Signaling by the Wnt family of secreted glycoproteins controls a wide range of cellular processes, including cell growth, cell polarity, cell differentiation and cell movement, during early development and tissue homeostasis (1). The canonical Wnt/β-catenin signaling functions by regulating the levels of transcriptional co-activator β-catenin (2). Without Wnt stimulation, cytoplasmic β-catenin is targeted for destruction by a complex consisting of the scaffold protein, Axin and APC and the serine/threonine kinases, CK1 and GSK3. Within this complex, CK1 and GSK3 sequentially phosphorylate β-catenin, which in turn undergoes β-TrCP-mediated ubiquitination and is degraded by the proteasome. Consequently, cytoplasmic β-catenin is kept at a low level, resulting in the transcription of Wnt target genes being repressed by the TCF/LEF family of proteins. When a Wnt ligand binds to Frizzled receptor and LRP6 co-receptor, GSK3-mediated phosphorylation of β-catenin is inhibited through the action of Dishevelled and the binding of Axin to LRP6. The stabilized β-catenin accumulates and moves to the nucleus, subsequently interacting with TCF/LEF factors and activating the expression of target genes.
Ubiquitination is a reversible posttranslational modification that affects the stability, positioning, and function of modified proteins. This modification is catalyzed by a three-enzyme cascade including a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). E3 ligases are crucial for determining the specificity of the ubiquitinated substrates (3). According to their structural characteristics, E3 ligases are classified into two major categories: the HECT domain family and the RING domain family (4). RNF152 is an E3 ubiquitin ligase with RING domain and single-pass transmembrane (TM) domain, which is mainly localized in lysosomes (5). RNF152 exhibits pro-apoptotic activity when overexpressed (5) and negatively controls mTORC1-mediated signaling by targeting RagA or Rheb GTPases for ubiquitination (6, 7). In addition, RNF152 positively regulates TLR/IL-1R-mediated inflammatory response by facilitating oligomerization of the adaptor protein MyD88, which is independent of its E3 ligase activity (8). In zebrafish embryos, RNF152 is involved in the development of the eyes and brains, and in Notch-Delta signaling (9).
In this study, we observed that RNF152 controls negatively Wnt/β-catenin signaling in
It has been shown that RNF152 inhibits cell proliferation and its expression is down-regulated in diverse types of cancers (10). However, not much is known about the biological activities of RNF152 in vertebrate early development. First, as a strategy to explore the developmental functions of RNF152, we overexpressed wild type (WT)
Prior to the loss-of-RNF152 function analysis in
For loss-of-function analysis of RNF152, we adopted an anti-sense morpholino oligo (MO)-mediated knockdown approach. For this, two different MOs (MO-a and MO-b) were designed to target distinct sequences around translation initiation sites of
Since overexpression of
We further tested whether the function of RNF152 is relevant to NC formation. As shown in Supplementary Fig. 3A, B, co-expression of
Using the animal cap assays, we also investigated the effects of knockdown of
Dishevelled (Dsh), a key mediator of Wnt/β-catenin signaling, undergoes dynamic oligomerization, resulting in formation of cytoplasmic puncta (12, 13). These puncta contain head-to-tail polymers, which correlate with the ability of Dsh to activate this pathway. The polymerization of Dsh at the plasma membrane provides a platform for the clustering of Wnt-LRP5/6-Fz to form signalosomes. It has been shown that RNF152 facilitates oligomerization of the adaptor protein MyD88 independently of its E3 ligase activity, resulting in activation of TLR/IL-1R-mediated inflammatory response (8). Given these findings, it is tempting to speculate that RNF152 might be involved in Dsh polymerization to negatively control Wnt/β-catenin signaling. To test this hypothesis, we first examined whether RNF152 would interact physically with Dsh. Notably, co-immunoprecipitation experiments demonstrated that the former associates with the latter in an E3 ligase-independent manner (Fig. 4A). In addition, we found that WT RNF152 markedly reduced the association of Myc-tagged and GFP-tagged Dsh in a dose-dependent fashion (Fig. 4B). RNF152(CS) also inhibited more strongly their interaction, indicating its E3 ligase-independent effect on Dsh oligomerization. We next tested the effects of overexpression of RNF152 on the formation of Dsh puncta in cells. As shown in Fig. 4C, GFP-Dsh exhibited a punctate cytoplasmic pattern in control animal cap cells. Co-expression of
In this study, we have demonstrated that RNF152 functions as a negative regulator of Wnt/β-catenin signaling in
RNF152 is an E3 ubiquitin ligase with a transmembrane (TM) domain which mediates its localization to lysosomes (5). Notably, the RNF152(CS) mutant but not the RNF152(dTM) can inhibit Wnt/β-catenin signaling (Fig. 1), suggesting that the membrane association but not the E3 ligase activity of RNF152 is critical for this inhibitory function. Similarly, the TM domain-mediated membrane localization of RNF152 but not its enzymatic activity is indispensable for its role in TLR/IL-1R-mediated signaling (8). In contrast, RNF152 acts on RagA and Rheb as a direct E3 ubiquitin ligase anchored to the lysosomal surface to negatively regulate mTORC1 activity (6, 7). As such, it is likely that RNF152 has E3 ligase-dependent and -independent functions, both of which require its membrane localization. Our results showed that RNF152(CS) mutant could inhibit Wnt-dependent transcriptions more strongly than WT RNF152 (Supplementary Fig. 3), even though their respective mRNAs were injected at the same concentration. RNF152 has been shown to ubiquitinate itself, which leads to its short half-life (14). The steady-state protein level of E3 ligase-defective RNF152 was increased compared to that of WT RNF152 (Fig. 4B). Thus, the stronger effects of RNF152(CS) on Wnt/β-catenin signaling may be due to abrogation of its autoubiquitination activity. RNF152 appears not to act as an E3 ligase to ubiquitinate a signaling mediator in the Wnt/β-catenin pathway. However, since the E3 ligase activity of RNF152 functions to shorten its own half-life, this enzymatic activity of RNF152 can affect the kinetics of Wnt/β-catenin signaling.
