SUMMARY
Cell fate decision upon prolonged mitotic arrest induced by microtubule targeting agents depends on the activity of the tumor suppressor and F-box protein FBXW7. FBXW7 promotes mitotic cell death and prevents premature escape from mitosis through mitotic slippage. Mitotic slippage is a process that can cause chemoresistance and tumor relapse. Therefore, understanding the mechanisms that regulate the balance between mitotic cell death and mitotic slippage is an important task. Here we report that FBXW7 protein levels markedly decline during extended mitotic arrest. FBXO45 binds to a conserved acidic N-terminal motif of FBXW7 specifically under a prolonged delay in mitosis, leading to ubiquitylation and subsequent proteasomal degradation of FBXW7 by the FBXO45-MYCBP2 E3 ubiquitin ligase. Moreover, we find that FBXO45-MYCBP2 counteracts FBXW7 in that it promotes mitotic slippage and prevents cell death in mitosis. Targeting this interaction represents a promising strategy to prevent chemotherapy resistance.
INTRODUCTION
The attachment of a single ubiquitin molecule or a polyubiquitin chain to a eukaryotic protein is an essential signaling event that can profoundly affect the fate of the target protein. For example, the ubiquitin-proteasome system controls protein degradation in a broad array of cellular processes (Hershko and Ciechanover, 1998). Cancer cells often contain mutations targeting ubiquitin-mediated proteolysis that lead to tumorigenesis. FBXW7, an F-box protein and substrate receptor for the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, acts as a tumor suppressor and is mutated or deleted in a variety of human cancers (Davis et al., 2014). FBXW7 exerts its anti-tumor activity through destruction of key oncoproteins, including JUN (Nateri et al., 2004) (Wei et al., 2005), MYC (Welcker et al., 2004) (Yada et al., 2004), Cyclin E (Koepp et al., 2001) (Moberg et al., 2001) (Strohmaier et al., 2001) and Notch1 (Hubbard et al., 1997) (Gupta-Rossi et al., 2001).
The abundance of F-box proteins including FBXW7 is controlled by ubiquitylation and degradation in an autocatalytic reaction within the SCF complex (Galan and Peter, 1999). FBXW7 autoubiquitylation can be regulated by Glomulin (GLMN), a protein that binds to RBX1 leading to inhibition of SCF activity, thereby increasing FBXW7 protein levels (Duda et al., 2012) (Tron et al., 2012). The only ubiquitin ligase known to date involved in regulating FBXW7 levels in neurons is Parkin. However, this regulation has only been observed for the FBXW7β isoform (Ekholm-Reed et al., 2013). It has also been shown that PLK2-kinase dependent phosphorylation of FBXW7 at S176 leads to its ubiquitin-mediated degradation and stabilization of Cyclin E (Cizmecioglu et al., 2012).
Accurate spindle function is crucial for a successful mitosis. Perturbation of microtubule dynamics leads to sustained activation of the spindle assembly checkpoint (SAC) (Rieder and Maiato, 2004). The SAC delays exit from mitosis by preventing the anaphase-promoting complex/cyclosome (APC/C) mediated proteolysis of Cyclin B1. Anti-cancer chemotherapeutics including vinca alkaloids or taxanes target microtubules and are successfully used in the clinics to treat multiple types of cancer. Anti-microtubule drugs cause an arrest or prolonged delay in mitosis followed by mitotic cell death. On the other hand, slow degradation of Cyclin B1 during a prolonged mitotic arrest can cause cells to prematurely exit from mitosis, a process called mitotic slippage (Brito and Rieder, 2006). During mitotic slippage, cells do not undergo proper chromosome segregation and cytokinesis. Most of the resulting tetraploid cells either undergo cell death after mitosis or arrest in interphase. However, depending on the p53 status of these cells, they may continue to proliferate as genomically unstable cells. This can lead to chemoresistance, thereby limiting the therapeutic use of anti-microtubule drugs (Frederiks et al., 2015) (Haschka et al., 2018). FBXW7 is a known regulator of mitotic cell fate. Upon mitotic arrest, FBXW7 promotes mitotic cell death and prevents mitotic slippage (Finkin et al., 2008) (Wertz et al., 2011). FBXO45 (FSN-1 in C. elegans; DFsn in Drosophila melanogaster) is an evolutionary conserved F-box protein. FBXO45 is atypical in that it binds to SKP1 via its F-box motif, but recruits an alternate RING-finger protein known as MYCBP2, a member of the PHR (PAM/Highwire/RPM-1) protein family and E3 ubiquitin ligase. MYCBP2 was shown to have important functions in developmental processes, such as axon termination and synapse formation, as well as axon degeneration (reviewed by (Grill et al., 2016) (Po et al., 2010)). FBXO45 contains a conserved F-box domain and a SPRY domain, which recruits substrates to the ubiquitin ligase complex (Chen et al., 2014) (Kugler et al., 2010). FBXO45-MYCBP2 has been linked to the proteasomal degradation of a few targets including the DLK-1 and p38 MAP kinase pathway (Nakata et al., 2005) and the nicotinamide-nucleotide adenylyltransferase family member NMNAT (Xiong et al., 2012).
