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ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled Sun-Hong Hwang a, b, Sunhoe Bang a, b, Kyung Shin Kang b, Deborah Kang b, Jongkyeong Chung a, b, * a
School of Biological Sciences, Seoul National University, 1 Gwanak-Ro, Gwanak-Gu, Seoul, 08826, Republic of Korea National Creative Research Initiatives Center for Energy Homeostasis Regulation, Institute of Molecular Biology and Genetics, Seoul National University, 1 Gwanak-Ro, Gwanak-Gu, Seoul, 08826, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 November 2018 Accepted 21 November 2018 Available online xxx
Wnt signaling pathway plays critical roles in body axes patterning, cell fate specification, cell proliferation, cell migration, stem cell maintenance, cancer development and etc. Deregulation of this pathway can be causative of cancer, metabolic disease and neurodegenerative disease such as Parkinson`s disease. Among the core components of Wnt signaling pathway, we discovered that Dishevelled (Dsh) interacts with ULK1 and is phosphorylated by ULK1. Unexpectedly, the knockdown of ULK1 elicited a marked increase in Wnt/b-catenin signaling. Multiple ULK1 phosphorylation sites existed on Dsh and many of them were located on the PDZ-DEP region. By using evolutionarily well conserved Drosophila Dsh, we found that S239, S247 and S254 in the PDZ-DEP region are involved in phosphorylation of Dsh by ULK1. Among these, S247 and S254 were conserved in human Dsh. When phospho-mimetic mutants (2D and 2E Dsh mutants) of these conserved residues were generated and expressed in the eyes of the fruit flies, the activity of Dsh was significantly decreased compared to wild type Dsh. Through additional alanine scanning, we further identified that S239, S247, S254, S266, S376, S554 and S555 on full length Dsh were phosphorylated by ULK1. In regards to the S266A mutation located in the PDZ domain among these phosphorylated residues, our results suggested that Dsh forms an SDS-resistant high molecular weight complex with b-catenin and TCF in the nucleus in an S266 phosphorylation-dependent manner. Based on these results, we propose that ULK1 plays a pivotal role in the regulation of Wnt/b-catenin signaling pathway by phosphorylating Dsh. © 2018 Elsevier Inc. All rights reserved.
Keywords: Wnt/b-catenin signaling Dishevelled Autophagy ULK1
1. Introduction Wnt induces a critical signaling pathway regulating animal development [1e5]. When the regulatory mechanism of the signaling pathway is defective, numerous human diseases, such as osteoarthritis, osteoporosis-pseudogliome syndrome (OPPG), familial exudative vitreoretinopathy (FEVR), tetra-amelia, polycystic kidneys, cardiac hypertrophy, schizophrenia, Alzheimer disease and cancer, are known to occur [2]. Wnt signaling pathway is divided into canonical Wnt signaling involving b-catenin, and non-canonical Wnt signaling without the involvement of b-catenin [4]. In the case of canonical Wnt signaling,
* Corresponding author. Institute of Molecular Biology and Genetics, Seoul National University, 1 Gwanak-Ro, Gwanak-Gu, Seoul, 08826, Republic of Korea. E-mail address: [email protected] (J. Chung).
Wnt ligand (such as Wnt3a) binds to Frizzled and LDL-receptor related protein 5/6 (LRP5/6), which inhibits the activity of the bcatenin destruction complex consisted of adenomatous polyposis coli (APC), Axin, glycogen synthase kinase 3b (GSK3b) and casein kinase 1a (CK1a) through Dishevelled (Dsh) [4]. Consequently, bcatenin accumulated in the cell moves into the nucleus and induces the transcription of the target genes such as Myc, cyclin D1 and Axin2 [4]. Wnt signaling pathway is consisted of a multistep signaling cascade, and the signaling components of the pathway are regulated by various intracellular factors, converging with diverse signaling pathways [4]. Dsh is a core component of Wnt signaling and participates in both canonical and non-canonical Wnt signaling [6]. When cells are under starvation of nutrients, ubiquitinationdependent degradation is induced on Dsh, and thus Dsh functions as a point of suppression in the signaling cascade [7]. In other words, understanding various upstream regulators for Dsh can lead
https://doi.org/10.1016/j.bbrc.2018.11.139 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: S.-H. Hwang et al., ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.139
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us to the profound comprehension of Wnt signaling pathway in different intracellular conditions [6]. Autophagy is a cellular catabolic process to degrade proteins and organelles using autophagosome [8e11]. UNC51-like kinase 1 (ULK1) is an important kinase as it initiates autophagy by forming a complex with ATG13 and FAK family kinase-interacting protein of 200 kDa (FIP200) [8]. In the autophagy machinery, ULK1 undergoes auto-phosphorylation and phosphorylates ATG13, FIP200, Beclin1 and other ATG genes to induce autophagy [12]. Moreover, as it has been found that ULK1 is also involved in other cellular activities, including membrane trafficking and innate immunity [13e16], discovering new targets of ULK1 and new roles of ULK1 in the cell have been growing in interest. 2. Materials and methods 2.1. Cell culture and transfection HEK293T cells were grown in Dulbecco's-modified Eagle's medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, MA) at 37 C in a humidified atmosphere with 5% CO2. Transfection of mammalian expression plasmid was performed by using polyethylenimine (Sigma, OH) according to the manufacturer's instruction. siRNAs (siScr and siULK1-#1, #2, and #3) were purchased from Bioneer in Korea. Co-transfection of siRNA and plasmids was performed by using Lipofectamine 2000 reagent (Invitrogen, MA) according to the manufacturer's instruction.
immunoblotting. Glutathione Sepharose 4B beads (GE Healthcare, MA) were used for GST pull-down assays. 2.5. Lambda protein phosphatase treatment After immunoprecipitation of mDvl1, the samples were washed twice with PBS and dephosphorylated with Lambda protein phosphatase (New England BioLabs, cat#P0753S, MA) by incubating for 1 h at 37 C. After completing dephosphorylation reaction, the samples were boiled in SDS-sample buffer for 10 min at 94 C. 2.6. Promoter assay HEK293T cells were seeded in triplicate on 12-well plates and transfected with pSuperTOP Flash (0.5 mg), pRL-TK (0.05 mg), and siRNAs. As a control, HEK293T cells were seeded in triplicate on 12well plates and transfected with pSuperFOP Flash (0.5 mg), pRL-TK (0.05 mg), and siRNAs. After 24 h of transfection, cells were treated with Wnt3a-conditioned medium (CM) for 24 h. The luciferase activities were measured by a dual luciferase assay kit (Promega, Madison, WI). Data were shown as TOP Flash/FOP Flash ratios. 2.7. Fly stocks UAS-Flag Dsh (WT, 2A, 2D, 2E) were generated by microinjection of pUAST vector-cloned DNA into w1118 embryos. Gmr-GAL4 was obtained from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN).
