary for sexual development, DEN1/DenA is required for asexual development. The CSN-DEN1/DenA interaction that affects DEN1/DenA protein levels presumably balances cellular deneddylase activity. A deneddylase disequilibrium impairs multicellular development and suggests that control of deneddylase activity is important for multicellular development.

DENEDDYLASE1 Deconjugates NEDD8 from Non-Cullin Protein Substrates in Arabidopsis thaliana

“…Although previous reports about a nonspecificity of the NEDD8 and ubiquitin conjugation machineries in certain experimental conditions had put into question the existence of non-cullin NEDD8 conjugates, our observation of an accumulation of such non-cullin NEDD8 conjugates in the den1 mutant clearly proves their existence. In that regard, our observation is comparable to similar observations with den1 mutants from A. nidulans and Drosophila where NEDD8 conjugate accumulations had also been reported but have remained biochemically unexplored (Chan et al, 2008;Christmann et al, 2013). Taken together, these observations clearly demonstrate that protein neddylation is a common posttranslational modification of many substrate proteins in the respective organisms.…”

“…Previous studies on DEN1 had indicated that this protein may also deneddylate cullins Christmann et al, 2013). In contrast, our den1 mutant and DEN1 protein analysis reveal that DEN1, although possessing cullin deneddylating activity in vitro, is not required for cullin deneddylation in planta, also not in the absence of the cullin deneddylating CSN subunit CSN5.…”

“…Regardless of the functional interplay between DEN1 and CSN5, which may be different between Arabidopsis, where we did not detect an interaction, and Aspergillus nidulans or mammalian cells, where such an interaction had been reported (Christmann et al, 2013), our data clearly indicate the presence of NEDD8-modified proteins other than cullins in planta. Although previous reports about a nonspecificity of the NEDD8 and ubiquitin conjugation machineries in certain experimental conditions had put into question the existence of non-cullin NEDD8 conjugates, our observation of an accumulation of such non-cullin NEDD8 conjugates in the den1 mutant clearly proves their existence.…”

“…The founding members of the DEN1 protein family were originally described as enzymes specific for the processing of NEDD8 precursors in mammalian systems, and subsequent studies had implicated DEN1 from animals, yeasts, and fungi also in the deneddylation of, e.g., cullins as bona fide NEDD8-conjugated proteins Wu et al, 2003;Reverter et al, 2005;Christmann et al, 2013). Our analysis of Arabidopsis DEN1 revealed that DEN1, although capable of processing the Arabidopsis NEDD8 precursors in vitro, is not solely responsible for precursor processing in planta since den1 loss-of-function mutants do not have any detectable defects in processing the Arabidopsis RUB precursors.…”

“…Since at least one report has so far described a physical interaction between DEN1 and CSN5 or the CSN complex (Christmann et al, 2013), we also examined a possible interaction between DEN1 and CSN5 in Arabidopsis. However, following immunoprecipitation of FLAG:DEN1 from plants, we did not detect CSN5 after probing DEN1 immunoprecipitates with a CSN5 antibody ( Figure 3E).…”