Here we report the identification of the E3 ligase FBXO45-MYCBP2 as a regulator of FBXW7 abundance during prolonged mitotic arrest induced by spindle poisons. FBXO45 binds to a conserved acidic N-terminal region of FBXW7. Depletion of FBXO45 leads to stabilization of FBXW7 protein upon mitotic arrest. Ubiquitylation of FBXW7 by FBXO45-MYCBP2 induces its proteasomal degradation inducing an increase in mitotic slippage and prevention of mitotic cell death.
RESULTS
The FBXO45-MYCBP2 complex binds to a conserved N-terminal motif in FBXW7α
The SCF-FBXW7 complex acts as a key factor determining sensitivity to antimitotic drugs in cancer by inducing mitotic cell death and preventing mitotic slippage (Allan et al., 2018) (Wertz et al., 2011) (Finkin et al., 2008). As mitotic slippage frequently occurs under prolonged mitotic arrest, it is conceivable that this could be induced by a decrease in FBXW7 protein levels. To address this, we analyzed protein levels of the ubiquitously expressed α-isoform of FBXW7 in mitotic HeLa cells at different time points after induction of mitotic arrest. We observed a slow but gradual decrease in the amount of FBXW7 protein during prolonged mitotic arrest (Figure 1A) similar to the decay of Cyclin B1 and MCL-1 under mitotic arrest (Brito and Rieder, 2006) (Harley et al., 2010), which was prevented by the addition of the proteasomal inhibitor MG132 (Figure S1A). We therefore anticipated that FBXW7 protein levels could be decreased in response to proteasomal protein degradation. To identify proteins that regulate SCF-FBXW7 protein levels we made use of an unbiased screen where we aimed at identifying FBXW7 binding proteins. We expressed Flag-FBXW7α in HEK-293T cells, FBXW7α complexes were immunoprecipitated and subsequent mass spectrometrical analysis was performed. Among the identified peptides were sequences corresponding to FBXO45 and MYCBP2, along with SCF components and known substrates of FBXW7 including MYC, Notch1/2 and members of the mediator complex (Table S1). Interestingly, FBXO45 and MYCBP2 have previously been identified in other screens for FBXW7 interacting proteins (Kourtis et al., 2015) (Huttlin et al., 2017). They have also been described to form an SCF-like E3 ubiquitin ligase complex (Liao et al., 2004).
We then analyzed whether FBXO45-MYCBP2 is the ubiquitin ligase that regulates FBXW7 protein levels under prolonged mitotic arrest. First, we aimed to confirm the binding between FBXW7 and FBXO45. We therefore expressed Flag-FBXO45 in HEK-293T cells and immunoprecipitated the protein with α-Flag-resin. We found that Flag-FBXO45 was able to co-immunoprecipitate endogenous FBXW7α (Figure 1B). FBXW7 occurs in three isoforms, FBXW7α, FBXW7β and FBXW7γ, that differ in subcellular localization and their N-terminal domain. FBXW7α is thought to perform most FBXW7 functions (Davis et al., 2014). To identify the binding site of FBXO45 within FBXW7 we generated different FBXW7α truncated versions and found that aa106-126 in the N-terminal domain of FBXW7α were required for FBXO45 binding (Figure S1B-D). Co-immunoprecipitation experiments using full-length Flag-FBXW7α or a Flag-FBXW7αΔ106-126 mutant showed that FBXW7αΔ106-126 failed to bind FBXO45 and MYCBP2 (Figure 1C). This could be confirmed by the fact that FBXO45 only interacted with FBXW7α but not with the other two isoforms, FBXW7β or FBXW7γ (Figure S1E), that lack the FBXW7α-specific N-terminal domain (Welcker and Clurman, 2008). Interestingly, the N-terminal stretch within the FBXW7α isoform (aa106-126) harbors conserved acidic amino acid residues as a potential FBXO45 interaction domain (Figure 1D). On other the hand, we identified the central domain of MYCBP2 (aa1951-2950) to be responsible for FBXW7α binding (Figure S1F-G). As this domain also contains the FBXO45 interaction site of MYCBP2, we hypothesized that FBXO45 could be the direct interaction partner of FBXW7α within the FBXO45-MYCBP2 complex. Analysis of binding between purified MBP-tagged N-terminal domain of FBXW7α (MBP-FBXW7α-N167) and in vitro translated [35S]-FBXO45 or [35S]-MYCBP2(1951-2950) showed that FBXW7α directly binds to FBXO45 but not to MYCBP2 (Figure 1E). Furthermore, using sequential co-immunoprecipitation experiments we showed that FBXW7α exists in a complex with FBXO45 and MYCBP2 (Figure 1F). Together, these results show that the FBXO45-MYCBP2 complex interacts with a conserved acidic stretch within the N-terminal domain of FBXW7α.
