Mol. Cells 2017; 40(6): 401-409
Published online June 14, 2017
https://doi.org/10.14348/molcells.2017.0032
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: leekh@kribb.re.kr (KHL); bykim@kribb.re.kr (BYK)
The primary cilium is a non-motile microtubule-based organelle that protrudes from the surface of most human cells and works as a cellular antenna to accept extracellular signals. Primary cilia assemble from the basal body during the resting stage (G0 phase) and simultaneously disassemble with cell cycle re-entry. Defective control of assembly or disassembly causes diverse human diseases including ciliopathy and cancer. To identify the effective compounds for studying primary cilium disassembly, we have screened 297 natural compounds and identified 18 and 17 primary cilium assembly and disassembly inhibitors, respectively. Among them, the application of KY-0120, identified as Brefeldin A, disturbed Dvl2-Plk1-mediated cilium disassembly via repression of the interaction of CK1?-Dvl2 and the expression of Plk1 mRNA. Therefore, our study may suggest useful compounds for studying the cellular mechanism of primary cilium disassembly to prevent ciliopathy and cancer.
Keywords brefeldin A, Dvl2, inhibitor, Plk1, primary cilium disassembly
Primary cilia are microtubule-based cellular sensing structures important for transducing extracellular signals and observed in most stromal and epithelial cells (Satir and Christensen, 2008; Seeley and Nachury, 2010). They are assembled in the G0 or G1 phase of the cell cycle and are disassembled as the cell cycle progresses to mitosis (Santos and Reiter, 2008; Seeley and Nachury, 2010). Numerous signaling receptors are present in the primary cilium, which transmit numerous external signals into the cell (Goetz and Anderson, 2010; Rohatgi et al., 2007). Despite many studies, research on the assembly and disassembly of primary cilia are only at the beginning. We found a primary cilium disassembly pathway regulated by the Wnt5a-casein kinase 1ɛ (CK1ɛ)-Dvl2-Plk1 axis in our previous study (Lee et al., 2012). Wnt5a stimulation activates CK1ɛ, phosphorylating the S143 and T224 residues of Dvl2, and Plk1 binds to each of these two residues, triggering primary cilium disassembly. We confirmed that the binding of phosphorylated Dvl2 to Plk1 is the most important factor of primary cilium disassembly (Lee et al., 2012). Abnormalities in the mechanisms of assembly or disassembly of primary cilia lead to diseases called ciliopathy. The pleiotropic effect of ciliopathy has been revealed by defects in multiple organs: eyes (retinitis pigmentosa), skeleton (polydactyly), kidney (polycystic kidney disease, PKD), adipose tissue (obesity), pancreas (diabetes), and CNS (Goetz and Anderson, 2010; Mockel et al., 2011; Nachury et al., 2007; Park et al., 2006). Recently, abnormality in the assembly and disassembly of primary cilia is also involved in carcinogenesis (Gradilone et al., 2013; Han et al., 2009; Kim et al., 2011; Moser et al., 2009; Wong et al., 2009). Over the years, various factors involved in the assembly and disassembly of primary cilia have been identified (Goetz and Anderson, 2010; Lee et al., 2012; Pugacheva et al., 2007), but research on identifying the underlying mechanism has only started now.
Brefeldin A is a small hydrophobic fungal metabolite that was initially isolated as an antibiotic from toxic fungi and that induces the redistribution of Golgi apparatus into the endoplasmic reticulum (ER) (Klausner et al., 1992). BFA inhibits ADP-ribosylation factor (Arf) and subsequently induces the collapse of Golgi-derived protein secretion. Therefore, BFA has recently been used as a therapeutic inhibitor of Arf-related viral/bacterial diseases (Colanzi et al., 2013; Tseng et al., 2014). In this study, we screened 297 fungus-derived natural compounds to isolate inhibitors of primary cilium disassembly and found KY-0120, identified as Brefeldin A (BFA), as an effective inhibitor of Dvl2-Plk1-mediated primary cilium disassembly. Therefore, our study may provide a novel pharmaceutical inhibitor to prevent ciliopathy and cancer.
A
For serum-starvation based ciliogenesis assay, hTERT-RPE cells were seeded on cover slips in 24 culture wells, cultured for 24 h and then serum-starved for 24 h. Compounds [KY-compounds or DMSO (Sigma, USA)] were treated simultaneously with serum-starvation. For serum-restimulation assay, less than 50% cells were seeded on cover slips in 24 culture wells, cultured for 24 h and then serum-starved for 48 h. The resulting cells were then re-stimulated with serum to induce primary cilia disassembly for 24 h. For the serum-restimulation assay, compounds [KY-compounds, DMSO (Sigma), BFA (Sigma), or Monensin (Sigma)] were treated 30 min before serum-restimulation. Resulting cells were fixed with 4% paraformaldehyde for 10 min at room temperature and immunostained with mouse anti-acetylated α-tubulin (Sigma, 1:200) and rabbit anti-γ-tubulin (Sigma, 1:200) antibodies. The number of cells bearing cilia was counted manually under a microscope. To measure the length of primary cilia, images were acquired by Zeiss Axi-oObserver. Z1 microscope at 1388 X 1040 pixels and 12-bit resolution, and analyzed by ZEN v2.1 (Carl Zeiss) software.
