A coated vesicle-associated kinase of 104 kDa (CVAK104) induces lysosomal degradation of frizzled 5 (Fzd5)

Abstract

Receptor internalization is recognized as an important mechanism for controlling numerous cell surface receptors. This event contributes not only to regulate signal transduction but also to adjust the amount of cell surface receptors. Frizzleds (Fzds) are seven-pass transmembrane receptor family proteins for Wnt ligands. Recent studies indicated that Fzd5 is internalized in response to Wnt stimulation to activate downstream signaling pathways. After internalization, it appears that Fzd5 is recycled back to the plasma membrane. However, whether internalized Fzd5 is sorted to lysosomes for protein degradation remains unclear. We here report that a coated vesicle-associated kinase of 104 kDa (CVAK104) selectively induces lysosomal degradation of Fzd5. We identify CVAK104 as a novel binding partner of Dishevelled (Dvl), a scaffold protein in the Wnt signaling pathway. Interestingly, we find that CVAK104 also interacts with Fzd5 but not with Fzd1 or Fzd4. CVAK104 selectively induces intracellular accumulation of Fzd5 via the clathrin-mediated pathway, which is suppressed by coexpression of a dominant negative form of Rab5. Fzd5 is subsequently degraded by a lysosomal pathway. Indeed, knockdown of endogenous CVAK104 by RNA interference results in an increase in the amount of Fzd5. In contrast, Wnt treatment induces Fzd5 internalization but does not stimulate its degradation. Overexpression or knockdown of CVAK104 results in a significant suppression or activation of the Wnt/beta-catenin pathway, respectively. These results suggest that CVAK104 regulates the amount of Fzd5 by inducing lysosomal degradation, which probably contributes to the suppression of the Wnt signaling pathway.

FIGURE 1.

Identification of CVAK104 as a novel Dvl-interacting protein. A, schematic diagrams of Dvl deletion constructs used in this study. Interactions with CVAK104 are summarized on the right of each construct. +, positive; −, negative. B, anti-FLAG immunoprecipitates of lysates obtained from cells stably expressing FLAG-tagged Dvl deletion constructs were subjected to SDS-PAGE, followed by silver staining. The arrowheads indicate proteins that were identified by mass spectrometry (top, CVAK104; bottom, NRX). C, lysates of MCF-7 cells were subjected to immunoprecipitation with anti-CVAK104 antibody. The precipitates were analyzed by immunoblotting with the indicated antibodies. Immunoprecipitations with rabbit IgG and without lysates were also performed as controls. D, Myc-CVAK104 and FLAG-tagged Dvl deletion constructs were expressed in COS-7 cells. The lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibody. The precipitates were analyzed by immunoblotting with the indicated antibodies. E, schematic diagrams of CVAK104 deletion constructs. Interactions with Dvl or Fzd5 are summarized on the right of each construct. +, positive; −, negative. F, Myc-tagged Dvl and FLAG-tagged CVAK104 deletion constructs were expressed in COS-7 cells. The lysates were subjected to immunoprecipitation with anti-FLAG antibody. The precipitates were analyzed by immunoblotting with the indicated antibodies.

FIGURE 2.

CVAK104 selectively induces the intracellular accumulation of Fzd5. A, 293 cells were transfected with Fzd-GFP alone. At 48 h after transfection, cells were subjected to an internalization assay and visualized by direct observation of GFP fluorescence. Scale bar, 5 μm. B, 293 cells were transfected with both Fzd-GFP and FLAG-CVAK104 and subjected to an internalization assay. Cells were visualized by direct observation of GFP fluorescence and immunostaining with anti-FLAG antibody. A merged image of the signals for Fzd5-GFP (green) and FLAG-CVAK104 (red) is also indicated. Scale bar, 5 μm. C, quantification of cells with internalized Fzd-GFP in A and B. All data are mean ± S.E. (n = 3). *, p < 0.01.

FIGURE 3.