FBXO45-MYCBP2 targets FBXW7α for degradation specifically during mitotic arrest
As FBXW7α is predominantly found in the nucleus (Kimura et al., 2003) whereas FBXO45 and MYCBP2 are cytosolic proteins (Chen et al., 2014) (Pierre et al., 2004) (Figure 2A), a possible interaction between these proteins may occur in mitosis upon nuclear envelope breakdown, when the contents of the cell nucleus are released into the cytoplasm. As FBXO45 is likely to be the substrate binding factor we analyzed the interaction between Flag-FBXO45 and endogenous FBXW7α in asynchronous and mitotic HeLa cells. The cell extracts for this experiment were generated under conditions where the nucleus remains intact. Interestingly, FBXW7α was specifically found in co-immunoprecipitation with Flag-FBXO45 in mitotic cells (Figure 2B). Furthermore, we noticed an increase in FBXW7α protein levels upon siRNA-mediated downregulation of FBXO45. This effect could only be observed in cells under a prolonged mitotic arrest but not in cells passing through an unperturbed mitosis. In addition, the effect was not observed in asynchronous cells (Figure 2C, Figure S2A-B). On the other hand, FBXW7 downregulation had no effect on FBXO45 protein levels (Figure 2C). To exclude off-target effects we used different FBXO45 and MYCBP2 siRNAs to reduce their expression in HeLa cells and confirmed that FBXW7α protein levels were upregulated in cells that were FBXO45 or MYCBP2-depleted under prolonged mitotic arrest (Figure S2C). The effect could be confirmed in U2OS cells using nocodazole and in HeLa cells using different inhibitors that cause a mitotic arrest (Figure S2D-I). We also aimed to confirm the observed regulation of FBXW7α protein levels by expression of a dominant-negative MYCBP2(1951-2950) fragment that binds FBXO45 and FBXW7α but lacks the RING domain (Figure S1F-G). Upon ectopical expression of Flag-MYCBP2(1951-2950) we found that FBXW7α protein levels were specifically upregulated in mitotic cells after treatment with nocodazole (Figure 2D). Taken together, FBXO45 binds FBXW7α in mitotic cells and the FBXO45-MYCBP2 complex regulates FBXW7α protein levels specifically during mitotic arrest.
FBXO45-MYCBP2 targets FBXW7α for ubiquitylation and degradation
To find out whether the regulation of FBXW7α by FBXO45-MYCBP2 is mediated by ubiquitylation we carried out in vivo ubiquitylation assays. We found that both overexpression of FBXO45 and MYCBP2 promote ubiquitylation of FBXW7α (Figure 3A). Deletion of the FBXO45 binding site in FBXW7α reduced the ubiquitylation of FBXW7α markedly in in vivo ubiquitylation assays using FBXW7αΔ106-126 expression (Figure 3B). To study the effect of FBXO45 on FBXW7α protein stability, we depleted FBXO45 in nocodazole-treated cells and added cycloheximide to block translation. Cells were then harvested at different time points after cycloheximide addition. In FBXO45-depleted cells FBXW7α was markedly stabilized (Figure 3C) suggesting that FBXO45 promotes degradation of FBXW7α upon mitotic arrest. In addition, Flag-FBXW7αΔ106-126 was stabilized in comparison to full-length Flag-FBXW7α. The stability of Flag-FBXW7α was increased by MG132 treatment (Figure 3D). From these experiments, we conclude that FBXO45-MYCBP2 mediated degradation of FBXW7α during prolonged mitosis depends on the proteasome.