2.2. Plasmid DNA 2.8. Statistics The plasmid encoding mDvl1 was a generous gift from Dr. Roel Nusse (Stanford University, CA). The plasmid encoding Dsh (LD20984) was purchased from Berkeley Drosophila Genome Project (BDGP). The cDNAs encoding mDvl1 and Dsh were cloned to pCMV10-3Flag vector (Sigma). The plasmids encoding Myc-ULK1, Myc-ATG13, Myc-FIP200 and Myc-ULK2 were generous gifts from Dr. Do-Hyung Kim (University of Minnesota, MN). The plasmids encoding Myc-mDvl2, pSuper TOP Flash, pSuper FOP Flash and pRL-TK were generous gifts from Dr. Eek-hoon Jho (University of Seoul, Korea). The plasmids encoding Myc-b-catenin S45Y and HATCF were generous gifts from Dr. Sung Hee Baek (Seoul National University, Korea). 2.3. Preparation of lysate, immunoprecipitation and immunoblotting For preparation of cell lysates, cells were washed with cold PBS and lysed with Buffer A (20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 2 mM EGTA, 50 mM b-glycerophosphate, 50 mM NaF, 2 mM DTT, 1 mM PMSF, 5 mg/ml leupeptin, 1 mg/ml pepstatin A and 1% Triton X-100). After cell lysis, cell lysates were centrifuged at 16,100 g for 30 min. The supernatant was subjected to immunoprecipitation and immunoblotting according to standard procedures. Cell pellets after elimination of supernatant were solubilized in SDS-sample buffer. The immunoblots were developed and visualized using LAS-4000 (Fujifilm, Japan). 2.4. Antibodies and reagents Mouse anti-Myc (Medical and Biological Laboratories, cat#M192-3, Japan), mouse anti-Flag (Medical and Biological Laboratories, cat#M185-3L, Japan), rat anti-HA (Roche Applied Science, IN), mouse anti-tubulin (DSHB, cat#E7, IA), mouse anti-GST (Upstate, cat#05e311, NY), and rabbit anti-ULK1 (Santacruz, cat#sc33182, CA) antibody were used for immunoprecipitation and/or
p values were obtained by one-way ANOVA Tukey's test, and <0.05 was considered to be significant. 3. Results 3.1. ULK1 interacts with mDvl1 ULK1 is a key autophagy-inducing kinase stimulated by starvation [17]. Recent research showed that ULK1 is also associated with innate immunity, vesicle trafficking, and endocytosis by phosphorylating a variety of downstream targets [13e15,18]. Therefore, it is important to reveal unknown targets of ULK1 to understand its functions in various cell signaling pathways. To find the novel interactors of ULK1, we used mass-spectrometry analysis. First, we overexpressed Myc-tagged ULK1 in HEK293T cells and performed immunoprecipitation. As a result, we detected putative interacting protein bands by silver staining and subsequently conducted MALDI-TOF analysis to identify the binding proteins to ULK1. Among these proteins, we identified the 85 kDa band as Dishevelled1 (Dvl1) (Fig. 1A). It was shown that ULK1 forms a protein complex that is composed of ULK1, ATG13 and FIP200, known as an autophagy initiation kinase complex [19]. To observe any functions of the components of the ULK1 complex on the interaction between ULK1 and Dvl1, we transiently expressed both mouse Dishevelled1 (mDvl1) and the components of the ULK1 complex in various combinations to detect the interaction between the ULK1 complex and mDvl1. Consistent with the previous mass-spectrometry data, the interaction between ULK1 and mDvl1 was detected, and this interaction was stronger when ATG13 was co-expressed (Fig. 1B). As reported, the fact that ATG13 stabilizes and activates the kinase activity of ULK1 can be the possible explanation for the enhanced interaction between ULK1 and mDvl1. However, the other component of the ULK1 complex, FIP200 did not seem to change
Please cite this article as: S.-H. Hwang et al., ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.139
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Fig. 1. ULK1 interacts with mDvl1. A, Silver stain of Myc-ULK1 co-immunoprecipitated proteins. HEK293T cells were transfected with Myc-ULK1. Cell lysates were immunoprecipitated by anti-Myc antibody. B, HEK293T cells were transfected with Flag-mDvl1, Myc-ULK1, Myc-ATG13 and Myc-FIP200 as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. Whole cell lysates (WCL) were used for detecting expression of ULK1, ATG13, FIP200 and mDvl1 by using anti-Myc and anti-Flag antibodies. Immunoprecipitated samples were detected with anti-Myc and anti-Flag antibodies. C, a schematic representation of the domain structure of ULK1. We made GST-fusion proteins for various ULK1 domains. D, HEK293T cells were transfected with Flag-mDvl1 and GST-ULK1 fragments as indicated. Cell lysates were subjected to GST pull-down. WCL were used for detecting expression of mDvl1 by using anti-Flag antibody. GST pull-down samples were detected for mDvl1 and ULK1 fragments using anti-Myc and anti-GST antibodies.
the level of the interaction between ULK1 and mDvl1 (Fig. 1B) [19]. ULK1 is composed of three domains: a kinase domain (KD), a serine/threonine-rich (S/T) domain and a C-terminus domain (CT) (Fig. 1C) [20]. To find which domain is responsible for binding to mDvl1, we made GST-fused ULK1 domain fragments. Among the fragments of ULK1, the KD and the S/T domain could interact with mDvl1 (Fig. 1D). 3.2. ULK1 inhibits the canonical Wnt/b-catenin signaling pathway and phosphorylates mDvl1 at the PDZ-DEP region We revealed that ULK1 interacts with mDvl1, a core component of Wnt signaling pathway. To examine the role of ULK1 in Wnt signaling pathway, we performed the TOP Flash promoter assay. In the experiment, we knocked down ULK1 using three independent ULK1 siRNAs. In Wnt3a-conditioned medium, canonical Wnt signaling pathway was activated, and all three siRNAs showed more than three times increased promoter activity than when siScramble (siScr) was applied (Fig. 2A). After the promoter assay, we used the same lysate to observe the level of ULK1 gene expression, and found that all three siULK1 effectively knocked down ULK1 (Fig. 2B). These results suggest that ULK1 is a negative regulator in Wnt/bcatenin signaling pathway. When co-expressed with ULK1, mDvl1 was upshifted in immunoblotting (Fig. 1B), which led us to suspect a presence of specific post-translational modification. Interestingly, mDvl1 was also upshifted when only the kinase domain of ULK1 was coexpressed (Fig. 1D). Therefore, we suspected that ULK1 directly phosphorylates mDvl1. To demonstrate, the construct of a kinasedead mutant form of ULK1, called ULK1 M92A (MA) [19], was used in the following experiment. When mDvl1 was coexpressed with ULK1 MA, no shift in mDvl1 was observed (Fig. 2C). Thus, the mobility shift of mDvl1 was dependent to the kinase activity of ULK1. To prove that the shift in mDvl1 is due to phosphorylation, we applied lambda protein phosphatase to the immunoprecipitated mDvl1. As expected, the treatment of
lambda protein phosphatase blocked the mobility shift of mDvl1 (Fig. 2C). In conclusion, the post-translational modification that took place on mDvl1 causing band shifts in immunoblot analyses was phosphorylation by ULK1. Although another kinase-dead form of ULK1, the K46I (KI) mutant [21], could not induce the band shift of mDvl1 as well, ULK2 that is most similar to ULK1 among the ULK family could induce the band shift of mDvl1 (Fig. 2D). The mDvl1 has three domains: the Dishevelled/Axin (DIX) domain, the PSD95/Dlg1/ZO-1 (PDZ) domain and the Dishevlled/ EGL-10/Pleckstrin (DEP) domain (Fig. 2F) [6]. To find the specific site of phosphorylation on mDvl1, fragmented mDvl1 in various combinations of the three domains were generated. Among them, the PDZ-DEP domain fragment was the smallest fragment showing band shifts in a ULK1 kinase activity-dependent manner (Fig. 2E). 3.3. ULK1 phosphorylates the PDZ-DEP region of Dsh at serine 254, 266 and 376 Since Atg1, a yeast ULK1 orthologue, prefers serine to threonine for phosphorylation [22], we substituted a serine with an alanine in the PDZ-DEP fragment of mDvl1. However, a single serine substitution did not eliminate the band shifts by ULK1 (data not shown). We concluded that multiple serines in mDvl1 were phosphorylated by ULK1. As mDvl1 has a poly-serine motif in the linker region, it was difficult to find changes in serine phosphorylation through alanine scanning analyses. Therefore, we took another approach for phosphorylation mapping. Since Dishevelled is a core element in Wnt signaling pathway, we assumed that the regulation of Dishevelled, such as phosphorylation by ULK1, should be highly conserved from Drosophila to humans [23]. Therefore, we performed the alanine scanning in Drosophila Dishevelled (Dsh), which lacks the polyserine region. Dsh also showed band shifts dependent on ULK1activity (Fig. 3A) and a much more prominent three-band-shift was detected with the PDZ-DEP fragment of Dsh (Fig. 3B). Hence,
Please cite this article as: S.-H. Hwang et al., ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.139
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Fig. 2. ULK1 inhibits canonical Wnt/b-catenin signaling pathway and phosphorylates mDvl1 at the PDZ-DEP region. A-B, HEK293T cells were transfected with pSuperTop and pRL-TK reporter plasmids with scramble (Scr) siRNA or ULK1 siRNA (#1, #2, and #3) as indicated. Cells were harvested 48 h after transfection, and luciferase activity was measured by the dual luciferase assay system. Wnt3a-CM was added to cells for 24 h before harvesting cells. Data represent average values from triplicate experiments. Cell lysates were used for detecting expression of ULK1 and tubulin by using anti-ULK1 and anti-tubulin antibodies. ***p < 0.0001. C, HEK293T cells were transfected with Flag-mDvl1, Myc-ULK1 wild type (WT) and Myc-ULK1 M92A (MA) as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL were used for detecting expression of mDvl1 and ULK1 using antiFlag and anti-Myc antibodies. Immunoprecipitated samples were treated with lambda protein phosphatase (PP) as indicated. After de-phosphorylation, immunoprecipitated samples were detected by anti-Flag antibody. D, HEK293T cells were transfected with Flag-mDvl1 and Myc-ULK1 WT, Myc-ULK1 K46I (KI), Myc-ULK1 M92A (MA) and Myc-ULK2 as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL were detected with anti-Myc antibody. Immunoprecipitated samples were detected with anti-Flag antibody. E, HEK293T cells were transfected with Flag-mDvl1 PDZ-DEP fragment, Myc-ULK1 and Myc-ULK1 K46I (KI) as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL were used for detecting expression of ULK1 using anti-Myc antibody. Immunoprecipitated samples were detected by anti-Flag antibody. F, a schematic representation of the domain structure of mDvl1.