To perform GST pull-down assay, bead-associated GST-CK1ɛ WT was purified from bacteria (BL21) and incubated with total cell lysates of Flag-Dvl2 overexpressed HEK293T cells for 5 hours. DMSO or BFA was added to the incubation mixture. The precipitates were washed with TBSN buffer [20 mM Tris-Cl (pH8.0), 150 mM NaCl, 1.5 mM EDTA, 0.5% NP-40, 5 mM EGTA, 0.5 mM Na3VO4, and 20 mM
For IP-kinase assays, HEK 293T cells transfected with Myc-tagged CK1ɛ (a gift of Jeffrey S. Rubin, NIH/NCI, MD) were harvested at 24 h after transfection and subjected to immunoprecipitation with anti-Myc antibody (Santa Cruz). Immunoprecipitates were reacted in a kinase cocktail [50 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 2 mM EGTA, 5 mM dithiothreitol, 0.5 mM Na3VO4, and 20 mM
HEK293T cells were co-transfected with HA-ubiquitin (Ub) and Flag-Plk1 construct (a gift of Kyung S. Lee, NIH/NCI, MD). The cells were then treated with either DMSO or BFA for 18 h before harvest. To prevent proteasomal degradation of Plk1, cells were also treated with 10 μM of MG132 for 3 h before harvest. After 24 h of transfection, cells were harvested and used for immunoprecipitation with anti-Flag antibody (Sigma).
Total RNA was extracted from hTERT-RPE cells treated with DMSO or BFA using RNeasyR mini kit (Qiagen). DMSO or BFA was treated 30 min before serum-restimulation at the indicated concentrations. cDNA was generated by SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Conventional RT-PCR was performed using a ProFlex™ Base Thermal Cycler (Applied Bio-systems) with the conditions of 95°C for 20 s, 62°C for 30 s, and 72°C for 45 s for a total of 25 cycles for Plk1, Dvl2, and GAPDH, followed by a 10 min final extension at 72°C. PCR products were electrophoresed in 3% agarose gel, stained with EcoDye™ Nucleic Acid Staining Solution (BIOFACT, Korea), and photographed. Real-time RT-PCR was performed in a final volume of 20 μl with 2 μl of cDNA, 10 pmol forward and 10 pmol reverse primer in 1X power SYBG green PCR master Mix (Applied Biosystems, USA) with the condition of 95°C for 15 s for denaturation, 55°C for 1 min for annealing and 72°C for 15 s extension using an QuantStudio™ 3 Real-Time PCR System (Applied Biosystems). The expression value of each gene was normalized by that of GAPDH. Final values were calculated using the ΔΔCt method. The results were analyzed using QuantStudio™ design & Analysis software v1.4 (Applied Biosystems). All the primers used in these experiments are summarized in
The same condition as serum-restimulation assay was used for FACS analyses. Resulting cells were trypsinized and subjected to propidium iodide staining using BD cycletest™ Plus DNA reagent kit (BD Biosciences, USA). FACS analyses were carried out using BD FACSCalibur™ Flow Cytometer (BD Biosciences) and data were analyzed by the CellQuest Pro v6.0 (BD Biosciences).
The structure elucidation of isolated Brefeldin A (collection No. KY-0120) was achieved by spectroscopic data measurements (1H and 13C NMR and MS). The NMR spectra were recorded on a Bruker AVANCE HD 800 NMR spectrometer (800 MHz for 1H and 200 MHz for 13C) at Korea Basic Science Institute (KBSI) in Ochang. Chemical shift values were referenced to the residual solvent signal (
We previously identified the Wnt5a-induced primary cilium disassembly pathway (Lee et al., 2012). Therefore, we attempted to isolate effective compounds for regulating the Wnt5a-CK1ɛ-Dvl2-Plk1-mediated primary cilium disassembly pathway. We used an in-house chemical library, KY compounds, which contains 297 natural compounds, as described in
We attempted to characterize KY-0120 and identified its structure as Brefeldin A (BFA) by NMR analysis (Fig. 2A and
BFA specific blockage of ciliary disassembly was further confirmed by using other Golgi-destroying drug, monensin (Nylander and Kalies, 1999; Rosa et al., 1992). Since monensin is widely used drug that inhibits protein secretion by Golgi disruption, we tested monensin for comparison. Surprisingly, monensin did not show the inhibitory effect on primary cilia disassembly which was seen by BFA. Even in high dose treatment of monensin, 100 μM, primary cilia disassembly was not blocked in comparison to BFA treatment (Fig. 2F). This result probably originated from the different mode of actions between these two drugs. In fact, BFA inhibits protein transport from the cis/medial Golgi complex to the ER, while monensin prevents protein secretion by inhibition of trans-Golgi function (Nylander and Kalies, 1999; Rosa et al., 1992).