FBXO45-MYCBP2 reduces the sensitivity of cells to spindle poisons
Previous studies have shown that FBXW7 regulates mitotic cell fate. In fact, FBXW7 promotes mitotic cell death and prevents mitotic slippage in cells that have been treated with anti-microtubule drugs to arrest them in mitosis (Finkin et al., 2008) (Wertz et al., 2011), which is in line with its function as a tumor suppressor. As we had found that FBXO45-MYCBP2 regulates FBXW7α protein levels during mitotic arrest (Figure 2), we asked whether the FBXO45-MYCBP2 complex as a regulator of FBXW7 would also affect mitotic cell fate. To test this, we analyzed mitotic cell fate by live-cell imaging (Figure S3A, Video S1-2). For our live-cell imaging analysis, we treated the cells with spindle poisons at concentrations that completely prevented cell division and caused either mitotic cell death or mitotic slippage (Brito et al., 2008). As shown in Figure 4A, reduction of FBXO45 led to an increase in mitotic cell death in cells that were arrested in mitosis with nocodazole. Similar effects were observed in cells arrested in mitosis by Taxol or vincristine treatment (Figure S3B-C). To exclude off-target effects we used siRNAs targeting different regions of FBXO45 mRNA (Figure 4A). In addition, expression of an siRNA-resistant version of Flag-FBXO45 was able to rescue the effect on mitotic cell fate (Figure 4B). Moreover, siRNA-mediated downregulation of MYCBP2 also caused an increase in mitotic cell death (Figure S3D). In contrast, FBXO45 and MYCBP2 depletion did not have an effect on progression of cells through an unperturbed mitosis (Figure S3E). To verify the results in an siRNA independent approach, we analyzed the effect of the dominant-negative MYCBP2(1951-2950) fragment on mitotic cell fate. As expected, overexpression of Flag-MYCBP2(1951-2950) increased mitotic cell death (Figure 4C). In addition, we performed a FACS analysis of the DNA content in cells arrested in mitosis. Mitotic slippage leads to the formation of tetraploid cells, which is reflected by an increase in the DNA content. FBXO45 depletion reduced the increase in DNA content, which confirms the negative effect of FBXO45 downregulation on mitotic slippage (Figure 4D, Figure S3F). Taken together, these results showed that the FBXO45-MYCBP2 complex negatively regulates mitotic cell death and increases mitotic slippage, suggesting that it has an opposing function during mitotic arrest compared to FBXW7. This is in line with the FBXO45-MYCBP2 complex being a negative regulator of FBXW7α during mitotic arrest. We therefore asked whether the observed effect of FBXO45-MYCBP2 on mitotic cell fate depends on FBXW7. In fact, FBXW7 depletion was able to rescue the effect of FBXO45, suggesting that FBXW7α is the downstream target of the FBXO45-MYCBP2 complex that mediates mitotic cell fate regulation (Figure 4E). Together, our data strongly suggest that FBXO45-MYCBP2-mediated degradation of FBXW7 under prolonged mitotic arrest induces mitotic slippage and prevents mitotic cell death.
DISCUSSION
The results presented here demonstrate that the FBXO45-MYCBP2 complex is a novel E3 ubiquitin ligase regulating the protein levels of FBXW7α during prolonged mitotic arrest induced by anti-microtubule drugs through ubiquitin mediated degradation. This degradation leads to a reduction of the sensitivity to microtubule-targeting drugs by causing a decrease in mitotic cell death and promoting mitotic slippage.
Prolonged mitotic arrest is caused by a sustained activation of the spindle assembly checkpoint (SAC). If the SAC cannot be satisfied and mitosis cannot be completed, there are two alternative cell fates. Cells either undergo mitotic cell death or perform mitotic slippage. Cell fate following mitotic arrest is regulated by two competing networks, namely Cyclin B1 degradation and pro-apoptotic caspase activation (Gascoigne and Taylor, 2008). Mitotic slippage is promoted by a slow degradation of Cyclin B1 in the presence of an active SAC during a protracted arrest in metaphase (Brito and Rieder, 2006). Cells undergo mitotic slippage and enter interphase without performing proper chromosome segregation or cytokinesis. The anaphase-promoting complex (APC/C) ubiquitin ligase is known to mediate the degradation of Cyclin B1, but recent data show that an additional E3 ubiquitin ligase, CRL2ZYG11, targets Cyclin B1 for degradation in a pathway that facilitates mitotic slippage (Balachandran et al., 2016). Besides cell fate regulation through Cyclin B1, MCL-1, a pro-survival member of the BCL-2 family, was shown to be involved in cell fate decision. MCL-1 is degraded by a ubiquitin-proteasome-dependent mechanism in response to the disruption of mitosis, resulting in cell death (Gascoigne and Taylor, 2008) (Harley et al., 2010) (Wertz et al., 2011).
The tumor suppressor FBXW7 also plays important roles in mitotic cell fate decision (Wertz et al., 2011) (Allan et al., 2018) (Finkin et al., 2008). However, whether or not FBXW7 has a direct role in regulating MCL-1 levels is a matter of debate (Wertz et al., 2011) (Sloss et al., 2016) (Allan et al., 2018) and therefore the identification of the substrate(s) of FBXW7 involved in regulating mitotic cell fate is an important task for future studies.
Our work identifies FBXO45 as a protein binding to a conserved acidic amino acid motif (aa106-126) of FBXW7α (Figure 1) to form a ternary complex with MYCBP2. Interestingly, MYCBP2 has been recently uncovered as a novel type of E3 ligase with esterification activity leading to ubiquitylation of serine and threonine residues in substrates (Pao et al., 2018), which is intrinsic to higher eukaryotes. In the future, it would therefore be interesting to see whether FBXW7 could be ubiquitylated on hydroxyl groups by MYCBP2.