Fig. 3. ULK1 phosphorylates the PDZ-DEP region of Dsh at serine 254, 266 and 376. A, HEK293T cells were transfected with Flag-Dsh, Myc-ULK1 WT and Myc-ULK1 K46I (KI) as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL were used for detecting expression of ULK1 by using anti-Myc antibody. Immunoprecipitated samples were detected by anti-Flag antibody. BeC, HEK293T cells were transfected with Flag-Dsh PDZ-DEP fragments (WT and mutants) and Myc-ULK1 as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL were used for detecting expression of ULK1 by using anti-Myc antibody. Immunoprecipitated samples were detected by anti-Flag antibody. D, Drosophila eyes expressing transgenes using the eye-specific GAL4 driver (Gmr-GAL4).
it was easier to find the serine residues responsible for the shifts with alanine scanning (Fig. 3B). Among the Dsh mutants in which serines were replaced to alanines in the PDZ-DEP region, S254A, S266A, and S376A mutants displayed a two-band-shift (Fig. 3B). When these serine sites were replaced concurrently to alanines as in the mutant 3A, the band shift was no more detected by ULK1 coexpression (Fig. 3C).
Among the three serine residues, serines 266 and 376 are conserved between fruit fly and mouse. Based on this, the functional importance of these two sites was studied through expressing Dsh with serine 266 and 376 mutations in the fruit fly eye. Transgenic flies expressing exogenous Dsh with mutations in the two serines to alanines (2A) and to their phosphomimetic forms, aspartic acids (2D) and glutamic acids (2E), were made
Please cite this article as: S.-H. Hwang et al., ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.139
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respectively. Upon activation of Wnt signaling in the fly, the eyes get smaller and the ommatidial arrays collapse [24]. When exogenous Dsh was expressed in the eyes of the flies using Gmr-GAL4, the eyes expressing Dsh WT and 2A mutant were severely damaged whereas the level of damage was much less severe in Dsh 2D and 2E mutants (Fig. 3D). In summary, phosphorylation of serine 266 and 376 on Dsh results in decreased activation of Wnt signaling pathway.
3.4. ULK1 inhibits formation of Dsh-containing protein complex through phosphorylation The protein band shift caused by ULK1 was reduced but still existed when the phosphorylation sites found in PDZ-DEP fragment were replaced in full length Dsh. Hence, we repeated the phosphorylation site mapping to find additional protein band shifts in larger fragments of Dsh, and finally in the mutant (7A) substituting a total of seven serines with alanines on S239, S247, S254, S266, S376, S554 and S555, no ULK1-dependent band shift was observed (Fig. 4A). Among these serine sites in the fly protein, we performed a mutagenesis on only the conserved serine positions in mDvl1 and discovered that band shifts caused by ULK1 were severely decreased (data not shown). However, from the fact that there was slight band shift remaining, additional phosphorylations that are not conserved between fruit fly and humans seem to exist in mDvl1. Both mDvl1 and Dsh exhibited an increase in band intensity when the band shift was induced by ULK1. Thus, we hypothesized that ULK1-induced phosphorylation may dissolve insoluble forms of Dsh proteins from cell lysate pellets, instead of increasing the stability of Dsh. In fact, ULK1 co-expression significantly decreased the amount of Dsh WT protein found in the pellet fraction.
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However, in the same condition, Dsh 7A mutant protein still remained in the pellet fraction (Fig. 4B). These results suggested that ULK1 functions as a kinase that directly phosphorylates and mobilizes Dsh proteins from the pellet to the cytosolic fraction. We suspected that the pellet fraction includes the nuclei and the Dsh mutant protein forms insoluble protein complexes with nuclear proteins. Surprisingly, among the 7 serines, when only serine 266 was mutated to alanine in Dsh, a thick protein band was observed over 200 kDa size (Fig. 4C). This high molecular weight protein complex was SDS-resistant and was also detected when Dsh WT was overexpressed, suggesting that the complex is better formed with Dsh S266A mutant than with WT. Previous data showed that Dsh forms a protein complex in the nucleus with b-catenin and TCF, and plays a critical role in the target gene induction for Wnt signaling pathway [25]. Indeed, we confirmed that there was an interaction between b-catenin, TCF and mDvl2 (self-oligomerization) in the high molecular weight complex (Fig. 4D, E, F). In this experiment, we used b-catenin with S45Y mutation which stabilizes the protein [26]. In conclusion, Dsh serine 266 phosphorylation by ULK1 inhibits intranuclear interaction between Dsh and other components of Wnt signaling pathway, such as b-catenin and TCF, suggesting that ULK1dependent phosphorylation of Dsh is critical for the regulation of Wnt signaling pathway.
4. Discussion 4.1. Dsh as the new target of ULK1 and the role of ULK1 in Wnt/bcatenin signaling pathway In this research, we have proposed that ULK1 is involved in Wnt/
b-catenin signaling pathway by phosphorylating Dsh. Hence, we
Fig. 4. ULK1 inhibits a formation of Dsh-containing complex through phosphorylation. A, HEK293T cells were transfected with Flag-Dsh (WT or 7A) and Myc-ULK1 as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL were used for detecting expression of ULK1 by using anti-Myc antibody. Immunoprecipitated samples were detected by anti-Flag antibody. B, HEK293T cells were transfected with Flag-Dsh (WT or 7A) and Myc-ULK1 as indicated. WCL were used for detecting expression of Dsh by using anti-Flag antibody and ULK1 by using anti-Myc antibody. Pellet samples were used for detecting expression of Dsh by using anti-Flag antibody and ULK1 by using anti-Myc antibody. C, HEK293T cells were transfected with Flag-Dsh (WT or 266A) as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. Immunoprecipitated samples were detected with anti-Flag antibody. D-F, HEK293T cells were transfected with Myc-mDvl2, Myc-b-catenin S45Y, HA-TCF and Flag-Dsh 266A as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL and immunoprecipitated samples were detected by anti-Myc antibody, anti-Flag antibody and anti-HA antibody as indicated. G, proposed model. A novel signaling mechanism to suppress Wnt/b-catenin signaling by ULK1.