To understand the mechanism underlying BFA’s inhibition of primary cilium disassembly, we applied the same strategy as that used for the isolation of KY-0120 shown in Fig. 1C. Treatment with BFA, similar to that with KY-0120, caused marked fast-migrating form of Dvl2 and reduction in Plk1 levels in the cilium disassembly condition (Fig. 3A). To clarify this, we used two different approaches for the two different proteins Dvl2 and Plk1. For Dvl2, we closely investigated phosphorylation regulation by CK1ɛ. Previous studies have shown that CK1-induced phosphorylation of Dvl2 generates a slow-migrating band, whereas dephosphorylation results in a fast-migrating form of Dvl2 (Bryja et al., 2007; Lee et al., 2012). Therefore, we first investigated whether BFA inhibits the activity of CK1ɛ directly. To this end, we performed an IP-kinase assay in the presence of DMSO control, CK1 inhibitor (D4476) (Rena et al., 2004), or BFA, as described in the Figure Legends and Materials & Methods section. As a result, the autophosphorylation signal of CK1ɛ was significantly decreased by treatment with D4476, a CK1 inhibitor, compared with that by treatment with the DMSO control. However, no difference was observed between treatment with the DMSO control and BFA (Fig. 3B). No significant difference was observed in both low- (0.1 μM) and high-concentration (100 μM) treatments with BFA in comparison to treatment with the DMSO control. Therefore, we changed the strategy to monitor the effect of BFA on the binding of CK1ɛ to Dvl2. For this, we performed both a GST pull-down assay (Fig. 3C) and an immunoprecipitation assay (Fig. 3D) in the presence of the DMSO control or the indicated concentrations of BFA. Compared with treatment with the DMSO control, treatment with BFA significantly reduced the binding of GST-CK1ɛ to Flag-Dvl2 (Fig. 3C). In line with this observation, the immunoprecipitation assay also showed the inhibitory effect of BFA on the binding between CK1ɛ and Dvl2 (Fig. 3D). These results suggest that BFA inhibits the phosphorylation of Dvl2 by interfering with the interaction between CK1ɛ and Dvl2, although BFA does not affect the activity of CK1ɛ itself.
For Plk1, we focused on the activation of the Plk1 degradation machinery or the inhibition of its expression. Because Plk1 was directly ubiquitinated by specific E3s and degraded by the ubiquitin-proteasome system (Charles et al., 1998; Shirayama et al., 1998), we then investigated whether Plk1 ubiquitination was enhanced by BFA. To this end, we employed an
We further confirmed the arrest of cell cycle progression by BFA during serum-starvation following serum-restimulation. FACS analyses revealed the blocking of cell cycle progression from the G1-phase to the S-phase of the cell cycle in the serum-restimulated condition by treatment with BFA (Fig. 5A). Additionally, in line with this observation, treatment with BFA increased cyclin E1 and decreased cyclin A, B1, and D1 levels (Fig. 5B). Presumably, the prevention of cilia disassembly by BFA inhibits cell cycle progression in serum-restimulation condition.
It has been reported that the mechanisms of primary cilium assembly and disassembly are closely associated with ciliopathy and cancer development (Goetz and Anderson, 2010; Gradilone et al., 2013; Han et al., 2009; Kim et al., 2011; Moser et al., 2009; Nachury et al., 2007; Park et al., 2006; Wong et al., 2009). Although the underlying mechanism of how those events are regulated remains unclear, the importance of the regulation of primary cilia for treating ciliopathy and cancer has recently emerged (Peluso et al., 2014; Xiang et al., 2014). Therefore, primary cilium regulation-based drug development is just the beginning. In this study, we identified an effective compound, KY-0120 (identified as BFA), from our natural compound library as a primary cilium disassembly inhibitor. BFA drastically reduced the phosphorylation of Dvl2 and the expression of Plk1. Additionally, BFA blocked cell cycle progression after serum-restimulation. Therefore, we speculate that the combination of those events by BFA may exert an inhibitory effect on primary cilium disassembly in the Wnt5a-CK1ɛ-Dvl2-Plk1-dependent primary cilium disassembly pathway. In our previous study, we demonstrated that the activation of CK1ɛ by Wnt5a stimulation phosphorylates S143 and T224 of Dvl2, and Plk1 binds to the phosphorylated residues, which eventually results in primary cilium disassembly (Lee et al., 2012). In this study, BFA was shown to interfere with the formation of the CK1ɛ-Dvl2 complex, which inhibits phosphorylation of Dvl2 by CK1ɛ and also inhibits the expression of Plk1 mRNA. Ultimately, this may result in the collapse of the primary cilium disassembly pathway induced by the Dvl2-Plk1 complex. Consistent with previous report that the blockage of cilia disassembly pathway caused longer cilia than normal condition (Lee et al., 2012), BFA treatment caused primary cilia elongation. The cilia elongation by BFA treatment is presumably due to the breakdown of the force balance between assembly and disassembly mechanism. BFA is known as an inhibitor of anterograde transport from ER to the Golgi apparatus, and the ER-Golgi system has been shown to have important functions in ciliogenesis (Hoffmeister et al., 2011). Therefore, we cannot rule out the possibility of BFA controlling ciliogenesis, by regulation of the ER-Golgi system, as well as primary cilium disassembly inhibition, by regulation of the Dvl2-Plk1 complex, as identified in this study. Since the transcriptional repressions by blockage of Golgi-dependent vesicular transport have been reported (Kingsbury and Cardenas, 2016; Pahl and Baeuerle, 1995; Wyrozumska et al., 2014), we speculate that BFA may inhibit the Plk1 mRNA expression via the blockage of vesicular trafficking-induced cytoplasmic-nuclear translocation of some transcription factor which induces Plk1 expression. However, the exact mechanism is needed to be elucidated in further study.