An interesting question that arises is why the regulation of FBXW7α by FBXO45-MYCBP2 can only be observed under a prolonged mitotic arrest but not in cells undergoing a normal mitosis (Figure 2C, Figure S2A-B, Figure S3E). As FBXW7α is a nuclear protein but FBXO45 is found in the cytoplasm (Figure 2A), the two proteins can only interact upon nuclear envelope breakdown (Figure 2B). However, since mitosis is a relatively short process in the eukaryotic cell cycle, the successful formation of a sustained interaction between FBXO45 and FBXW7α may require a longer arrest in mitosis.
In the future, the development of a specific inhibitor to block the FBXO45-MYCBP2-FBXW7α axis might actively promote mitotic cell death and prevent mitotic slippage. Therefore, treatment of cancer patients with this inhibitor after or in combination with anti-microtubule drugs could be a promising approach in order to increase the efficiency of chemotherapeutic treatment.
MATERIALS AND METHODS
Cell culture and transfection
Human cell lines were grown in a humidified atmosphere with 5% CO2 at 37°C. HeLa, U2OS and U2OS Flp-In-T-Rex cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) with 1 g/l glucose. HEK-293T cells were grown in DMEM with 4.5 g/l glucose. The media were supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich or Thermo Scientific). HeLa GFP-α-tubulin/RFP-H2B cells (D. Gerlich, ETH Zürich) were cultured in DMEM containing 1 g/l glucose, 10% FBS, 500 μg/ml G418 and 0.5 μg/ml puromycin. The T-Rex system was used to generate U2OS Flp-In-T-Rex Flag-FBXW7α, U2OS Flp-In-T-Rex Flag-FBXW7αΔ106-126 and U2OS Flp-In-T-Rex Flag-MYCBP2(1951-2950) stable cell lines according to the manufacturer’s instructions (Life Technologies).
Cell culture work was performed under sterile conditions. Cell line authentication was regularly performed by Multiplexion in Heidelberg. HEK-293T and HeLa cells were transiently transfected with plasmid DNA using polyethylenimine (PEI, Polysciences) at a final concentration of 5 μg/ml. The cells were incubated at 37°C and harvested 24-48 h after transfection. If the cells were harvested 48 h after transfection, the transfection mixture was removed 6-24 h after transfection and replaced by fresh growth medium.
For the transfection of HeLa or U2OS cells with siRNA, the transfection reagent Lipofectamine 2000 (Invitrogen) was used according to the manufacturer’s instructions. Cells were transfected with 30 nM of siRNA for 72 h. For rescue experiments, 50 ng of plasmid were co-transfected with 40 nM of siRNA for 72 h.
The following siRNA sequences were used:
GL2 (firefly luciferase, control), 5’-CGUACGCGGAAUACUUCGAtt-3’;
FBXO45 #1, 5’-GGAGAAAGAAUUCGAGU CAtt-3’;
FBXO45 #2, 5’-ACACAUGGUUAUUGCGUAUtt-3’;
FBXW7, 5’-ACAGGACAGUGUUUACAAAtt-3’;
MYCBP2 #1, 5’-CCCGAGAUCUUGGGAAUAAtt-3’.
FBXO45 #3 (200933, L-023542-01-0005) and MYCBP2 #2 (23077, L-006951-00-0005) were purchased as SMARTpools from Dharmacon. If not stated otherwise, FBXO45 siRNA #2 was used for FBXO45 depletion and MYCBP2 siRNA #2 was transfected in order to downregulate MYCBP2.
Cell cycle synchronization
In order to arrest HeLa or U2OS cells in G1/S phase, the cells were treated with 2 mM thymidine for 18 h. In order to arrest HeLa or U2OS cells in mitosis, cells were first synchronized in G1/S phase by treatment with 2 mM thymidine for 17 h. The cells were released into the cell cycle by washing three times with PBS and incubation with fresh growth medium for 5 h. Finally, the cells were treated with 250 ng/ml nocodazole, 500-1000 nM Taxol, 500 nM vincristine, 5 μM STLC or 100 nM BI2536 for 16-17 h. Mitotic cells were collected by a mitotic shake-off.
MG132 and cycloheximide treatment
In order to inhibit proteasomal degradation in HEK-293T cells, the cells were treated with 10 μM MG132 (Sigma-Aldrich) for 4-5 h before harvesting. In order to inhibit protein synthesis in HeLa cells, they were treated with 100 μg/ml cycloheximide (Sigma-Aldrich). For a cycloheximide chase assay, the cells were harvested at different time points after cycloheximide treatment.