Please cite this article as: S.-H. Hwang et al., ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.139
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have provided evidence to support the idea. First, ULK1 knockdown cells displayed increased activation of Wnt/b-catenin signaling pathway. Second, ULK1 interacted with and phosphorylated Dsh. Third, the phosphomimetic form of Drosophila Dsh showed lower activity of Wnt signaling in Drosophila. Fourth, ULK1 mobilized Dsh from the pellet fraction to the soluble cell lysates. Fifth, among the 7 serine residues that ULK1 phosphorylated, replacement of serine to alanine on the residue 266 in the PDZ domain of Dsh resulted in increased formation of the SDS-resistant high molecular weight complex by inducing stronger interactions between Dsh and both b-catenin and TCF. Collectively, our data established a novel regulatory mechanism for Wnt/b-catenin signaling pathway by ULK1dependent phosphorylation of Dsh (Fig. 4G), implicating the critical role of ULK1-activating signals such as nutrient starvation. 4.2. A new therapeutic target for tumors with aberrant Wnt signaling Wnt signaling is highly activated in many types of cancer, especially in colon cancer [2]. Therefore, specific inhibitors for Wnt signaling are potentially promising drugs for cancer treatments. Earlier researches have already reported the role of autophagy in tumor development [27]. For example, autophagy inhibits tumorigenesis by degrading misfolded proteins, defective mitochondria, and aggregated proteins, and thus relieves the cells from stress conditions [27]. Therefore, not only the function of ULK1 in autophagy but also the phosphorylation activity on Dsh may be promising therapeutic targets for the tumorigenesis with aberrant Wnt signaling if a specific activator of ULK1 is developed. Since rapalogs induces ULK1 [28], it will be worthwhile to examine whether the drugs are effective in treating various Wnt-dependent tumors. Abbreviations The abbreviations used are: Dsh, Dishevelled; mDvl1, mouse Dishevelled1; ULK1, UNC51-like kinase 1; FIP200, FAK family kinase-interacting protein of 200 kDa; KD, kinase domain; S/T, serine/threonine-rich domain; CT, C-terminus domain; siScr, siScramble; DIX, Dishevelled/Axin domain; PDZ, PSD95/Dlg1/ZO-1 domain; DEP, Dishevlled/EGL-10/Pleckstrin domain; Wnt3a-CM, Wnt3a-conditioned medium; PP, Lambda protein phosphatase. Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article. Author contributions The authors have made the following declarations about their contributions: Conceived and designed the experiment: S.H.H. and J.C. Performed the experiments: S.H.H. and S.H.B. Analyzed the data: S.H.H., S.H.B. and J.C. Wrote the paper: S.H.H., K.S.K., D.K. and J.C. Acknowledgments J.C. was supported by a grant (HI17C0328) of the Korea Health Technology R&D project through the Korea Health Industry Development Institute, funded by the Ministry of Health and Welfare. S.H.H., S.H.B. and J.C. were supported by BK21 Plus Program from the Ministry of Education, Republic of Korea. S.H.H. was supported by Global Ph.D. Fellowship Program from National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning, Republic of Korea. mDvl1 was a generous gift from Dr. Roel Nusse (Stanford
University, CA). Myc-ULK1, Myc-ATG13, Myc-FIP200 and Myc-ULK2 were generous gifts from Dr. Do-Hyung Kim (University of Minnesota, MN). Myc-mDvl2, pSuperTOP Flash, pSuperFOP Flash, pRLTK and Wnt3a-conditioned medium were generous gifts from Dr. Eek-hoon Jho (University of Seoul, Korea). Myc-b-catenin S45Y and HA-TCF were generous gifts from Dr. Sung Hee Baek (Seoul National University, Korea). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.139. References [1] R. van Amerongen, R. Nusse, Towards an integrated view of Wnt signaling in development, Development 136 (2009) 3205e3214. [2] A. Klaus, W. Birchmeier, Wnt signalling and its impact on development and cancer, Nat. Rev. Canc. 8 (2008) 387e398. [3] J.R.K. Seifert, M. Mlodzik, Frizzled/PCP signalling: a conserved mechanism regulating cell polarity and directed motility, Nat. Rev. Genet. 8 (2007) 126e138. [4] C. Niehrs, The complex world of WNT receptor signalling, Nat. Rev. Mol. Cell Biol. 13 (2012) 767e779. [5] W. Kim, M. Kim, E.H. Jho, Wnt/beta-catenin signalling: from plasma membrane to nucleus, Biochem. J. 450 (2013) 9e21. [6] C. Gao, Y.-G. Chen, Dishevelled: the hub of Wnt signaling, Cell. Signal. 22 (2010) 717e727. [7] C. Gao, W. Cao, L. Bao, et al., Autophagy negatively regulates Wnt signalling by promoting Dishevelled degradation, Nat. Cell Biol. 12 (2010). 781-U738. [8] C. He, D.J. Klionsky, Regulation mechanisms and signaling pathways of autophagy, Annu. Rev. Genet., pp. 67-93. [9] O. Florey, M. Overholtzer, Autophagy proteins in macroendocytic engulfment, Trends Cell Biol. 22 (2012) 374e380. [10] S. Shimizu, Biological roles of alternative autophagy, Mol. Cell. 41 (2018) 50e54. [11] M.S. Lee, Overview of the minireviews on autophagy, Mol. Cell. 41 (2018) 1e2. [12] D. Papinski, C. Kraft, Regulation of autophagy by signaling through the atg1/ ULK1 complex, J. Mol. Biol. 428 (2016) 1725e1741. [13] J.H. Joo, B. Wang, E. Frankel, et al., The noncanonical role of ULK/ATG1 in ERto-golgi trafficking is essential for cellular homeostasis, Mol. Cell 62 (2016) 491e506. [14] H. Konno, K. Konno, G.N. Barber, Cyclic dinucleotides trigger ULK1 (ATG1) phosphorylation of STING to prevent sustained innate immune signaling, Cell 155 (2013) 688e698. [15] D. Saleiro, S. Mehrotra, B. Kroczynska, et al., Central role of ULK1 in type I interferon signaling, Cell Rep. 11 (2015) 605e617. [16] J. Kim, Y.M. Lim, M.S. Lee, The role of autophagy in systemic metabolism and human-type diabetes, Mol. Cell. 41 (2018) 11e17. [17] N. Mizushima, The role of the Atg1/ULK1 complex in autophagy regulation, Curr. Opin. Cell Biol. 22 (2010) 132e139. [18] X. Zhou, J.R. Babu, S. da Silva, et al., Unc-51-like kinase 1/2-mediated endocytic processes regulate filopodia extension and branching of sensory axons, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 5842e5847. [19] C.H. Jung, C.B. Jun, S.-H. Ro, et al., ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery, Mol. Biol. Cell 20 (2009) 1992e2003. [20] J. Kim, M. Kundu, B. Viollet, et al., AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1, Nat. Cell Biol. 13 (2011) 132eU171. [21] E.Y.W. Chan, A. Longatti, N.C. McKnight, et al., Kinase-inactivated ULK proteins inhibit autophagy via their conserved C- terminal domains using an atg13independent mechanism, Mol. Cell Biol. 29 (2009) 157e171. [22] D. Papinski, M. Schuschnig, W. Reiter, et al., Early steps in autophagy depend on direct phosphorylation of Atg9 by the Atg1 kinase, Mol. Cell 53 (2014) 471e483. [23] B.T. MacDonald, K. Tamai, X. He, Wnt/beta-Catenin signaling: components, mechanisms, and diseases, Dev. Cell 17 (2009) 9e26. [24] A. Penton, A. Wodarz, R. Nusse, A mutational analysis of dishevelled in Drosophila defines novel domains in the dishevelled protein as well as novel suppressing alleles of axin, Genetics 161 (2002) 747e762. [25] X.-q. Gan, J.-y. Wang, Y. Xi, et al., Nuclear Dvl, c-Jun, beta-catenin, and TCF form a complex leading to stabilization of beta-catenin-TCF interaction, JCB (J. Cell Biol.) 180 (2008) 1087e1100. [26] M.A. Buendia, Genetic alterations in hepatoblastoma and hepatocellular carcinoma: common and distinctive aspects, Med. Pediatr. Oncol. 39 (2002) 530e535. [27] R. Mathew, V. Karantza-Wadsworth, E. White, Role of autophagy in cancer, Nat. Rev. Canc. 7 (2007) 961e967. [28] Y.C. Kim, K.L. Guan, mTOR: a pharmacologic target for autophagy regulation, J. Clin. Invest. 125 (2015) 25e32.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled Sun-Hong Hwang a, b, Sunhoe Bang a, b, Kyung Shin Kang b, Deborah Kang b, Jongkyeong Chung a, b, * a
School of Biological Sciences, Seoul National University, 1 Gwanak-Ro, Gwanak-Gu, Seoul, 08826, Republic of Korea National Creative Research Initiatives Center for Energy Homeostasis Regulation, Institute of Molecular Biology and Genetics, Seoul National University, 1 Gwanak-Ro, Gwanak-Gu, Seoul, 08826, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 November 2018 Accepted 21 November 2018 Available online xxx
Wnt signaling pathway plays critical roles in body axes patterning, cell fate specification, cell proliferation, cell migration, stem cell maintenance, cancer development and etc. Deregulation of this pathway can be causative of cancer, metabolic disease and neurodegenerative disease such as Parkinson`s disease. Among the core components of Wnt signaling pathway, we discovered that Dishevelled (Dsh) interacts with ULK1 and is phosphorylated by ULK1. Unexpectedly, the knockdown of ULK1 elicited a marked increase in Wnt/b-catenin signaling. Multiple ULK1 phosphorylation sites existed on Dsh and many of them were located on the PDZ-DEP region. By using evolutionarily well conserved Drosophila Dsh, we found that S239, S247 and S254 in the PDZ-DEP region are involved in phosphorylation of Dsh by ULK1. Among these, S247 and S254 were conserved in human Dsh. When phospho-mimetic mutants (2D and 2E Dsh mutants) of these conserved residues were generated and expressed in the eyes of the fruit flies, the activity of Dsh was significantly decreased compared to wild type Dsh. Through additional alanine scanning, we further identified that S239, S247, S254, S266, S376, S554 and S555 on full length Dsh were phosphorylated by ULK1. In regards to the S266A mutation located in the PDZ domain among these phosphorylated residues, our results suggested that Dsh forms an SDS-resistant high molecular weight complex with b-catenin and TCF in the nucleus in an S266 phosphorylation-dependent manner. Based on these results, we propose that ULK1 plays a pivotal role in the regulation of Wnt/b-catenin signaling pathway by phosphorylating Dsh. © 2018 Elsevier Inc. All rights reserved.