Plk1 has been reported to play various roles in mitosis, such as in mitotic entry, G2/M checkpoint regulation, spindle assembly, chromosome segregation, and cytokinesis (Barr et al., 2004; Petronczki et al., 2008; von Schubert et al., 2015). Therefore, the inhibitory effect of BFA on the expression of Plk1 during cilia disassembly period, which is confirmed in this study, may be able to function not only in primary cilium disassembly but also in mitotic regulation. In particular, the overexpression of Plk1 has been identified as a major cause of cancer (Knecht, 1999; Strebhardt and Ullrich, 2006), suggesting that the inhibition of the expression of Plk1 by treatment with BFA may be applied as a therapeutic option against the development of Plk1-induced cancers. Dvl2 is known as a scaffold protein that plays an important role in both Wnt canonical and non-canonical pathways (MacDonald et al., 2009; Singh et al., 2010). Several kinases that regulate Dvl2 function have been reported, among which phosphorylation of Dvl2 by the CK1 family has been shown to be an important mechanism for controlling Wnt signaling (Kishida et al., 2001; Peters et al., 1999). These findings suggest the possibility that the inhibition of binding between CK1ɛ and Dvl2 by BFA may not only result in deficiency of primary cilium disassembly but also result in regulatory effects on Wnt canonical and Wnt-PCP signaling pathways.
We also confirmed that treatment with BFA arrested the progression of the cell cycle from the G1-phase to the S-phase during the serum-restimulation period. This suggests the inhibition of primary cilium disassembly by blocking G1-phase to S-phase progression and implies two possibilities: BFA directly acts on cell cycle progression or it indirectly inhibits cell cycle progression by regulating Dvl2 or Plk1. Presumably, it is possible that the cells treated with BFA in the absence of serum fail to leave the G0 phase even after serum restimulation. Our findings may provide a powerful tool for studying ciliogenesis and suggest a pharmaceutical inhibitor to treat cilium-related diseases including cancer.
(A) The first round of compound library screening. A total of 297 fungal metabolites were screened for ciliogenesis. hTERT-RPE cells were serum-depleted for 24 h, and 10 μM of each compound was applied to hTERT-RPE cells simultaneously with serum-depletion. After 24 h of serum-starvation, cells were fixed and subjected to immunocytochemistry. (See
(A) Structural elucidation of KY-0120. The structure of KY-0120 was defined by NMR analyses (See
(A) BFA reduces the phosphorylation of Dvl2 and the amount of Plk1 during primary cilium disassembly. hTERT-RPE cells were treated with either the DMSO control or BFA as described in
(A) BFA does not work in the ubiquitination of Plk1. HEK293T cells were co-transfected with HA-Ub and Flag-Plk1. The DMSO control or BFA was applied for 18 h. To prevent protein degradation, MG132 was applied to cells 3 h prior to cell harvest. Flag-Plk1 was immunoprecipitated with anti-Flag antibody and precipitates were subjected to immunoblotting analysis with the indicated antibodies. (B, C) BFA reduces the expression of Plk1 mRNA. hTERT-RPE cells were treated with BFA as indicated in
BFA increases the G1 population of the cells. The same cells as in
Mol. Cells 2017; 40(6): 401-409
Published online June 30, 2017 https://doi.org/10.14348/molcells.2017.0032
Copyright © The Korean Society for Molecular and Cellular Biology.
Uijeong Lee1,2,4, Sun-Ok Kim1,4, Jeong-Ah Hwang1,3,4, Jae-Hyuk Jang1,2, Sangkeun Son1,2, In-Ja Ryoo1, Jong Seog Ahn1,2, Bo Yeon Kim1,2,*, and Kyung Ho Lee1,*
1World Class Institute (WCI), Anticancer Agent Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Chungbuk 28116, Korea, 2Department of Biomolecular Science, University of Science and Technology, Daejeon 34113, Korea, 3Research Institute of Medical Sciences, Department of Physiology, College of Medicine, Chungnam National University, Daejeon 34134, Korea
Correspondence to:*Correspondence: leekh@kribb.re.kr (KHL); bykim@kribb.re.kr (BYK)
The primary cilium is a non-motile microtubule-based organelle that protrudes from the surface of most human cells and works as a cellular antenna to accept extracellular signals. Primary cilia assemble from the basal body during the resting stage (G0 phase) and simultaneously disassemble with cell cycle re-entry. Defective control of assembly or disassembly causes diverse human diseases including ciliopathy and cancer. To identify the effective compounds for studying primary cilium disassembly, we have screened 297 natural compounds and identified 18 and 17 primary cilium assembly and disassembly inhibitors, respectively. Among them, the application of KY-0120, identified as Brefeldin A, disturbed Dvl2-Plk1-mediated cilium disassembly via repression of the interaction of CK1?-Dvl2 and the expression of Plk1 mRNA. Therefore, our study may suggest useful compounds for studying the cellular mechanism of primary cilium disassembly to prevent ciliopathy and cancer.