CVAK104-induced Fzd5 accumulation occurs via the clathrin-dependent pathway. A, 293 cells were transfected with Fzd5-GFP and FLAG-CVAK104. At 48 h after transfection, cells were subjected to an internalization assay and visualized by direct observation of GFP fluorescence and staining with anti-EEA1 antibody. A merged image of the signals for GFP (green) and EEA1 (red) is also indicated. Scale bar, 5 μm. B, 293 cells expressing Fzd5-GFP and FLAG-CVAK104 were pretreated with DMSO, MDC, or nystatin for 1 h and were then subjected to an internalization assay in the presence of inhibitors. Scale bar, 5 μm. C, quantification of cells with internalized Fzd5-GFP in B. All data are mean ± S.E. (n = 3). *, p < 0.01. D, 293 cells expressing Fzd5-GFP and FLAG-CVAK104 with or without Myc-Rab5 S34N were subjected to an internalization assay and then visualized by direct observation of GFP fluorescence and immunostained with anti-FLAG antibody. Scale bar, 5 μm.

FIGURE 4.

CVAK104 selectively interacts with Fzd5. A, lysates of COS-7 cells expressing Fzd-GFP alone or with FLAG-CVAK104 were subjected to immunoprecipitation (IP) with anti-FLAG antibody. The precipitates were analyzed by immunoblotting with the indicated antibodies. B, lysates of COS-7 cells expressing Fzd-GFP with FLAG-tagged CVAK104 deletion constructs were subjected to immunoprecipitation with anti-FLAG antibody. The precipitates were analyzed by immunoblotting with the indicated antibodies. C, lysates of COS-7 cells expressing FLAG-CVAK104, Fzd5-GFP, and Myc-Dvl wild type (WT) or ΔDIX2 (Mut) were subjected to immunoprecipitation with anti-FLAG antibody. The precipitates were analyzed by immunoblotting with the indicated antibodies. D, lysates of COS-7 cells expressing FLAG-CVAK104, Myc-Dvl, and Fzd5-GFP wild type or K525A (KA) were subjected to immunoprecipitation with anti-FLAG antibody. The precipitates were analyzed by immunoblotting with the indicated antibodies. E, 293 cells were transfected with FLAG-CVAK104 and Fzd5-GFP. At 24 h after transfection, cells were treated with Wnt3a-conditioned medium (CM) for 6 h, and then the lysates were subjected to immunoprecipitation with anti-FLAG antibody. The precipitates were analyzed by immunoblotting with the indicated antibodies. F, 293 cells were transfected with FLAG-CVAK104 and Myc-Dvl. At 24 h after transfection, cells were treated with Wnt3a CM as described in E, and then immunoprecipitation and immunoblotting analyses were performed.

FIGURE 5.

Lysosomal degradation of Fzd5. A, 293 cells expressing Fzd5-GFP were treated with DMSO, chloroquine (100 μm), or MG132 (20 μm) for 10 h. The cells were fixed and visualized by direct observation of GFP fluorescence. Scale bar, 5 μm. B, 293 cells stably expressing Fzd-GFP were transfected with an empty (−) or FLAG-CVAK104 expression vector (+), and then cell lysates were analyzed by immunoblotting with the indicated antibodies. C, 293 cells stably expressing Fzd5-GFP were transfected with an empty (−) or FLAG-CVAK104 expression vector (+) or treated with control (−) or Wnt3a CM (+). The lysates were subjected to immunoblotting analysis with the indicated antibodies. D, 293 cells stably expressing Fzd5-GFP were transfected with an empty (−) or FLAG-CVAK104 expression (+) vector. Cells were treated with 100 μm chloroquine (Chlo) or 20 μm MG132 (MG) for 10 h. The lysates were subjected to immunoblotting with the indicated antibodies. E, 293 cells stably expressing Fzd5-GFP were transfected with control or CVAK104-siRNAs. At 48 h after transfection, the lysates were subjected to immunoblotting with the indicated antibodies.

FIGURE 6.

CVAK104 negatively regulates the Wnt/β-catenin pathway. A, 293 cells were transfected with an empty (−) or FLAG-CVAK104 expression vector (+) with the reporter plasmids. At 48 h after transfection, cells were treated with control (−) or Wnt3a CM (+) for 10 h, and then reporter gene expression assays were performed. -Fold activation values were measured relative to the levels of luciferase activity in cells transfected with an empty vector. All data are mean ± S.E. (n = 3). *, p < 0.01. B, 293 cells were transfected with control or CVAK104-siRNAs. At 48 h after transfection, cells were treated with Wnt3a CM, and then reporter gene expression assays were performed as described in A. All data are mean ± S.E. (n = 3). *, p < 0.01. C, the lysates of cells treated as in A were subjected to immunoblotting with the indicated antibodies. D, the lysates of cells treated as in B were subjected to immunoblotting with the indicated antibodies.