Keywords: Wnt/b-catenin signaling Dishevelled Autophagy ULK1
1. Introduction Wnt induces a critical signaling pathway regulating animal development [1e5]. When the regulatory mechanism of the signaling pathway is defective, numerous human diseases, such as osteoarthritis, osteoporosis-pseudogliome syndrome (OPPG), familial exudative vitreoretinopathy (FEVR), tetra-amelia, polycystic kidneys, cardiac hypertrophy, schizophrenia, Alzheimer disease and cancer, are known to occur [2]. Wnt signaling pathway is divided into canonical Wnt signaling involving b-catenin, and non-canonical Wnt signaling without the involvement of b-catenin [4]. In the case of canonical Wnt signaling,
* Corresponding author. Institute of Molecular Biology and Genetics, Seoul National University, 1 Gwanak-Ro, Gwanak-Gu, Seoul, 08826, Republic of Korea. E-mail address: [email protected] (J. Chung).
Wnt ligand (such as Wnt3a) binds to Frizzled and LDL-receptor related protein 5/6 (LRP5/6), which inhibits the activity of the bcatenin destruction complex consisted of adenomatous polyposis coli (APC), Axin, glycogen synthase kinase 3b (GSK3b) and casein kinase 1a (CK1a) through Dishevelled (Dsh) [4]. Consequently, bcatenin accumulated in the cell moves into the nucleus and induces the transcription of the target genes such as Myc, cyclin D1 and Axin2 [4]. Wnt signaling pathway is consisted of a multistep signaling cascade, and the signaling components of the pathway are regulated by various intracellular factors, converging with diverse signaling pathways [4]. Dsh is a core component of Wnt signaling and participates in both canonical and non-canonical Wnt signaling [6]. When cells are under starvation of nutrients, ubiquitinationdependent degradation is induced on Dsh, and thus Dsh functions as a point of suppression in the signaling cascade [7]. In other words, understanding various upstream regulators for Dsh can lead
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Please cite this article as: S.-H. Hwang et al., ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.139
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us to the profound comprehension of Wnt signaling pathway in different intracellular conditions [6]. Autophagy is a cellular catabolic process to degrade proteins and organelles using autophagosome [8e11]. UNC51-like kinase 1 (ULK1) is an important kinase as it initiates autophagy by forming a complex with ATG13 and FAK family kinase-interacting protein of 200 kDa (FIP200) [8]. In the autophagy machinery, ULK1 undergoes auto-phosphorylation and phosphorylates ATG13, FIP200, Beclin1 and other ATG genes to induce autophagy [12]. Moreover, as it has been found that ULK1 is also involved in other cellular activities, including membrane trafficking and innate immunity [13e16], discovering new targets of ULK1 and new roles of ULK1 in the cell have been growing in interest. 2. Materials and methods 2.1. Cell culture and transfection HEK293T cells were grown in Dulbecco's-modified Eagle's medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, MA) at 37 C in a humidified atmosphere with 5% CO2. Transfection of mammalian expression plasmid was performed by using polyethylenimine (Sigma, OH) according to the manufacturer's instruction. siRNAs (siScr and siULK1-#1, #2, and #3) were purchased from Bioneer in Korea. Co-transfection of siRNA and plasmids was performed by using Lipofectamine 2000 reagent (Invitrogen, MA) according to the manufacturer's instruction.
immunoblotting. Glutathione Sepharose 4B beads (GE Healthcare, MA) were used for GST pull-down assays. 2.5. Lambda protein phosphatase treatment After immunoprecipitation of mDvl1, the samples were washed twice with PBS and dephosphorylated with Lambda protein phosphatase (New England BioLabs, cat#P0753S, MA) by incubating for 1 h at 37 C. After completing dephosphorylation reaction, the samples were boiled in SDS-sample buffer for 10 min at 94 C. 2.6. Promoter assay HEK293T cells were seeded in triplicate on 12-well plates and transfected with pSuperTOP Flash (0.5 mg), pRL-TK (0.05 mg), and siRNAs. As a control, HEK293T cells were seeded in triplicate on 12well plates and transfected with pSuperFOP Flash (0.5 mg), pRL-TK (0.05 mg), and siRNAs. After 24 h of transfection, cells were treated with Wnt3a-conditioned medium (CM) for 24 h. The luciferase activities were measured by a dual luciferase assay kit (Promega, Madison, WI). Data were shown as TOP Flash/FOP Flash ratios. 2.7. Fly stocks UAS-Flag Dsh (WT, 2A, 2D, 2E) were generated by microinjection of pUAST vector-cloned DNA into w1118 embryos. Gmr-GAL4 was obtained from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN).
