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Loss of the Mammalian APC/C Activator FZR1 Shortens G1 and Lengthens S Phase but Has Little Effect on Exit from Mitosis

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Loss of the Mammalian APC/C Activator FZR1 Shortens G1 and Lengthens S Phase but Has Little Effect on Exit from Mitosis 4208 Research Article Loss of the mammalian APC/C activator FZR1 shortens G1 and lengthens S phase but has little effect on exit from mitosis Reinhard Sigl1, Cornelia Wandke1, Veronika Rauch1, Jane Kirk2, Tim Hunt2 and Stephan Geley1,* 1Division of Molecular Pathophysiology, Biocenter, Innsbruck Medical University, Innsbruck, Austria 2Clare Hall Laboratories, Cancer Research UK, South Mimms, England, UK *Author for correspondence ([email protected]) Accepted 10 September 2009 Journal of Cell Science 122, 4208-4217 Published by The Company of Biologists 2009 doi:10.1242/jcs.054197 Summary The anaphase-promoting complex/cyclosome (APC/C) is knockdown of p53 in U2OS cells with inducible FZR1 siRNA essential for progression through mitosis. At anaphase onset, also failed to restore their proliferative capacity. Thus, the the APC/C requires the activator protein CDC20 to target proliferation defects are a direct consequence of the genetic securin and cyclin B1 for proteasome-dependent degradation, damage inflicted by loss of FZR1 function and are largely but then depends on the CDC20-related protein FZR1 (also independent of p53. In summary, mammalian FZR1 is not known as CDH1) to remain active until the onset of the next S required for the completion of mitosis, but is an important phase. To investigate the role of FZR1 in mammalian cells, we regulator of G1 phase and is required for efficient DNA used RNAi in human cell lines and conditional gene targeting replication in human and mouse somatic cells. in mouse embryonic fibroblasts. In neither case was FZR1 required for exit from mitosis, but in cells lacking FZR1, the Supplementary material available online at G1 phase was shortened and the S phase was prolonged. In http://jcs.biologists.org/cgi/content/full/122/22/4208/DC1 several normal and transformed human cell lines, loss of FZR1 function induced DNA-damage responses and impaired Key words: FZR1, APC/C, Mitosis, Proteolysis, Cyclin, Ubiquitin, proliferation independently of the p53 status. Constitutive p53, Replication, DNA damage Introduction al., 2005). In contrast to APC/C-CDC20, the FZR1-associated form Journal of Cell Science The anaphase-promoting complex/cyclosome (APC/C) is a large of the APC/C has a different substrate spectrum. In addition to D- multisubunit ubiquitin protein ligase that is essential for sister box-containing proteins, the APC/C-FZR1 also targets other proteins chromatid separation and exit from mitosis. Because these events with different degrons, such as the KEN box present in CDC20 are crucial for equational chromosome segregation, the activity of (Pfleger and Kirschner, 2000). The switch from CDC20- to FZR1- the APC/C is tightly controlled by several mechanisms, including activated APC/C occurs during anaphase B (Hagting et al., 2002) cyclin-dependent kinase (CDK)-dependent phosphorylation and the and it is believed that FZR1, by targeting Aurora kinase A and PLK1, binding of cell division control protein 20 (CDC20). Once activated, contributes significantly to APC/C-dependent proteolysis during exit the APC/C recognises its substrates via specific motifs, such as the from mitosis (Pines, 2006). destruction boxes (D-box) present in cyclin B1 and securin. Its After completion of mitosis, the APC/C remains active until the activity against these essential substrates is, however, constrained next S phase (Brandeis and Hunt, 1996) and several APC/C by the mitotic checkpoint to prevent premature sister chromatid substrates either remain or become unstable in G1 phase. Degradation separation and exit from mitosis, which can generate aneuploid of some of these substrates is important for maintenance of G1 phase progeny (reviewed by Peters, 2006). In contrast to the ubiquitylation and avoidance of premature entry into S phase. These include the of securin and cyclin B, which depends on the completion of mitotic cyclins, which continue to be translated after exit from chromosome bi-orientation, the APC/C-dependent degradation of mitosis, but are unstable in G1 phase (Almeida et al., 2005), and cyclin A occurs as soon as cells enter mitosis, despite the activity the F-box protein SKP2, which controls the abundance of the CDK of the mitotic checkpoint (den Elzen and Pines, 2001; Geley et al., inhibitor p27Kip1 (Wei et al., 2004). Recently, it has been shown that 2001). These differences are poorly understood, but recent evidence the tumour suppressor RB1 (pRB) cooperates with FZR1 in targeting points to significant differences in the molecular recognition of SKP2 for polyubiquitylation (Binne et al., 2007). Thus, in addition different substrates (Kimata et al., 2008; Matyskiela and Morgan, to its role in destabilising late mitotic substrates, FZR1 seems to 2009; Wolthuis et al., 2008). have an important