As shown in this work, the self-association of Dsh proteins and the formation of their cytoplasmic puncta were impeded in RNF152-overexpressed cells. This inhibitory effect on Dsh polymerization could also be seen in cells overexpressing RNF152(CS). These results suggest that RNF152 might control negatively Dsh polymerization in an E3 ligase-independent fashion. Dsh polymerization provides a dynamic scaffold with a high concentration of binding sites for its partners such as Frizzled receptors and Axin to form signalosome (13). This signalosome assembly leads to the inhibition of GSK3-mediated phosphorylation of β-catenin and, as a consequence, to stabilization of β-catenin. Thus, high levels of RNF152 activity would disrupt signalosome formation, thereby repressing Wnt/β-catenin signaling. As a mechanism of action, RNF152 might regulate the equilibrium between diffuse Dsh monomers and Dsh polymers in the cytoplasm or the intermolecular interactions between Dsh proteins, which remains to be investigated in the future. It has been shown that Dsh puncta do not colocalize with endo-cytic markers or lipid dyes or associate with membranes (15). In contrast, RNF152 colocalizes with early endosomes and late endosomes as well as lysosomes as revealed by confocal microscopy (8). Future experiments are warranted to elucidate how the formation of Dsh puncta is relevant to membrane-anchored RNF152.
This study was compliant with all relevant ethical regulations regarding animal research. All animal experiments were approved by the Institutional Animal Care and Use Committee at the Asan Institute for Life Sciences, Asan Medical Center.
For RT-PCR analysis, total RNA was extracted from embryos or animal caps with TRIzol reagent and treated with RNase-free DNase I to remove genomic DNA. Approximately 5 μg of total RNA was reverse-transcribed using random hexamers and M-MLV reverse transcriptase (Promega). PCRs were carried out in a standard 50 μl of PCR with Taq polymerase. The numbers of PCR cycles for each primer set were determined empirically to maintain amplification in the linear range. The following primers were used:
For coimmunoprecipitation of proteins, cultured cells were lysed in Triton X-100 lysis buffer (50 mM Tris (pH 7.6), 1% Triton X-100, 50 mM NaCl, 1 mM EDTA, 1 mM sodium vanadate, 10 mM NaF, 1 mM PMSF, 20 μg/ml aprotinin, 20 μg/ml leupeptin). Proteins were collected from cell lysates using Protein G Mag Sepharose (GE Healthcare)-bound antibodies overnight at 4°C on a rotating platform. The eluted immunoprecipitates and protein lysates were separated by 6-10% SDS-PAGE. Western blotting was carried out according to a standard protocol with anti-β-catenin (1:1000, Santa Cruz), anti-Myc (1:1000, Santa Cruz), anti-Flag (1:1000, Sigma), anti-GFP (1:1000, Santa Cruz) and anti-β-actin (1:1000, Santa Cruz) antibodies.
For reporter assays in
The subcellular localization of GFP-Dsh was examined using an assay described in (19). Briefly, four-cell stage embryos were injected into the animal pole region of all blastomeres with GFP-Dsh (1 ng) with or without mRNAs as indicated. Animal cap explants were excised at late blastula stages, fixed in 4% paraformaldehyde in PBS for 2 h, rinsed in PBS and directly mounted for GFP-tagged proteins. The image was acquired on a confocal laser-scanning microscope (Zeiss).
Statistical significance was determined by using
Additional materials and methods can be found in the Supplementary data.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2020R1F1A1075945).
The authors have no conflicting interests.