2.2. Plasmid DNA 2.8. Statistics The plasmid encoding mDvl1 was a generous gift from Dr. Roel Nusse (Stanford University, CA). The plasmid encoding Dsh (LD20984) was purchased from Berkeley Drosophila Genome Project (BDGP). The cDNAs encoding mDvl1 and Dsh were cloned to pCMV10-3Flag vector (Sigma). The plasmids encoding Myc-ULK1, Myc-ATG13, Myc-FIP200 and Myc-ULK2 were generous gifts from Dr. Do-Hyung Kim (University of Minnesota, MN). The plasmids encoding Myc-mDvl2, pSuper TOP Flash, pSuper FOP Flash and pRL-TK were generous gifts from Dr. Eek-hoon Jho (University of Seoul, Korea). The plasmids encoding Myc-b-catenin S45Y and HATCF were generous gifts from Dr. Sung Hee Baek (Seoul National University, Korea). 2.3. Preparation of lysate, immunoprecipitation and immunoblotting For preparation of cell lysates, cells were washed with cold PBS and lysed with Buffer A (20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 2 mM EGTA, 50 mM b-glycerophosphate, 50 mM NaF, 2 mM DTT, 1 mM PMSF, 5 mg/ml leupeptin, 1 mg/ml pepstatin A and 1% Triton X-100). After cell lysis, cell lysates were centrifuged at 16,100 g for 30 min. The supernatant was subjected to immunoprecipitation and immunoblotting according to standard procedures. Cell pellets after elimination of supernatant were solubilized in SDS-sample buffer. The immunoblots were developed and visualized using LAS-4000 (Fujifilm, Japan). 2.4. Antibodies and reagents Mouse anti-Myc (Medical and Biological Laboratories, cat#M192-3, Japan), mouse anti-Flag (Medical and Biological Laboratories, cat#M185-3L, Japan), rat anti-HA (Roche Applied Science, IN), mouse anti-tubulin (DSHB, cat#E7, IA), mouse anti-GST (Upstate, cat#05e311, NY), and rabbit anti-ULK1 (Santacruz, cat#sc33182, CA) antibody were used for immunoprecipitation and/or
p values were obtained by one-way ANOVA Tukey's test, and <0.05 was considered to be significant. 3. Results 3.1. ULK1 interacts with mDvl1 ULK1 is a key autophagy-inducing kinase stimulated by starvation [17]. Recent research showed that ULK1 is also associated with innate immunity, vesicle trafficking, and endocytosis by phosphorylating a variety of downstream targets [13e15,18]. Therefore, it is important to reveal unknown targets of ULK1 to understand its functions in various cell signaling pathways. To find the novel interactors of ULK1, we used mass-spectrometry analysis. First, we overexpressed Myc-tagged ULK1 in HEK293T cells and performed immunoprecipitation. As a result, we detected putative interacting protein bands by silver staining and subsequently conducted MALDI-TOF analysis to identify the binding proteins to ULK1. Among these proteins, we identified the 85 kDa band as Dishevelled1 (Dvl1) (Fig. 1A). It was shown that ULK1 forms a protein complex that is composed of ULK1, ATG13 and FIP200, known as an autophagy initiation kinase complex [19]. To observe any functions of the components of the ULK1 complex on the interaction between ULK1 and Dvl1, we transiently expressed both mouse Dishevelled1 (mDvl1) and the components of the ULK1 complex in various combinations to detect the interaction between the ULK1 complex and mDvl1. Consistent with the previous mass-spectrometry data, the interaction between ULK1 and mDvl1 was detected, and this interaction was stronger when ATG13 was co-expressed (Fig. 1B). As reported, the fact that ATG13 stabilizes and activates the kinase activity of ULK1 can be the possible explanation for the enhanced interaction between ULK1 and mDvl1. However, the other component of the ULK1 complex, FIP200 did not seem to change
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Fig. 1. ULK1 interacts with mDvl1. A, Silver stain of Myc-ULK1 co-immunoprecipitated proteins. HEK293T cells were transfected with Myc-ULK1. Cell lysates were immunoprecipitated by anti-Myc antibody. B, HEK293T cells were transfected with Flag-mDvl1, Myc-ULK1, Myc-ATG13 and Myc-FIP200 as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. Whole cell lysates (WCL) were used for detecting expression of ULK1, ATG13, FIP200 and mDvl1 by using anti-Myc and anti-Flag antibodies. Immunoprecipitated samples were detected with anti-Myc and anti-Flag antibodies. C, a schematic representation of the domain structure of ULK1. We made GST-fusion proteins for various ULK1 domains. D, HEK293T cells were transfected with Flag-mDvl1 and GST-ULK1 fragments as indicated. Cell lysates were subjected to GST pull-down. WCL were used for detecting expression of mDvl1 by using anti-Flag antibody. GST pull-down samples were detected for mDvl1 and ULK1 fragments using anti-Myc and anti-GST antibodies.
the level of the interaction between ULK1 and mDvl1 (Fig. 1B) [19]. ULK1 is composed of three domains: a kinase domain (KD), a serine/threonine-rich (S/T) domain and a C-terminus domain (CT) (Fig. 1C) [20]. To find which domain is responsible for binding to mDvl1, we made GST-fused ULK1 domain fragments. Among the fragments of ULK1, the KD and the S/T domain could interact with mDvl1 (Fig. 1D). 3.2. ULK1 inhibits the canonical Wnt/b-catenin signaling pathway and phosphorylates mDvl1 at the PDZ-DEP region We revealed that ULK1 interacts with mDvl1, a core component of Wnt signaling pathway. To examine the role of ULK1 in Wnt signaling pathway, we performed the TOP Flash promoter assay. In the experiment, we knocked down ULK1 using three independent ULK1 siRNAs. In Wnt3a-conditioned medium, canonical Wnt signaling pathway was activated, and all three siRNAs showed more than three times increased promoter activity than when siScramble (siScr) was applied (Fig. 2A). After the promoter assay, we used the same lysate to observe the level of ULK1 gene expression, and found that all three siULK1 effectively knocked down ULK1 (Fig. 2B). These results suggest that ULK1 is a negative regulator in Wnt/bcatenin signaling pathway. When co-expressed with ULK1, mDvl1 was upshifted in immunoblotting (Fig. 1B), which led us to suspect a presence of specific post-translational modification. Interestingly, mDvl1 was also upshifted when only the kinase domain of ULK1 was coexpressed (Fig. 1D). Therefore, we suspected that ULK1 directly phosphorylates mDvl1. To demonstrate, the construct of a kinasedead mutant form of ULK1, called ULK1 M92A (MA) [19], was used in the following experiment. When mDvl1 was coexpressed with ULK1 MA, no shift in mDvl1 was observed (Fig. 2C). Thus, the mobility shift of mDvl1 was dependent to the kinase activity of ULK1. To prove that the shift in mDvl1 is due to phosphorylation, we applied lambda protein phosphatase to the immunoprecipitated mDvl1. As expected, the treatment of
lambda protein phosphatase blocked the mobility shift of mDvl1 (Fig. 2C). In conclusion, the post-translational modification that took place on mDvl1 causing band shifts in immunoblot analyses was phosphorylation by ULK1. Although another kinase-dead form of ULK1, the K46I (KI) mutant [21], could not induce the band shift of mDvl1 as well, ULK2 that is most similar to ULK1 among the ULK family could induce the band shift of mDvl1 (Fig. 2D). The mDvl1 has three domains: the Dishevelled/Axin (DIX) domain, the PSD95/Dlg1/ZO-1 (PDZ) domain and the Dishevlled/ EGL-10/Pleckstrin (DEP) domain (Fig. 2F) [6]. To find the specific site of phosphorylation on mDvl1, fragmented mDvl1 in various combinations of the three domains were generated. Among them, the PDZ-DEP domain fragment was the smallest fragment showing band shifts in a ULK1 kinase activity-dependent manner (Fig. 2E). 3.3. ULK1 phosphorylates the PDZ-DEP region of Dsh at serine 254, 266 and 376 Since Atg1, a yeast ULK1 orthologue, prefers serine to threonine for phosphorylation [22], we substituted a serine with an alanine in the PDZ-DEP fragment of mDvl1. However, a single serine substitution did not eliminate the band shifts by ULK1 (data not shown). We concluded that multiple serines in mDvl1 were phosphorylated by ULK1. As mDvl1 has a poly-serine motif in the linker region, it was difficult to find changes in serine phosphorylation through alanine scanning analyses. Therefore, we took another approach for phosphorylation mapping. Since Dishevelled is a core element in Wnt signaling pathway, we assumed that the regulation of Dishevelled, such as phosphorylation by ULK1, should be highly conserved from Drosophila to humans [23]. Therefore, we performed the alanine scanning in Drosophila Dishevelled (Dsh), which lacks the polyserine region. Dsh also showed band shifts dependent on ULK1activity (Fig. 3A) and a much more prominent three-band-shift was detected with the PDZ-DEP fragment of Dsh (Fig. 3B). Hence,
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Fig. 2. ULK1 inhibits canonical Wnt/b-catenin signaling pathway and phosphorylates mDvl1 at the PDZ-DEP region. A-B, HEK293T cells were transfected with pSuperTop and pRL-TK reporter plasmids with scramble (Scr) siRNA or ULK1 siRNA (#1, #2, and #3) as indicated. Cells were harvested 48 h after transfection, and luciferase activity was measured by the dual luciferase assay system. Wnt3a-CM was added to cells for 24 h before harvesting cells. Data represent average values from triplicate experiments. Cell lysates were used for detecting expression of ULK1 and tubulin by using anti-ULK1 and anti-tubulin antibodies. ***p < 0.0001. C, HEK293T cells were transfected with Flag-mDvl1, Myc-ULK1 wild type (WT) and Myc-ULK1 M92A (MA) as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL were used for detecting expression of mDvl1 and ULK1 using antiFlag and anti-Myc antibodies. Immunoprecipitated samples were treated with lambda protein phosphatase (PP) as indicated. After de-phosphorylation, immunoprecipitated samples were detected by anti-Flag antibody. D, HEK293T cells were transfected with Flag-mDvl1 and Myc-ULK1 WT, Myc-ULK1 K46I (KI), Myc-ULK1 M92A (MA) and Myc-ULK2 as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL were detected with anti-Myc antibody. Immunoprecipitated samples were detected with anti-Flag antibody. E, HEK293T cells were transfected with Flag-mDvl1 PDZ-DEP fragment, Myc-ULK1 and Myc-ULK1 K46I (KI) as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL were used for detecting expression of ULK1 using anti-Myc antibody. Immunoprecipitated samples were detected by anti-Flag antibody. F, a schematic representation of the domain structure of mDvl1.
