The SCFSkp2 Ubiquitin Ligase Complex Interacts with the Human Replication Licensing Factor Cdt1 and Regulates Cdt1 Degradation

Abstract

DNA replication initiation is tightly controlled so that each origin only fires once per cell cycle. Cell cycle-dependent Cdt1 degradation plays an essential role in DNA replication control, as overexpression of Cdt1 leads to re-replication. In this study, we investigated the mechanisms of Cdt1 degradation in mammalian cells. We showed that the F-box protein Skp2 specifically interacted with human Cdt1 in a phosphorylation-dependent manner. The SCF Skp2 complex ubiquitinated Cdt1 both in vivo and in vitro. Down-regulation of Skp2 or disruption of the interaction between Cdt1 and Skp2 resulted in a stabilization and accumulation of Cdt1. These results suggest that the SCF Skp2 -mediated ubiquitination pathway may play an important role in the cell cycle-dependent Cdt1 degradation in mammalian cells.In all eukaryotic cells, DNA replication is tightly controlled to ensure that initiation of replication occurs only once per cell cycle (1-3). The key to this regulation is the formation of prereplication complexes (pre-RCs) 1 by loading MCM proteins to replication origins (4, 5). Studies from yeast and Xenopus showed that Cdc6 (termed as Cdc18 in Schizosaccharomyces pombe) and Cdt1 are required for MCM proteins to associate with chromatin (6 -14). In fission yeast, the protein levels of both Cdc18 and Cdt1 are tightly controlled during the cell cycle and Cdc18 and Cdt1 only appear in G 1 , which is important to prevent re-formation of pre-RCs after DNA is replicated in S phase (15)(16)(17)(18). Cdc18 is targeted for ubiquitination-mediated degradation when cells enter S phase of the cell cycle (17).In mammalian cells, Cdc6 protein levels remain almost constant throughout G 1 , S, and G 2 phases (19 -24). After the onset of S phase, Cdc6 is phosphorylated and exported to the cytoplasm (20,23,25,26). In contrast to Cdc6, the protein level of Cdt1 fluctuates during the cell cycle (27). It accumulates in early G 1 phase of the cell cycle when replication is licensed and disappears at the onset of S phase. The correlation of Cdt1 accumulation and assembly of pre-RCs in early G 1 phase suggests that Cdt1 may play an important role in the control of replication licensing in mammalian cells. This idea is further supported by the observation that overexpression of Cdt1 promotes DNA re-replication in p53 deficient cells (28).It has been shown that in mammalian cells, while Cdt1 protein level varies during the cell cycle, the mRNA level of Cdt1 remains relatively constant at different cell cycle stages (27). In the presence of proteasome inhibitors, Cdt1 accumulates in S phase when it is normally absent. This suggests that proteasome-dependent degradation may regulate the Cdt1 level during the cell cycle in mammalian cells. However, the detailed mechanisms of Cdt1 degradation remain unknown.The ubiquitin-dependent proteolytic pathway plays an important role for protein degradation (29). The conjugation of polyubiquitin chains to substrates requires enzymes, E1 (a ubiquitin-activating enzyme), E2 (a ubiquitin-conjugating enzyme...

A spontaneous Cdt1 mutation in 129 mouse strains reveals a regulatory domain restraining replication licensing

“…We then directly tested the sensitivity of the Cdt1 PEST variants to SCF Skp2 -dependent degradation in vivo. As previously observed 14 , overexpression of SKP2 (which ectopically activates SCF Skp2 ) downregulated WT Cdt1 expression ( Supplementary Fig. S5B).…”

“…Nevertheless, we directly evaluated the possible implication of the PEST domain in the ubiquitinmediated degradation of Cdt1 by its known E3 ligases, SCF Skp2 , CRL4 Cdt2 and APC Cdh1 . SCF Skp2 has an important role in regulating Cdt1 levels, notably in S phase 14 . Efficient association of Cdt1 with this ligase requires the presence of a cyclin-binding motif (RxL) and a CDK phosphorylation site (T29) on Cdt1 (ref.…”

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“…The key event in suppressing relicensing of origins is the inactivation of the MCM loading factor Cdt1 through two mechanisms [57]. First, Cdt1 undergoes cell cycledependent proteolysis during S and G 2 [58]. Second, residual Cdt1 is inhibited by the binding of a small regulatory protein called geminin, which is expressed at high levels during the S, G 2 and M phases [59][60][61].…”

The Emerging Role of the COP9 Signalosome in Cancer

“…[10][11][12]. This function has been proposed to be mediated by the catalytic subunit CSN5 but requires the assembly of the entire CSN holocomplex (10)(11)(12)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). The deneddylation of Cullins is required for Cullin-mediated degradation of E3 substrates.…”