Live-cell imaging
For the analysis of mitotic cell fate or unperturbed mitotic progression, live-cell imaging was performed. For mitotic cell fate analysis, U2OS cells were transfected with 30 nM siRNA targeting GL2, FBXW7, FBXO45 or MYCBP2. 48 h after transfection, 2.5 × 104 cells were seeded into Ibidi dish chambers. 72 h after transfection, the cells were treated with 250 ng/ml nocodazole, 1 μM Taxol or 500 nM vincristine. 4 h after the spindle poison treatment, the cells were monitored by a 10x/0.3 EC PlnN Ph1 DICI objective on a Zeiss Cell Observer Z1 inverted microscope (AxioCam MRm camera system) with incubation at 5% CO2 and 37°C. Multi-tile phase-contrast images were taken every 10 min for 48 h using the Zeiss ZEN blue software. Data analysis was performed with ImageJ Fiji software. Cell death was defined by cell morphology and cessation of movement. Mitotic slippage was defined by mitotic exit without cell division. For the analysis of unperturbed mitotic progression, HeLa GFP-α-tubulin/RFP-H2B cells were transfected with 30 nM siRNA targeting GL2 or FBXO45 and MYCBP2. Analysis was performed as described previously (Kratz et al., 2016). 48 h after transfection, 2.5 ×x 104 cells were seeded into Ibidi dish chambers and arrested in G1/S phase by thymidine treatment. 24 h after thymidine addition, the cells were released from the arrest and analyzed by live-cell imaging using LED module Colibri.2 with 470 nm for GFP and 590 nm for RFP fluorochrome excitation.
Preparation of protein extracts from mammalian cells
For the preparation of cell extracts, cell pellets were resuspended in 3-5 volumes of RIPA, NP40 or Urea lysis buffer. RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 1% NP40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1 mM DTT, 10 μg/ml TPCK, 5 μg/ml TLCK, 0.1 mM Na3VO4, 1 μg/ml Aprotinin, 1 μg/ml Leupeptin, 10 μg/ml Trypsin inhibitor from soybean) was used for the analysis of protein levels in whole cell extracts and for in vivo ubiquitylation assays. NP40 lysis buffer (40 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 10 mM β-glycerophosphate, 5 mM NaF, 0.5% NP40, 1 mM DTT, 10 μg/ml TPCK, 5 μg/ml TLCK, 0.1 mM Na3VO4, 1 μg/ml Aprotinin, 1 μg/ml Leupeptin, 10 μg/ml Trypsin inhibitor from soybean) was used for immunoprecipitations. Urea lysis buffer (8 M Urea, 30 mM imidazole, 0. 1 M phosphate buffer pH 8.0) was used for in vivo ubiquitylation assays. The cell lysates were incubated on ice for 30 min with short vortexing every 5-10 min. The lysates were cleared by centrifugation at maximal speed and 4°C in an Eppendorf 5415 R centrifuge for 15 min. The supernatants were transferred to fresh reaction tubes. Extracts of cytoplasmic and nuclear cell fractions were prepared using the CelLytic NuCLEAR Extraction Kit (Sigma) according to the manufacturer’s instructions.
The protein concentration of a cell extract was determined according to the Bradford method in a Bio-Rad Protein assay. For SDS-PAGE, the cell extracts were mixed with equal volumes of 2x Laemmli buffer and incubated at 95°C for 5 min.
Western blotting
The following antibodies were used for Western blotting: Rabbit α-FBXW7α antibody (A301-720A, 1:15000) was obtained from Bethyl. Rabbit α-FBXO45 (NBP1-91891, 1:500) was purchased from Novus. Rabbit α-MYCBP2 was a kind gift from K. Scholich (Dorr et al., 2015). Mouse α-Flag (M2, F3165, 1:5000), mouse α-tubulin (B-5-1-2, 1:10000) and mouse α-Vinculin (hVIN-1, 1:5000) were obtained from Sigma-Aldrich. Rabbit α-Cyclin B1 has been described before (Hoffmann et al., 1994). Mouse α-HA (16B12, 1:1000) was purchased from Babco. Mouse α-Myc (9E10, 1:500), rabbit α-SKP1 (H-163, 1:1000) and mouse α-ubiquitin (P4D1, 1:1000) were obtained from Santa Cruz. Mouse α-Nucleophosmin (NPM, 32-5200, 1:1000) was ordered from Zymed. Rabbit α-MCL-1 (4572, 1:1000) was obtained from Cell Signaling. Goat α-mouse IgG HRP (1:5000) was purchased from Novus. Donkey α-rabbit IgG HRP (1:5000) was obtained from Jackson Laboratories.