Keywords: brefeldin A, Dvl2, inhibitor, Plk1, primary cilium disassembly
Primary cilia are microtubule-based cellular sensing structures important for transducing extracellular signals and observed in most stromal and epithelial cells (Satir and Christensen, 2008; Seeley and Nachury, 2010). They are assembled in the G0 or G1 phase of the cell cycle and are disassembled as the cell cycle progresses to mitosis (Santos and Reiter, 2008; Seeley and Nachury, 2010). Numerous signaling receptors are present in the primary cilium, which transmit numerous external signals into the cell (Goetz and Anderson, 2010; Rohatgi et al., 2007). Despite many studies, research on the assembly and disassembly of primary cilia are only at the beginning. We found a primary cilium disassembly pathway regulated by the Wnt5a-casein kinase 1ɛ (CK1ɛ)-Dvl2-Plk1 axis in our previous study (Lee et al., 2012). Wnt5a stimulation activates CK1ɛ, phosphorylating the S143 and T224 residues of Dvl2, and Plk1 binds to each of these two residues, triggering primary cilium disassembly. We confirmed that the binding of phosphorylated Dvl2 to Plk1 is the most important factor of primary cilium disassembly (Lee et al., 2012). Abnormalities in the mechanisms of assembly or disassembly of primary cilia lead to diseases called ciliopathy. The pleiotropic effect of ciliopathy has been revealed by defects in multiple organs: eyes (retinitis pigmentosa), skeleton (polydactyly), kidney (polycystic kidney disease, PKD), adipose tissue (obesity), pancreas (diabetes), and CNS (Goetz and Anderson, 2010; Mockel et al., 2011; Nachury et al., 2007; Park et al., 2006). Recently, abnormality in the assembly and disassembly of primary cilia is also involved in carcinogenesis (Gradilone et al., 2013; Han et al., 2009; Kim et al., 2011; Moser et al., 2009; Wong et al., 2009). Over the years, various factors involved in the assembly and disassembly of primary cilia have been identified (Goetz and Anderson, 2010; Lee et al., 2012; Pugacheva et al., 2007), but research on identifying the underlying mechanism has only started now.
Brefeldin A is a small hydrophobic fungal metabolite that was initially isolated as an antibiotic from toxic fungi and that induces the redistribution of Golgi apparatus into the endoplasmic reticulum (ER) (Klausner et al., 1992). BFA inhibits ADP-ribosylation factor (Arf) and subsequently induces the collapse of Golgi-derived protein secretion. Therefore, BFA has recently been used as a therapeutic inhibitor of Arf-related viral/bacterial diseases (Colanzi et al., 2013; Tseng et al., 2014). In this study, we screened 297 fungus-derived natural compounds to isolate inhibitors of primary cilium disassembly and found KY-0120, identified as Brefeldin A (BFA), as an effective inhibitor of Dvl2-Plk1-mediated primary cilium disassembly. Therefore, our study may provide a novel pharmaceutical inhibitor to prevent ciliopathy and cancer.
A
For serum-starvation based ciliogenesis assay, hTERT-RPE cells were seeded on cover slips in 24 culture wells, cultured for 24 h and then serum-starved for 24 h. Compounds [KY-compounds or DMSO (Sigma, USA)] were treated simultaneously with serum-starvation. For serum-restimulation assay, less than 50% cells were seeded on cover slips in 24 culture wells, cultured for 24 h and then serum-starved for 48 h. The resulting cells were then re-stimulated with serum to induce primary cilia disassembly for 24 h. For the serum-restimulation assay, compounds [KY-compounds, DMSO (Sigma), BFA (Sigma), or Monensin (Sigma)] were treated 30 min before serum-restimulation. Resulting cells were fixed with 4% paraformaldehyde for 10 min at room temperature and immunostained with mouse anti-acetylated α-tubulin (Sigma, 1:200) and rabbit anti-γ-tubulin (Sigma, 1:200) antibodies. The number of cells bearing cilia was counted manually under a microscope. To measure the length of primary cilia, images were acquired by Zeiss Axi-oObserver. Z1 microscope at 1388 X 1040 pixels and 12-bit resolution, and analyzed by ZEN v2.1 (Carl Zeiss) software.
