Transactivation of Schizosaccharomyces pombe cdt2+ stimulates a Pcu4–Ddb1–CSN ubiquitin ligase

Abstract

Cullin-4 forms a scaffold for multiple ubiquitin ligases. In Schizosaccharomyces pombe, the Cullin-4 homologue (Pcu4) physically associates with Ddb1 and the COP9 signalosome (CSN). One target of this complex is Spd1. Spd1 regulates ribonucleotide reductase (RNR) activity. Spd1 degradation during S phase, or following DNA damage of G2 cells, results in the nuclear export of the small RNR subunit. We demonstrate that Cdt2, an unstable WD40 protein, is a regulatory subunit of Pcu4-Ddb1-CSN ubiquitin ligase. cdt2 deletion stabilises Spd1 and prevents relocalisation of the small RNR subunit from the nucleus to the cytoplasm. cdt2 þ is periodically transcribed by the Cdc10/DSC1 transcription factor during S phase and transiently transcribed following DNA damage of G2 cells, corresponding to Spd1 degradation profiles. Cdt2 co-precipitates with Spd1, and Cdt2 overexpression results in constitutive Spd1 degradation. We propose that Cdt2 incorporation into the Pcu4-Ddb1-CSN complex prompts Spd1 targeting and subsequent degradation and that Cdt2 is a WD40 repeat adaptor protein for Cullin-4-based ubiquitin ligase.

DTL/CDT2 is essential for both CDT1 regulation and the early G2/M checkpoint

“…We favor the former model because there is strong evidence that the CUL4-DDB1 ligase plays multiple roles in the DNA damage response. We, and others, have shown that the DNA-damage-induced degradation of CDT1 or Spd1 is absolutely dependent on the human (CUL4-DDB1-DTL) or S. pombe (Pcu4-Ddb1-Cdt2) versions of the DTL-containing complex (Liu et al 2005;Higa et al 2006;Jin et al 2006). Additionally, CUL4-DDB1 ligases that include two other substrate recognition proteins, CSA or DDB2, are also modulated by DNA damage and play essential roles in the repair of ultraviolet (UV)-damaged DNA.…”

“…Given these facts, we hypothesized that DTL is required for CDT1 inhibition during S phase. This hypothesis was supported by the recent finding that Cdt2 (the S. pombe DTL homolog) is required for the Pcu4-Ddb1 E3 ubiquitin ligase (the S. pombe equivalent of CUL4-DDB1) to ubiquitinate an inhibitor of ribonucleotide reductase called Spd1 during S phase and after DNA damage (Liu et al 2005). Therefore, we hypothesized that DTL is a component of the CUL4-DDB1 complex required for CDT1 ubiquitination.…”

“…In S. pombe, IR exposure causes an increase in Cdt2 protein levels within 1 h (Liu et al 2005); therefore, we tested whether DNA damage increased the abundance of DTL in human cells or stimulated the interaction of DTL with the CUL4-DDB1 complex. Exposing HeLa cells to IR 1 h before the immunoprecipitations did not cause an appreciable change in DTL protein levels overall or in the extent of interaction between DTL and CUL4-DDB1 (Fig.…”

The COP9 signalosome-mediated deneddylation is stimulated by caspases during apoptosis

“…As it has been shown, CSN-mediated deneddylation prevents the assembly of a specific CRL [4]. In cooperation with the UPS the CSN participates in processes such as DNA repair [7], cell cycle [8], angiogenesis [9] and development [10][11][12]. However, its role in apoptosis is unknown.…”

A functional genome-wide genetic screening identifies new pathways controlling the G1/S transcriptional wave

“…21 Spd1 and Epe1 accumulate in cells lacking Ddb1, Cdt2 or with defective COP9, inducing defects in cell cycle and in heterochromatin silencing. Since the defects in cell cycle are largely rescued by deletion of the ribonucleotide reductase inhibitor, Spd1, 22,23 we hypothesized that these strains could have an increased basal activation of the DNA-synthesis checkpoint. One of the consequences of activating Cds1, the effector kinase of this checkpoint, is the phosphorylation of Yox1.…”