Immunoprecipitation
Immunoprecipitations of Flag-tagged proteins were performed using α-Flag M2 affinity beads (Sigma-Aldrich). For each immunoprecipitation reaction, 10-40 μl of the α-Flag M2 affinity bead suspension was used. The beads were washed twice with TBS, once with glycine buffer (0.1 M glycine-HCl pH 3.5) and three times with TBS. Buffers and beads were kept on ice and centrifugations were performed at 5000 rpm and 4°C for 2 min in an Eppendorf 5415 R centrifuge. HEK-293T or HeLa cell extracts were prepared with NP40 lysis buffer and 6-15 mg of each extract were transferred to the prepared beads. Each reaction was filled up to a final volume of 1 ml using NP40 lysis buffer. The reactions were incubated overnight on a rotating wheel at 4°C. After the incubation, the beads were washed 3-5 times with NP40 lysis buffer. Immunoprecipitated proteins were eluted from the beads by competition with 100 μl of a 3× Flag peptide solution (100-500 ng/μl in NP40 lysis buffer). The elution was carried out on ice for 30 min with short vortexing every 5-10 min. After the elution, the beads were centrifuged at 5000 rpm and 4°C for 2 min. 90 μl of the supernatant were transferred to a fresh reaction tube and 30 μl of 4× Laemmli buffer were added. After incubation at 95°C for 2 min, 20-40% of the immunoprecipitation sample were analyzed by SDS-PAGE and Western blotting.
In order to analyze whether proteins exist in ternary complexes, sequential immunoprecipitations were performed. In the first step of the experiment, 15-20 mg of HEK-293T cell extracts were used for an immunoprecipitation directed against a Flag-tagged MYCBP2 fragment. The immunoprecipitation was performed as described above. Instead of mixing the eluate with Laemmli buffer, it was used for a second immunoprecipitation directed against HA-FBXO45. HA-FBXO45 was immunoprecipitated with 20 μl of α-HA agarose beads (Sigma-Aldrich) according to the manufacturer’s instructions. Finally, the beads were incubated with 30 μl of 2× Laemmli buffer at 95°C for 5 min. The supernatant was analyzed by SDS-PAGE and Western blotting.
Mass spectrometry analysis
Immunoprecipitation samples were analyzed by SDS-PAGE and stained by Colloidal Coomassie. Analysis was performed by M. Schnölzer (DKFZ Protein Analysis Facility, Heidelberg). Briefly, whole gel lanes were cut into slices. Proteins were reduced and alkylated by incubation with 10 mM DTT in 40 mM NH4HCO3 for 1 h at 56°C in the dark and incubation with 55 mM iodoacetamide in 40 mM NH4HCO3 for 30 min at 25°C. After washing of the slices with H2O and 50% acetonitrile, they were dried with 100% acetonitrile. Proteins were digested in-gel with trypsin (0.17 μg in 10 μl 40 mM NH4HCO3, Promega) at 37°C over night. Tryptic peptides were extracted with 50% acetonitrile/0.1% TFA and 100% acetonitrile. Supernatants were lyophilized and redissolved in 0.1% TFA/5% hexafluoroisopropanol. Solutions were analyzed by nanoLC-ESI-MS/MS. Peptides were separated with a nanoAcquity UPLC system (Waters GmbH). Peptides were loaded on a C18 trap column with a particle size of 5 μm (Waters GmbH). Liquid chromatography was carried out on a BEH130 C18 column with a particle size of 1.7 μm (Waters GmbH). A 1 h gradient was applied for protein identification. The nanoUPLC system was connected to an LTQ Orbitrap XL mass spectrometer (Thermo Scientific). Data were acquired with the Xcalibur software 2.1 (Thermo Scientific). The SwissProt database (taxonomy human) was used for database searches with the MASCOT search engine (Matrix Science). Data from individual gel slices were merged. Peptide mass tolerance was 5 ppm, fragment mass tolerance was 0.4 Da and significance threshold was p<0.01.
In vivo ubiquitylation assays
HEK-293T cells transiently transfected with the indicated plasmids were treated with 10 μM MG132 4-5 h before harvesting. Cells were harvested and cell extracts were prepared with RIPA lysis buffer containing 10 mM of the DUB inhibitor N-ethylmaleimide (NEM). 2-3 mg of protein extract were used for each sample. Flag-FBXW7α was immunoprecipitated with α-Flag affinity beads. Immunoprecipitation and washing steps were performed with RIPA lysis buffer containing 10 mM NEM. Alternatively, endogenous FBXW7α was immunoprecipitated by incubation of each protein extract with 1 μg of FBXW7α antibody on a rotating wheel at 4°C overnight. After the incubation with the antibody, the extracts were incubated with 15 μl of protein A sepharose beads (GE Healthcare) on a rotating wheel at 4°C for 1 h. The beads were washed four times with RIPA lysis buffer containing 10 mM NEM. Finally, the beads were incubated in 30 μl of 2x Laemmli buffer at 95°C for 5 min. 25 μl of the supernatant were analyzed by SDS-PAGE and Western blotting.