To perform GST pull-down assay, bead-associated GST-CK1ɛ WT was purified from bacteria (BL21) and incubated with total cell lysates of Flag-Dvl2 overexpressed HEK293T cells for 5 hours. DMSO or BFA was added to the incubation mixture. The precipitates were washed with TBSN buffer [20 mM Tris-Cl (pH8.0), 150 mM NaCl, 1.5 mM EDTA, 0.5% NP-40, 5 mM EGTA, 0.5 mM Na3VO4, and 20 mM
For IP-kinase assays, HEK 293T cells transfected with Myc-tagged CK1ɛ (a gift of Jeffrey S. Rubin, NIH/NCI, MD) were harvested at 24 h after transfection and subjected to immunoprecipitation with anti-Myc antibody (Santa Cruz). Immunoprecipitates were reacted in a kinase cocktail [50 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 2 mM EGTA, 5 mM dithiothreitol, 0.5 mM Na3VO4, and 20 mM
HEK293T cells were co-transfected with HA-ubiquitin (Ub) and Flag-Plk1 construct (a gift of Kyung S. Lee, NIH/NCI, MD). The cells were then treated with either DMSO or BFA for 18 h before harvest. To prevent proteasomal degradation of Plk1, cells were also treated with 10 μM of MG132 for 3 h before harvest. After 24 h of transfection, cells were harvested and used for immunoprecipitation with anti-Flag antibody (Sigma).
Total RNA was extracted from hTERT-RPE cells treated with DMSO or BFA using RNeasyR mini kit (Qiagen). DMSO or BFA was treated 30 min before serum-restimulation at the indicated concentrations. cDNA was generated by SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Conventional RT-PCR was performed using a ProFlex™ Base Thermal Cycler (Applied Bio-systems) with the conditions of 95°C for 20 s, 62°C for 30 s, and 72°C for 45 s for a total of 25 cycles for Plk1, Dvl2, and GAPDH, followed by a 10 min final extension at 72°C. PCR products were electrophoresed in 3% agarose gel, stained with EcoDye™ Nucleic Acid Staining Solution (BIOFACT, Korea), and photographed. Real-time RT-PCR was performed in a final volume of 20 μl with 2 μl of cDNA, 10 pmol forward and 10 pmol reverse primer in 1X power SYBG green PCR master Mix (Applied Biosystems, USA) with the condition of 95°C for 15 s for denaturation, 55°C for 1 min for annealing and 72°C for 15 s extension using an QuantStudio™ 3 Real-Time PCR System (Applied Biosystems). The expression value of each gene was normalized by that of GAPDH. Final values were calculated using the ΔΔCt method. The results were analyzed using QuantStudio™ design & Analysis software v1.4 (Applied Biosystems). All the primers used in these experiments are summarized in
The same condition as serum-restimulation assay was used for FACS analyses. Resulting cells were trypsinized and subjected to propidium iodide staining using BD cycletest™ Plus DNA reagent kit (BD Biosciences, USA). FACS analyses were carried out using BD FACSCalibur™ Flow Cytometer (BD Biosciences) and data were analyzed by the CellQuest Pro v6.0 (BD Biosciences).
The structure elucidation of isolated Brefeldin A (collection No. KY-0120) was achieved by spectroscopic data measurements (1H and 13C NMR and MS). The NMR spectra were recorded on a Bruker AVANCE HD 800 NMR spectrometer (800 MHz for 1H and 200 MHz for 13C) at Korea Basic Science Institute (KBSI) in Ochang. Chemical shift values were referenced to the residual solvent signal (
We previously identified the Wnt5a-induced primary cilium disassembly pathway (Lee et al., 2012). Therefore, we attempted to isolate effective compounds for regulating the Wnt5a-CK1ɛ-Dvl2-Plk1-mediated primary cilium disassembly pathway. We used an in-house chemical library, KY compounds, which contains 297 natural compounds, as described in
We attempted to characterize KY-0120 and identified its structure as Brefeldin A (BFA) by NMR analysis (Fig. 2A and
BFA specific blockage of ciliary disassembly was further confirmed by using other Golgi-destroying drug, monensin (Nylander and Kalies, 1999; Rosa et al., 1992). Since monensin is widely used drug that inhibits protein secretion by Golgi disruption, we tested monensin for comparison. Surprisingly, monensin did not show the inhibitory effect on primary cilia disassembly which was seen by BFA. Even in high dose treatment of monensin, 100 μM, primary cilia disassembly was not blocked in comparison to BFA treatment (Fig. 2F). This result probably originated from the different mode of actions between these two drugs. In fact, BFA inhibits protein transport from the cis/medial Golgi complex to the ER, while monensin prevents protein secretion by inhibition of trans-Golgi function (Nylander and Kalies, 1999; Rosa et al., 1992).
