Cop9/signalosome subunits and Pcu4 regulate ribonucleotide reductase by both checkpoint-dependent and -independent mechanisms

Abstract

The signalosome is implicated in regulating cullin-dependent ubiquitin ligases. We find that two signalosome subunits, Csn1 and Csn2, are required to regulate ribonucleotide reductase (RNR) through the degradation of a small protein, Spd1, that acts to anchor the small RNR subunit in the nucleus. Spd1 destruction correlates with the nuclear export of the small RNR subunit, which, in turn, correlates with a requirement for RNR in replication and repair. Spd1 degradation is promoted by two separate CSN-dependent mechanisms. During unperturbed S phase, Spd1 degradation is independent of checkpoint proteins. In irradiated G2 cells, Spd1 degradation requires the DNA damage checkpoint. The signalosome copurifies with Pcu4 (cullin 4). Pcu4, Csn1, and Csn2 promote the degradation of Spd1, identifying a new function for the signalosome as a regulator of Pcu4-containing E3 ubiquitin ligase. The COP9 signalosome (CSN) complex was originally identified as a negative regulator of photomorphogenesis in plants (for review, see Schwechheimer and Deng 2001). Subsequently, it was purified from human cell extracts during attempts to isolate the 19S regulatory lid complex of the proteosome (Seeger et al. 1998). The human signalosome consists of eight core subunits, each sharing significant homology with a corresponding subunit in the regulatory 19S lid complex of the proteosome . The purified CSN complex can cleave the ubiquitin-like Nedd8 protein from cullins Wee et al. 2002). Csn5 contains a putative metalloprotease motif that is presumed to mediate deneddylation activity (Cope et al. 2002). Cullins are subunits of E3 ubiquitin ligases (Feldman et al. 1997;Skowyra et al. 1997), and deneddylation of cullins decreases SCF E3 ubiquitin ligase activity (Osaka et al. 2000). SCF E3 complexes typically consist of a cullin, the Rbx1 RING domain protein that binds an E2 enzyme Skowyra et al. 1999), and an adapter protein, Skp1, that binds an F-box protein that determines the substrate specificity (Skowyra et al. 1997).In Arabidopsis, the signalosome is involved in the degradation of the two bZIP transcription factors (Hy5, HyH) that lie at the top of a transcriptional cascade required to induce ∼ 30% of Arabidopsis genes during photomorphogenesis (Holm et al. 2002). An E2-like protein, Cop10, and an E3 RING protein, Cop1, are also required to degrade Hy5 and HyH, which occurs when seedlings are germinated in the dark (Osterlund et al. 2000;Holm et al. 2002;Suzuki et al. 2002). The biochemical role of the signalosome is unknown, although a correlation with Cop1 nuclear localization (von Arnim et al. 1997) and the associations between the signalosome and E3 ubiquitin ligases Schwechheimer et al. 2002) suggest a regulatory role in ubiquitination that may be linked to subcellular localization (Chamovitz et al. 1996;Hellmann and Estelle 2002).A highly conserved signalosome complex was identified in the fission yeast Schizosaccharomyces pombe (Mundt et al. 1999) and subsequently shown to be required to remove the Nedd8 ubiquitin-like protein fr...

Nedd8 on cullin: building an expressway to protein destruction

“…However, UV treatment was found to cause the association between phosphorylated RNA polymerase II and the CSA-DDB1-CUL4A-ROC1/Rbx1-CSN complex. Consistent with these observations, a complex containing spCUL4/Pcu4, DDB1 homologue Ddb1 and CSN was isolated in fission yeast (Liu et al, 2003).…”

CSN1 inhibits c-Jun phosphorylation and down-regulates ectopic expression of JNK1

“…However, this cannot exclude the possibility that small amount of CSN1 disassociated from or unassociated with the complex may exist transiently under certain conditions. The finding that CSN1 and CSN2 can function independently of other CSNs in fission yeast is consistent with this speculation (Mundt et al, 1999;Liu et al, 2003). By native gel electrophoresis, Fukumoto et al, (2005) showed that CSN complexes are more dynamic and heterogeneous than previously believed.…”

IncreasedRrm2gene dosage reduces fragile site breakage and prolongs survival of ATR mutant mice

“…S. cerevisiae Dif1 or Schizosaccharomyces pombe Spd1 retain Rnr2 in the nucleus, preventing its interaction with cytoplasmic Rnr1. Like Crt1 or Sml1, deletion of spd1 or dif1 increases RNR activity and suppresses the lethality of checkpoint mutants (Liu et al 2003;Lee et al 2008;Wu and Huang 2008). Even though no clear orthologs for yeast RNR inhibitory proteins have been found in mammals, evidence suggests that the connection between ATR and RNR is conserved to some extent.…”

“…S. cerevisiae Dif1 or Schizosaccharomyces pombe Spd1 retain Rnr2 in the nucleus, preventing its interaction with cytoplasmic Rnr1. Like Crt1 or Sml1, deletion of spd1 or dif1 increases RNR activity and suppresses the lethality of checkpoint mutants (Liu et al 2003;Lee et al 2008;Wu and Huang 2008 (Bester et al 2011;Danilova et al 2014). Furthermore, CHK1 activation by topoisomerase inhibitors induces the expression of RRM2 through E2F-dependent transcription (Zhang et al 2009).…”