Fig. 3. ULK1 phosphorylates the PDZ-DEP region of Dsh at serine 254, 266 and 376. A, HEK293T cells were transfected with Flag-Dsh, Myc-ULK1 WT and Myc-ULK1 K46I (KI) as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL were used for detecting expression of ULK1 by using anti-Myc antibody. Immunoprecipitated samples were detected by anti-Flag antibody. BeC, HEK293T cells were transfected with Flag-Dsh PDZ-DEP fragments (WT and mutants) and Myc-ULK1 as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL were used for detecting expression of ULK1 by using anti-Myc antibody. Immunoprecipitated samples were detected by anti-Flag antibody. D, Drosophila eyes expressing transgenes using the eye-specific GAL4 driver (Gmr-GAL4).
it was easier to find the serine residues responsible for the shifts with alanine scanning (Fig. 3B). Among the Dsh mutants in which serines were replaced to alanines in the PDZ-DEP region, S254A, S266A, and S376A mutants displayed a two-band-shift (Fig. 3B). When these serine sites were replaced concurrently to alanines as in the mutant 3A, the band shift was no more detected by ULK1 coexpression (Fig. 3C).
Among the three serine residues, serines 266 and 376 are conserved between fruit fly and mouse. Based on this, the functional importance of these two sites was studied through expressing Dsh with serine 266 and 376 mutations in the fruit fly eye. Transgenic flies expressing exogenous Dsh with mutations in the two serines to alanines (2A) and to their phosphomimetic forms, aspartic acids (2D) and glutamic acids (2E), were made
Please cite this article as: S.-H. Hwang et al., ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.139
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respectively. Upon activation of Wnt signaling in the fly, the eyes get smaller and the ommatidial arrays collapse [24]. When exogenous Dsh was expressed in the eyes of the flies using Gmr-GAL4, the eyes expressing Dsh WT and 2A mutant were severely damaged whereas the level of damage was much less severe in Dsh 2D and 2E mutants (Fig. 3D). In summary, phosphorylation of serine 266 and 376 on Dsh results in decreased activation of Wnt signaling pathway.
3.4. ULK1 inhibits formation of Dsh-containing protein complex through phosphorylation The protein band shift caused by ULK1 was reduced but still existed when the phosphorylation sites found in PDZ-DEP fragment were replaced in full length Dsh. Hence, we repeated the phosphorylation site mapping to find additional protein band shifts in larger fragments of Dsh, and finally in the mutant (7A) substituting a total of seven serines with alanines on S239, S247, S254, S266, S376, S554 and S555, no ULK1-dependent band shift was observed (Fig. 4A). Among these serine sites in the fly protein, we performed a mutagenesis on only the conserved serine positions in mDvl1 and discovered that band shifts caused by ULK1 were severely decreased (data not shown). However, from the fact that there was slight band shift remaining, additional phosphorylations that are not conserved between fruit fly and humans seem to exist in mDvl1. Both mDvl1 and Dsh exhibited an increase in band intensity when the band shift was induced by ULK1. Thus, we hypothesized that ULK1-induced phosphorylation may dissolve insoluble forms of Dsh proteins from cell lysate pellets, instead of increasing the stability of Dsh. In fact, ULK1 co-expression significantly decreased the amount of Dsh WT protein found in the pellet fraction.
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However, in the same condition, Dsh 7A mutant protein still remained in the pellet fraction (Fig. 4B). These results suggested that ULK1 functions as a kinase that directly phosphorylates and mobilizes Dsh proteins from the pellet to the cytosolic fraction. We suspected that the pellet fraction includes the nuclei and the Dsh mutant protein forms insoluble protein complexes with nuclear proteins. Surprisingly, among the 7 serines, when only serine 266 was mutated to alanine in Dsh, a thick protein band was observed over 200 kDa size (Fig. 4C). This high molecular weight protein complex was SDS-resistant and was also detected when Dsh WT was overexpressed, suggesting that the complex is better formed with Dsh S266A mutant than with WT. Previous data showed that Dsh forms a protein complex in the nucleus with b-catenin and TCF, and plays a critical role in the target gene induction for Wnt signaling pathway [25]. Indeed, we confirmed that there was an interaction between b-catenin, TCF and mDvl2 (self-oligomerization) in the high molecular weight complex (Fig. 4D, E, F). In this experiment, we used b-catenin with S45Y mutation which stabilizes the protein [26]. In conclusion, Dsh serine 266 phosphorylation by ULK1 inhibits intranuclear interaction between Dsh and other components of Wnt signaling pathway, such as b-catenin and TCF, suggesting that ULK1dependent phosphorylation of Dsh is critical for the regulation of Wnt signaling pathway.
4. Discussion 4.1. Dsh as the new target of ULK1 and the role of ULK1 in Wnt/bcatenin signaling pathway In this research, we have proposed that ULK1 is involved in Wnt/
b-catenin signaling pathway by phosphorylating Dsh. Hence, we
Fig. 4. ULK1 inhibits a formation of Dsh-containing complex through phosphorylation. A, HEK293T cells were transfected with Flag-Dsh (WT or 7A) and Myc-ULK1 as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL were used for detecting expression of ULK1 by using anti-Myc antibody. Immunoprecipitated samples were detected by anti-Flag antibody. B, HEK293T cells were transfected with Flag-Dsh (WT or 7A) and Myc-ULK1 as indicated. WCL were used for detecting expression of Dsh by using anti-Flag antibody and ULK1 by using anti-Myc antibody. Pellet samples were used for detecting expression of Dsh by using anti-Flag antibody and ULK1 by using anti-Myc antibody. C, HEK293T cells were transfected with Flag-Dsh (WT or 266A) as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. Immunoprecipitated samples were detected with anti-Flag antibody. D-F, HEK293T cells were transfected with Myc-mDvl2, Myc-b-catenin S45Y, HA-TCF and Flag-Dsh 266A as indicated. Cell lysates were immunoprecipitated by anti-Flag antibody. WCL and immunoprecipitated samples were detected by anti-Myc antibody, anti-Flag antibody and anti-HA antibody as indicated. G, proposed model. A novel signaling mechanism to suppress Wnt/b-catenin signaling by ULK1.