role in controlling G1 phase. By maintaining the As cyclins are degraded, CDK activity declines and phosphatases APC/C activity during exit from mitosis and in early G1 phase, FZR1 convert the APC/C back to its hypophosphorylated interphase state, could also contribute to the establishment of pre-replicative which is activated by Fizzy-related (FZR1, also known as CDH1), complexes (pre-RCs) on origins of DNA replication by providing a a CDC20-related protein (Kramer et al., 1998). Similarly to CDC20, time window with low CDK activity (reviewed by Diffley, 2004). FZR1 contains seven WD40 repeats, which form a seven-bladed At the end of G1 phase, the APC/C is inactivated by FBXO5 propeller that is involved in substrate and APC/C binding (Kraft et (Emi1), which binds to and inactivates FZR1, allowing cyclin A to FZR1 function in mitosis and G1 phase 4209 accumulate and activate CDK2. Active CDK2, in turn, contributes conditional gene ablation in the mouse. In both systems, we found to the inactivation of the APC/C by inhibitory phosphorylation of that cells had no obvious problems in returning to interphase after FZR1 (Kramer et al., 2000). In addition, APC/C activity in G1 phase the end of mitosis. Cells lacking FZR1, however, had an unusually is self-limited by autoubiquitylation and degradation of UBCH10, short G1 phase and proliferated poorly. one of the E2 enzymes of APC/C (Rape and Kirschner, 2004). Knockdown of UBCH10 has recently been shown to shorten G1 Results phase by increasing cyclin A levels, suggesting that UBCH10 is Human FZR1 is not required for exit from mitosis but controls the essential E2 for FZR1-APC/C in G1 phase (Rape and Kirschner, the onset of DNA replication 2004; Walker et al., 2008). To study whether FZR1 is essential for exit from mitosis, we used FZR1 and its orthologues have been studied in several transient RNAi to knockdown FZR1 in human U2OS organisms and were found to be important for establishing or osteosarcoma cells, which reduced FZR1 levels to more than 95% maintaining G1 phase, but are dispensable for mitosis (Blanco et as determined by immunoblotting and densitometry scanning. Fig. al., 2000; Jacobs et al., 2002; Sudo et al., 2001; Visintin et al., 1A shows that knockdown of FZR1 stabilised several known FZR1 1997). By contrast, analysis of mouse embryonic fibroblasts substrates, such as hKid (KIF22), Aurora kinase A (AURKA) and (MEFs) derived from FZR1-deficient knockout mice has provided B (AURKB). However, CDC20 and PLK1 were only slightly evidence that FZR1 is involved in regulating exit from mitosis stabilised in FZR1 RNAi cells released from a nocodazole (noc) (Garcia-Higuera et al., 2008; Li et al., 2008), whereas FZR1 RNAi- block. Their overall levels in RNAi cells were higher and both based studies in human cells have been controversial (Floyd et proteins re-accumulated faster in late-G1 phase than in controls. al., 2008; Engelbert et al., 2008). In FZR1 RNAi cells, the early-G1 phase decline of CDC25A and To clarify the role of FZR1 during exit from mitosis, we used UBCH10 was prevented, but as cells progressed towards S phase, two experimental systems using RNAi in human cells and the protein levels of both proteins dropped. SKP2 declined in G1-S Journal of Cell Science Fig. 1. FZR1 RNAi in human cells does not affect exit from mitosis. (A)U2OS cells transfected with control and FZR1 siRNAs were synchronised in mitosis (M) and released for analysis of APC/C substrates by immunoblotting. FZR1 substrates are indicated by a, mitotic APC/C substrates by b, and G1-phase substrates by c. (B)Signals were scanned and plotted normalised to GAPDH values. (C)Length of mitosis determined by videomicroscopy of U2OS FZR1 RNAi or control cells. (D)U2OS cyclin B1-GFP cells were induced for 24 hours with 1g/ml dox, transfected with FZR1 siRNA for 24 hours and monitored by fluorescence microscopy. Arrows highlight a dividing cell, arrowheads G1-phase cells. Scale bar: 20m. Time is hours:minutes. (E)Stable inducible FZR1 RNAi cells were induced for 3 days using 1g/ml dox, arrested in mitosis, harvested, released and analysed for cyclin B1, FZR1 and GAPDH. (F)FZR1 RNAi cells treated with or without doxycyline (dox) were released from a mitotic block, stained for AURKB, - tubulin and DNA and evaluated for bi-nucleation. 4210 Journal of Cell Science 122 (22) phase independently of FZR1 levels (Fig. 1Ac,1B) but the overall amount of SKP2 was about ten times higher in FZR1 RNAi cells. In contrast to the above FZR1 substrates, the degradation of cyclin A and cyclin B1, geminin and securin was not affected in mitosis (Fig. 1B), but these proteins re-accumulated ~2 hours sooner in G1 phase in FZR1-knockdown cells (Fig. 1B). In summary, knockdown of FZR1 only affected the degradation of late mitotic and G1-phase APC/C substrates. But although late mitotic substrates, such as AURKA and PLK1 were stabilised, the length of mitosis was not significantly prolonged, as judged as the time from cell rounding to initiation of cytokinesis by live-cell phase-contrast time-lapse microscopy (Fig.
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