SCFβ-TRCP E3 ubiquitin ligase targets the tumor suppressor ZNRF3 for ubiquitination and degradation

Abstract

Wnt signaling has emerged as a major regulator of tissue development by governing the self-renewal and maintenance of stem cells in most tissue types. As a key upstream regulator of the Wnt pathway, the transmembrane E3 ligase ZNRF3 has recently been established to play a role in negative regulation of Wnt signaling by targeting Frizzled (FZD) receptor for ubiquitination and degradation. However, the upstream regulation of ZNRF3, in particular the turnover of ZNRF3, is still unclear. Here we report that ZNRF3 is accumulated in the presence of proteasome inhibitor treatment independent of its E3-ubiquitin ligase activity. Furthermore, the Cullin 1-specific SCF complex containing β-TRCP has been identified to directly interact with and ubiquitinate ZNRF3 thereby regulating its protein stability. Similar with the degradation of β-catenin by β-TRCP, ZNRF3 is ubiquitinated by β-TRCP in both CKI-phosphorylation- and degron-dependent manners. Thus, our findings not only identify a novel substrate for β-TRCP oncogenic regulation, but also highlight the dual regulation of Wnt signaling by β-TRCP in a context-dependent manner where β-TRCP negatively regulates Wnt signaling by targeting β-catenin, and positively regulates Wnt signaling by targeting ZNRF3.

Keywords: CKI; Wnt; ZNRF3; ubiquitination; β-TRCP.

Figure 1

ZNRF3 protein level is regulated by Cullin 1 based E3 ligases, in a 26S proteasome dependent manner. (A and B) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HeLa cells transfected with WT or RING-domain deleted (ΔRING) ZNRF3. Resulting cells were treated with MG132 (10 μmol/L) (A) or MLN4924 (10 μmol/L) (B) for 12 h before harvesting for IB analysis. (C–E) IB analysis of immunoprecipitations (IP) and WCL derived from HeLa cells co-transfected ZNRF3 with various Cullin family proteins (C), Skp1 (D) or Rbx1 (E) after treated with MG132 (10 μmol/L) for 12 h before being harvested for IB analysis. (F) IB analysis of WCL derived from U2OS cells lentivirally infected with control (sh-Scr) or multiple independent shRNAs against Cullin 1 (sh-Cullin1). Infected cells were selected with 1 μg/mL puromycin for 72 h to eliminate non-infected cells before harvesting. (G and H) IB analysis of WCL derived from the cell lines in (F) treated with CHX (100 mg/L) for different time point (G), relative protein levels were quantified and plotted in (H)

Figure 2

β-TRCP directly interacts with and promotes the degradation of ZNRF3. (A–C) IB analysis of IP product and WCL derived from HEK293T (A and C) or HeLa (B) cells transfected with indicated constructs, resulting cells were then treated with MG132 (10 μmol/L) for 12 h before harvesting. (D and E) IB analysis of WCL derived from HeLa cells co-transfected ZNRF3 with different TRCP encoding constructs, treated without (D) or with MG132 (10 μmol/L) (E) for 12 h before harvesting. (F and G) IB analysis of WCL derived from HeLa (F) or U2OS (G) cells lentivirally infected with control (sh-Scr) or independent shRNAs against β-TRCP1 (sh-TRCP1). Infected cells were selected with 1 μg/mL puromycin for 72 h to eliminate non-infected cells before harvesting. (H and I) IB analysis of WCL derived from cells generating in (F) and treated with CHX (100 mg/L) for different time points (H). The relative protein levels were quantified and plotted in (I). (J) IB analysis of ubiquitin-products and WCL derived from HEK293 cells transfected with indicated constructs. Where indicated Nickel-beads were used to pull down His-tagged Ub proteins

Figure 3

CKI synergizes with β-TRCP to mediate ZNRF3 degradation. (A–D) IB analysis of IP and WCL derived from HEK293 (A and C) or HeLa (B and D) cells transfected with indicated constructs, cell lysates were treated with or without phosphatase (PPase) for 30 min (A) or cells were treated with MG132 for 12 h (C) before harvesting. (E) IB analysis of WCL derived from HeLa cells infected with lentivirus for control (sh-Scr) or multiple independent shRNAs against CKIδ (sh-CKIδ). Infected cells were selected with 1 μg/mL puromycin for 72 h to eliminate non-infected cells before harvesting. (F) IB analysis of WCL derived from HeLa cells treated with different concentrations of CKI inhibitor D4476 overnight before harvesting. (G) IB analysis of ubiquitin-products and WCL derived from HEK293 cells transfected with indicated constructs treated with or without CKI inhibitors. Where indicated Nickel-beads were used to pull down His-tagged Ub proteins. (H–I) IB analysis of WCL derived from HeLa cells transfected with indicated constructs and treated with CHX (100 mg/L) for indicated time points (H), the relative protein levels were quantified and plotted in (I)

Figure 4

β-TRCP promotes the degradation of ZNRF3 in a degron-dependent manner. (A) A schematic illustration of the domain structure and putative β-TRCP-degron motifs in ZNRF3, as well as the sequence alignment of ZNRF3 among different species to illustrate evolutionary conservation of degrons. Where indicated, the canonical β-TRCP-degron motifs are shown. (B and C) IB analysis of IP and WCL derived from HeLa cells transfected with indicated constructs. (D) IB analysis of ubiquitin-products and WCL derived from HEK293 cells transfected with indicated constructs. Where indicated Nickel-beads were used to pull down His-tagged Ub proteins. (E and F) IB analysis of WCL derived from HeLa cells transfected with indicated constructs and treated with CHX (100 μmol/L) for indicated time points (E), the relative protein levels were quantified and plotted in (F)

Figure 5

Proposed models for the context-dependent roles of β-TRCP in regulating the Wnt signaling. (A) Under Wnt off conditions, β-TRCP formed a degradation complex to recognize and degrade CKIα/GSK3-mediated phosphorylated form of β-catenin, to repress β-catenin targeted genes, and maintain Wnt in silent status. (B) Under Wnt on conditions, the activated FZD/LRP5/6 receptor destructs β-catenin degradation complex and releases β-catenin downstream targeted genes. Simultaneously, as a feedback regulator of Wnt pathway, ZNRF3 degrades FZD to alleviate Wnt signal. Meantime, β-TRCP1 could promote the degradation of CKI-mediated phosphorylated form of ZNRF3, to sustain the Wnt pathway