To understand the mechanism underlying BFA’s inhibition of primary cilium disassembly, we applied the same strategy as that used for the isolation of KY-0120 shown in Fig. 1C. Treatment with BFA, similar to that with KY-0120, caused marked fast-migrating form of Dvl2 and reduction in Plk1 levels in the cilium disassembly condition (Fig. 3A). To clarify this, we used two different approaches for the two different proteins Dvl2 and Plk1. For Dvl2, we closely investigated phosphorylation regulation by CK1ɛ. Previous studies have shown that CK1-induced phosphorylation of Dvl2 generates a slow-migrating band, whereas dephosphorylation results in a fast-migrating form of Dvl2 (Bryja et al., 2007; Lee et al., 2012). Therefore, we first investigated whether BFA inhibits the activity of CK1ɛ directly. To this end, we performed an IP-kinase assay in the presence of DMSO control, CK1 inhibitor (D4476) (Rena et al., 2004), or BFA, as described in the Figure Legends and Materials & Methods section. As a result, the autophosphorylation signal of CK1ɛ was significantly decreased by treatment with D4476, a CK1 inhibitor, compared with that by treatment with the DMSO control. However, no difference was observed between treatment with the DMSO control and BFA (Fig. 3B). No significant difference was observed in both low- (0.1 μM) and high-concentration (100 μM) treatments with BFA in comparison to treatment with the DMSO control. Therefore, we changed the strategy to monitor the effect of BFA on the binding of CK1ɛ to Dvl2. For this, we performed both a GST pull-down assay (Fig. 3C) and an immunoprecipitation assay (Fig. 3D) in the presence of the DMSO control or the indicated concentrations of BFA. Compared with treatment with the DMSO control, treatment with BFA significantly reduced the binding of GST-CK1ɛ to Flag-Dvl2 (Fig. 3C). In line with this observation, the immunoprecipitation assay also showed the inhibitory effect of BFA on the binding between CK1ɛ and Dvl2 (Fig. 3D). These results suggest that BFA inhibits the phosphorylation of Dvl2 by interfering with the interaction between CK1ɛ and Dvl2, although BFA does not affect the activity of CK1ɛ itself.
For Plk1, we focused on the activation of the Plk1 degradation machinery or the inhibition of its expression. Because Plk1 was directly ubiquitinated by specific E3s and degraded by the ubiquitin-proteasome system (Charles et al., 1998; Shirayama et al., 1998), we then investigated whether Plk1 ubiquitination was enhanced by BFA. To this end, we employed an
We further confirmed the arrest of cell cycle progression by BFA during serum-starvation following serum-restimulation. FACS analyses revealed the blocking of cell cycle progression from the G1-phase to the S-phase of the cell cycle in the serum-restimulated condition by treatment with BFA (Fig. 5A). Additionally, in line with this observation, treatment with BFA increased cyclin E1 and decreased cyclin A, B1, and D1 levels (Fig. 5B). Presumably, the prevention of cilia disassembly by BFA inhibits cell cycle progression in serum-restimulation condition.
It has been reported that the mechanisms of primary cilium assembly and disassembly are closely associated with ciliopathy and cancer development (Goetz and Anderson, 2010; Gradilone et al., 2013; Han et al., 2009; Kim et al., 2011; Moser et al., 2009; Nachury et al., 2007; Park et al., 2006; Wong et al., 2009). Although the underlying mechanism of how those events are regulated remains unclear, the importance of the regulation of primary cilia for treating ciliopathy and cancer has recently emerged (Peluso et al., 2014; Xiang et al., 2014). Therefore, primary cilium regulation-based drug development is just the beginning. In this study, we identified an effective compound, KY-0120 (identified as BFA), from our natural compound library as a primary cilium disassembly inhibitor. BFA drastically reduced the phosphorylation of Dvl2 and the expression of Plk1. Additionally, BFA blocked cell cycle progression after serum-restimulation. Therefore, we speculate that the combination of those events by BFA may exert an inhibitory effect on primary cilium disassembly in the Wnt5a-CK1ɛ-Dvl2-Plk1-dependent primary cilium disassembly pathway. In our previous study, we demonstrated that the activation of CK1ɛ by Wnt5a stimulation phosphorylates S143 and T224 of Dvl2, and Plk1 binds to the phosphorylated residues, which eventually results in primary cilium disassembly (Lee et al., 2012). In this study, BFA was shown to interfere with the formation of the CK1ɛ-Dvl2 complex, which inhibits phosphorylation of Dvl2 by CK1ɛ and also inhibits the expression of Plk1 mRNA. Ultimately, this may result in the collapse of the primary cilium disassembly pathway induced by the Dvl2-Plk1 complex. Consistent with previous report that the blockage of cilia disassembly pathway caused longer cilia than normal condition (Lee et al., 2012), BFA treatment caused primary cilia elongation. The cilia elongation by BFA treatment is presumably due to the breakdown of the force balance between assembly and disassembly mechanism. BFA is known as an inhibitor of anterograde transport from ER to the Golgi apparatus, and the ER-Golgi system has been shown to have important functions in ciliogenesis (Hoffmeister et al., 2011). Therefore, we cannot rule out the possibility of BFA controlling ciliogenesis, by regulation of the ER-Golgi system, as well as primary cilium disassembly inhibition, by regulation of the Dvl2-Plk1 complex, as identified in this study. Since the transcriptional repressions by blockage of Golgi-dependent vesicular transport have been reported (Kingsbury and Cardenas, 2016; Pahl and Baeuerle, 1995; Wyrozumska et al., 2014), we speculate that BFA may inhibit the Plk1 mRNA expression via the blockage of vesicular trafficking-induced cytoplasmic-nuclear translocation of some transcription factor which induces Plk1 expression. However, the exact mechanism is needed to be elucidated in further study.