For denaturing ubiquitylation assays, cell extracts were prepared using Urea lysis buffer. Cell extracts were sonified, centrifuged and incubated with Nickel-NTA agarose beads on a rotating wheel at RT for 2 h. Afterwards, the beads were washed four times with Urea lysis buffer and proteins were finally eluted using 30 μl of 2x Laemmli buffer containing 200 mM imidazole. 25 μl of the supernatant were analyzed by SDS-PAGE and Western blotting.
Expression and purification of MBP-tagged proteins
Chemically competent E. coli Rosetta (DE3) were transformed with pMAL-MBP-FBXW7α-N167. A single colony was used to inoculate 10 ml of LB medium containing 100 μg/ml ampicillin and 0.2% glucose. The culture was incubated over night at 30°C with constant shaking at 180 rpm. The overnight culture was used to inoculate 1 l of LB medium containing 100 μg/ml ampicillin and 0.2% glucose. The culture was incubated at 37°C with constant shaking at 180 rpm. When an OD600 of 0.5 was reached, the culture was cooled down on ice at 4°C and protein expression was induced by the addition of 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The culture was further incubated over night at 18°C with constant shaking at 180 rpm. Bacteria were harvested by centrifugation at 5000 rpm and 4°C for 15 min (F10-6×500Y rotor, Piramoon). The pellet was resuspended in 25 ml of cold column buffer. Cell lysis was performed with a high pressure homogenizer (15000-17000 psi/1030-1170 bar for one pass, EmulsiFlex C5, Avestin). The lysate was centrifuged at 20000 g and 4°C for 20 min (WX Ultra 80, Thermo Scientific). The supernatant was applied onto a column with 1 ml of equilibrated amylose resin (NEB). The column with the beads and the extract was incubated on a rotating wheel at 4°C for 1 h. Afterwards, the beads were washed three times with 15 ml of column buffer. Finally, the proteins bound to the beads were eluted with 5 ml of column buffer containing 10 mM maltose. 10 fractions of 500 μl each were collected. Protein containing fractions were identified by spotting the fractions on nitrocellulose and staining with Ponceau S solution. Protein containing fractions were pooled and MBP-FBXW7α-N167 was further purified with a preparative Superdex 200 column in 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM β-mercaptoethanol, 5% glycerol. Purified MBP-FBXW7α-N167 fractions were analyzed by SDS-PAGE and Colloidal Coomassie staining. Protein containing fractions were aliquoted, frozen in liquid nitrogen and stored at −80°C.
In vitro transcription and translation and in vitro binding assays
For in vitro binding assays, FBXO45 and MYCBP2(1951-2950) cDNA sequences in pCMV-3Tag1A backbones were transcribed and translated in vitro using the TNT T3 coupled reticulocyte system (Promega) according to the manufacturer’s instructions. The proteins were synthesized in the presence of 20 μCi [35S]-methionine so that synthesized proteins were radioactively labelled. 20 μl of the in vitro translated proteins were incubated with 10 μg of MBP-FBXW7α-N167 or MBP alone coupled to 10 μl of amylose beads in a final volume of 500 μl NP40 lysis buffer on a rotating wheel at 4°C for 2 h. The beads were washed five times with 800 μl of NP40 lysis buffer. Finally, the beads were incubated with 30 μl of 2× Laemmli buffer at 95°C for 5 min. Input and pull-down samples were analyzed by SDS-PAGE and stained with Colloidal Coomassie. The gel was then incubated with Amersham Amplify Fluorographic reagent (GE Healthcare) for 30 min with gentle shaking. Afterwards, the gel was dried at 80°C for 1 h in a vacuum dryer and analyzed by autoradiography.
AUTHOR CONTRIBUTION
IH and KTR designed the experiments. KTR, YTK and BV performed the experiments. IH and KTR wrote the paper.
DECLARATION OF INTERESTS
The authors declare no conflict of interests
Video S1: Related to Figure 4
Exemplary video of a U2OS cell undergoing mitotic cell death upon prolonged mitotic arrest. Mitotic arrest was caused by treatment with nocodazole.
Video S2: Related to Figure 4
Exemplary video of a U2OS cell undergoing mitotic slippage upon prolonged mitotic arrest. Mitotic arrest was caused by treatment with nocodazole.
ACKNOWLEDGMENTS
We thank D. Gerlich, G. Melino, A. Peschiaroli and K. Scholich for providing reagents and cell lines. We acknowledge the DKFZ Mass Spectrometry and Microscopy Core Facilities for providing equipment and excellent technical assistance. We thank Bettina Dörr for expert technical assistance. We acknowledge the members of our lab for critically reading the manuscript.