Please cite this article as: S.-H. Hwang et al., ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.139
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have provided evidence to support the idea. First, ULK1 knockdown cells displayed increased activation of Wnt/b-catenin signaling pathway. Second, ULK1 interacted with and phosphorylated Dsh. Third, the phosphomimetic form of Drosophila Dsh showed lower activity of Wnt signaling in Drosophila. Fourth, ULK1 mobilized Dsh from the pellet fraction to the soluble cell lysates. Fifth, among the 7 serine residues that ULK1 phosphorylated, replacement of serine to alanine on the residue 266 in the PDZ domain of Dsh resulted in increased formation of the SDS-resistant high molecular weight complex by inducing stronger interactions between Dsh and both b-catenin and TCF. Collectively, our data established a novel regulatory mechanism for Wnt/b-catenin signaling pathway by ULK1dependent phosphorylation of Dsh (Fig. 4G), implicating the critical role of ULK1-activating signals such as nutrient starvation. 4.2. A new therapeutic target for tumors with aberrant Wnt signaling Wnt signaling is highly activated in many types of cancer, especially in colon cancer [2]. Therefore, specific inhibitors for Wnt signaling are potentially promising drugs for cancer treatments. Earlier researches have already reported the role of autophagy in tumor development [27]. For example, autophagy inhibits tumorigenesis by degrading misfolded proteins, defective mitochondria, and aggregated proteins, and thus relieves the cells from stress conditions [27]. Therefore, not only the function of ULK1 in autophagy but also the phosphorylation activity on Dsh may be promising therapeutic targets for the tumorigenesis with aberrant Wnt signaling if a specific activator of ULK1 is developed. Since rapalogs induces ULK1 [28], it will be worthwhile to examine whether the drugs are effective in treating various Wnt-dependent tumors. Abbreviations The abbreviations used are: Dsh, Dishevelled; mDvl1, mouse Dishevelled1; ULK1, UNC51-like kinase 1; FIP200, FAK family kinase-interacting protein of 200 kDa; KD, kinase domain; S/T, serine/threonine-rich domain; CT, C-terminus domain; siScr, siScramble; DIX, Dishevelled/Axin domain; PDZ, PSD95/Dlg1/ZO-1 domain; DEP, Dishevlled/EGL-10/Pleckstrin domain; Wnt3a-CM, Wnt3a-conditioned medium; PP, Lambda protein phosphatase. Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article. Author contributions The authors have made the following declarations about their contributions: Conceived and designed the experiment: S.H.H. and J.C. Performed the experiments: S.H.H. and S.H.B. Analyzed the data: S.H.H., S.H.B. and J.C. Wrote the paper: S.H.H., K.S.K., D.K. and J.C. Acknowledgments J.C. was supported by a grant (HI17C0328) of the Korea Health Technology R&D project through the Korea Health Industry Development Institute, funded by the Ministry of Health and Welfare. S.H.H., S.H.B. and J.C. were supported by BK21 Plus Program from the Ministry of Education, Republic of Korea. S.H.H. was supported by Global Ph.D. Fellowship Program from National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning, Republic of Korea. mDvl1 was a generous gift from Dr. Roel Nusse (Stanford
University, CA). Myc-ULK1, Myc-ATG13, Myc-FIP200 and Myc-ULK2 were generous gifts from Dr. Do-Hyung Kim (University of Minnesota, MN). Myc-mDvl2, pSuperTOP Flash, pSuperFOP Flash, pRLTK and Wnt3a-conditioned medium were generous gifts from Dr. Eek-hoon Jho (University of Seoul, Korea). Myc-b-catenin S45Y and HA-TCF were generous gifts from Dr. Sung Hee Baek (Seoul National University, Korea). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.139. References [1] R. van Amerongen, R. Nusse, Towards an integrated view of Wnt signaling in development, Development 136 (2009) 3205e3214. [2] A. Klaus, W. Birchmeier, Wnt signalling and its impact on development and cancer, Nat. Rev. Canc. 8 (2008) 387e398. [3] J.R.K. Seifert, M. Mlodzik, Frizzled/PCP signalling: a conserved mechanism regulating cell polarity and directed motility, Nat. Rev. Genet. 8 (2007) 126e138. [4] C. Niehrs, The complex world of WNT receptor signalling, Nat. Rev. Mol. Cell Biol. 13 (2012) 767e779. [5] W. Kim, M. Kim, E.H. Jho, Wnt/beta-catenin signalling: from plasma membrane to nucleus, Biochem. J. 450 (2013) 9e21. [6] C. Gao, Y.-G. Chen, Dishevelled: the hub of Wnt signaling, Cell. Signal. 22 (2010) 717e727. [7] C. Gao, W. Cao, L. Bao, et al., Autophagy negatively regulates Wnt signalling by promoting Dishevelled degradation, Nat. Cell Biol. 12 (2010). 781-U738. [8] C. He, D.J. Klionsky, Regulation mechanisms and signaling pathways of autophagy, Annu. Rev. Genet., pp. 67-93. [9] O. Florey, M. Overholtzer, Autophagy proteins in macroendocytic engulfment, Trends Cell Biol. 22 (2012) 374e380. [10] S. Shimizu, Biological roles of alternative autophagy, Mol. Cell. 41 (2018) 50e54. [11] M.S. Lee, Overview of the minireviews on autophagy, Mol. Cell. 41 (2018) 1e2. [12] D. Papinski, C. Kraft, Regulation of autophagy by signaling through the atg1/ ULK1 complex, J. Mol. Biol. 428 (2016) 1725e1741. [13] J.H. Joo, B. Wang, E. Frankel, et al., The noncanonical role of ULK/ATG1 in ERto-golgi trafficking is essential for cellular homeostasis, Mol. Cell 62 (2016) 491e506. [14] H. Konno, K. Konno, G.N. Barber, Cyclic dinucleotides trigger ULK1 (ATG1) phosphorylation of STING to prevent sustained innate immune signaling, Cell 155 (2013) 688e698. [15] D. Saleiro, S. Mehrotra, B. Kroczynska, et al., Central role of ULK1 in type I interferon signaling, Cell Rep. 11 (2015) 605e617. [16] J. Kim, Y.M. Lim, M.S. Lee, The role of autophagy in systemic metabolism and human-type diabetes, Mol. Cell. 41 (2018) 11e17. [17] N. Mizushima, The role of the Atg1/ULK1 complex in autophagy regulation, Curr. Opin. Cell Biol. 22 (2010) 132e139. [18] X. Zhou, J.R. Babu, S. da Silva, et al., Unc-51-like kinase 1/2-mediated endocytic processes regulate filopodia extension and branching of sensory axons, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 5842e5847. [19] C.H. Jung, C.B. Jun, S.-H. Ro, et al., ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery, Mol. Biol. Cell 20 (2009) 1992e2003. [20] J. Kim, M. Kundu, B. Viollet, et al., AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1, Nat. Cell Biol. 13 (2011) 132eU171. [21] E.Y.W. Chan, A. Longatti, N.C. McKnight, et al., Kinase-inactivated ULK proteins inhibit autophagy via their conserved C- terminal domains using an atg13independent mechanism, Mol. Cell Biol. 29 (2009) 157e171. [22] D. Papinski, M. Schuschnig, W. Reiter, et al., Early steps in autophagy depend on direct phosphorylation of Atg9 by the Atg1 kinase, Mol. Cell 53 (2014) 471e483. [23] B.T. MacDonald, K. Tamai, X. He, Wnt/beta-Catenin signaling: components, mechanisms, and diseases, Dev. Cell 17 (2009) 9e26. [24] A. Penton, A. Wodarz, R. Nusse, A mutational analysis of dishevelled in Drosophila defines novel domains in the dishevelled protein as well as novel suppressing alleles of axin, Genetics 161 (2002) 747e762. [25] X.-q. Gan, J.-y. Wang, Y. Xi, et al., Nuclear Dvl, c-Jun, beta-catenin, and TCF form a complex leading to stabilization of beta-catenin-TCF interaction, JCB (J. Cell Biol.) 180 (2008) 1087e1100. [26] M.A. Buendia, Genetic alterations in hepatoblastoma and hepatocellular carcinoma: common and distinctive aspects, Med. Pediatr. Oncol. 39 (2002) 530e535. [27] R. Mathew, V. Karantza-Wadsworth, E. White, Role of autophagy in cancer, Nat. Rev. Canc. 7 (2007) 961e967. [28] Y.C. Kim, K.L. Guan, mTOR: a pharmacologic target for autophagy regulation, J. Clin. Invest. 125 (2015) 25e32.
Please cite this article as: S.-H. Hwang et al., ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.139