Plk1 has been reported to play various roles in mitosis, such as in mitotic entry, G2/M checkpoint regulation, spindle assembly, chromosome segregation, and cytokinesis (Barr et al., 2004; Petronczki et al., 2008; von Schubert et al., 2015). Therefore, the inhibitory effect of BFA on the expression of Plk1 during cilia disassembly period, which is confirmed in this study, may be able to function not only in primary cilium disassembly but also in mitotic regulation. In particular, the overexpression of Plk1 has been identified as a major cause of cancer (Knecht, 1999; Strebhardt and Ullrich, 2006), suggesting that the inhibition of the expression of Plk1 by treatment with BFA may be applied as a therapeutic option against the development of Plk1-induced cancers. Dvl2 is known as a scaffold protein that plays an important role in both Wnt canonical and non-canonical pathways (MacDonald et al., 2009; Singh et al., 2010). Several kinases that regulate Dvl2 function have been reported, among which phosphorylation of Dvl2 by the CK1 family has been shown to be an important mechanism for controlling Wnt signaling (Kishida et al., 2001; Peters et al., 1999). These findings suggest the possibility that the inhibition of binding between CK1ɛ and Dvl2 by BFA may not only result in deficiency of primary cilium disassembly but also result in regulatory effects on Wnt canonical and Wnt-PCP signaling pathways.
We also confirmed that treatment with BFA arrested the progression of the cell cycle from the G1-phase to the S-phase during the serum-restimulation period. This suggests the inhibition of primary cilium disassembly by blocking G1-phase to S-phase progression and implies two possibilities: BFA directly acts on cell cycle progression or it indirectly inhibits cell cycle progression by regulating Dvl2 or Plk1. Presumably, it is possible that the cells treated with BFA in the absence of serum fail to leave the G0 phase even after serum restimulation. Our findings may provide a powerful tool for studying ciliogenesis and suggest a pharmaceutical inhibitor to treat cilium-related diseases including cancer.
(A) The first round of compound library screening. A total of 297 fungal metabolites were screened for ciliogenesis. hTERT-RPE cells were serum-depleted for 24 h, and 10 μM of each compound was applied to hTERT-RPE cells simultaneously with serum-depletion. After 24 h of serum-starvation, cells were fixed and subjected to immunocytochemistry. (See
(A) Structural elucidation of KY-0120. The structure of KY-0120 was defined by NMR analyses (See
(A) BFA reduces the phosphorylation of Dvl2 and the amount of Plk1 during primary cilium disassembly. hTERT-RPE cells were treated with either the DMSO control or BFA as described in
(A) BFA does not work in the ubiquitination of Plk1. HEK293T cells were co-transfected with HA-Ub and Flag-Plk1. The DMSO control or BFA was applied for 18 h. To prevent protein degradation, MG132 was applied to cells 3 h prior to cell harvest. Flag-Plk1 was immunoprecipitated with anti-Flag antibody and precipitates were subjected to immunoblotting analysis with the indicated antibodies. (B, C) BFA reduces the expression of Plk1 mRNA. hTERT-RPE cells were treated with BFA as indicated in
BFA increases the G1 population of the cells. The same cells as in
Jun Bae Park, Hayeong Park, Jimin Son, Sang-Jun Ha, and Hyun-Soo Cho
Mol. Cells 2019; 42(8): 597-603 https://doi.org/10.14348/molcells.2019.0114Ju Hwan Cho, and Joong-Soo Han
Mol. Cells 2017; 40(11): 805-813 https://doi.org/10.14348/molcells.2017.0241Kyung S. Lee, Jung-Eun Park, Young Hwi Kang, Tae-Sung Kim, and Jeong K. Bang
Mol. Cells 2014; 37(4): 286-294 https://doi.org/10.14348/molcells.2014.0002
(A) The first round of compound library screening. A total of 297 fungal metabolites were screened for ciliogenesis. hTERT-RPE cells were serum-depleted for 24 h, and 10 μM of each compound was applied to hTERT-RPE cells simultaneously with serum-depletion. After 24 h of serum-starvation, cells were fixed and subjected to immunocytochemistry. (See
(A) Structural elucidation of KY-0120. The structure of KY-0120 was defined by NMR analyses (See
(A) BFA reduces the phosphorylation of Dvl2 and the amount of Plk1 during primary cilium disassembly. hTERT-RPE cells were treated with either the DMSO control or BFA as described in
(A) BFA does not work in the ubiquitination of Plk1. HEK293T cells were co-transfected with HA-Ub and Flag-Plk1. The DMSO control or BFA was applied for 18 h. To prevent protein degradation, MG132 was applied to cells 3 h prior to cell harvest. Flag-Plk1 was immunoprecipitated with anti-Flag antibody and precipitates were subjected to immunoblotting analysis with the indicated antibodies. (B, C) BFA reduces the expression of Plk1 mRNA. hTERT-RPE cells were treated with BFA as indicated in
BFA increases the G1 population of the cells. The same cells as in