A Coated Vesicle-associated Kinase of 104 kDa (CVAK104) Induces Lysosomal Degradation of Frizzled 5 (Fzd5)

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/β-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.Internalization of cell surface receptors is an important event to regulate signal transduction from the extracellular environment (1, 2). This event contributes to control the amount of receptors at the plasma membrane. Internalization mainly occurs via the clathrin-dependent pathway. It is characterized by the recruitment of adaptor protein (AP),² such as AP-2, and the assembly of a clathrin coat, which helps the inward budding of clathrin-coated vesicles (3). Internalized receptors are transported to early endosomes, from where they are either recycled back to the plasma membrane or directed to degradative components, such as lysosomes. Rab5, a member of the Rab family GTPase proteins that exert regulatory functions in the endocytic and exocytic trafficking, regulates the fusion of plasma membrane-derived vesicles with early endosomes and homotypic fusion among early endosomes (4).Accumulating data indicate that numerous regulatory proteins also play important roles in endocytic processes. Coated vesicle-associated kinase of 104 kDa (CVAK104) is one of these accessory proteins, which was recently discovered by mass spectroscopy analysis of AP preparations form bovine brain (5). Several groups reported that CVAK104 interacts with clathrin (5–7). In addition, CVAK104 binds to AP-2 and phosphorylates the β subunit of AP-2 in vitro, suggesting a role in the clathrin-mediated endocytosis (5). Furthermore, it was recently demonstrated that CVAK104 also functions in trafficking between the trans-Golgi network and endosomes. For example, knockdown of CVAK104 by small interfering RNAs (siRNAs) results in missorting of the lysosomal enzyme cathepsin D (6). CVAK104 also regulates sorting of t-SNARE proteins from the trans-Golgi network to late endosomes in which they function as an adaptor for docking and fusion of vesicles (7). These reports suggest an importance of CVAK104 in intracellular trafficking that occurs after endocytosis. The Wnt signaling pathway is evolutionarily conserved from nematodes to mammals and is involved in embryonic development and various human diseases, including cancer (8–10). In this signaling pathway, Dishevelled (Dvl) functions as an essential signal transducer from the Wnt receptors to downstream components. Dvl is composed of three conserved domains: an N-terminal Dishevelled-Axin (DIX) domain, a PSD95/Dlg/ZD1 (PDZ) domain in the middle, and a C-terminal Dishevelled-Egl10-pleckstrin (DEP) domain. It is well known that these three domains are required for protein-protein interaction to transduce signals to downstream targets. Dvl also possesses a region harboring positively charged (basic) amino acid residues (termed the basic region) (11–14). It is reported that the basic region is also required for interaction with several downstream signaling components. Indeed, Frat1 and NRX (nucleoredoxin) interact with Dvl through the basic region and the PDZ domain (15, 16). Furthermore, Par1 binds only to the basic region (17). These results suggest that the basic region plays a critical role in the function of Dvl.Frizzled (Fzd) receptors are seven-pass transmembrane proteins. The Fz genes were first identified in Drosophila in a screen for mutations that disrupt the polarity of epidermal cells in the adult fly (18). Ten genes encoding Fzds have been identified in the human genome (19), and the overall structure of Fzd receptors is well conserved among the 10 proteins and also throughout evolution (20, 21). Accumulating evidence indicates that Fzd receptors are internalized in response to their Wnt ligands. Wnt5a induces the internalization of Fzd4 (22). Wnt3a induces the internalization of Fzd5 via the clathrin-dependent pathway (23). In addition, Wnt11 cooperates with atypical receptor-related tyrosine kinase to promote the internalization of Fzd7 via the β-arrestin-2-dependent pathway (24). These ligand-dependent internalizations of Fzd receptors are required for activating signaling pathways. Recent studies also demonstrate that Dvl not only functions as a signal transducer but also plays important roles in internalization of the Fzd receptor. It has been reported that Dvl recruits β-arrestin-2 to internalize Fzd4 in response to Wnt5a treatment (22) and that interaction between Dvl and AP-2 is needed to stimulate internalization of Fzd4 (25). After internalization, cell surface receptors are generally recycled back to the plasma membrane or sorted to lysosomes for protein degradation. It has also been reported that Fzd5 internalized in a ligand-dependent manner appears to be recycled back to the plasma membrane, because internalized Fzd5 co-localizes with Rab11, which plays an important role in the recycling process (23). However, whether receptor degradation, another common consequence after receptor internalization, occurs in the case of Fzd5 still remains unknown.In this study, we search for in vivo Dvl binding partners and identify CVAK104 as a novel Dvl-interacting protein. We also find that CVAK104 interacts with Fzd5 and that expression of CVAK104 induces intracellular accumulation of Fzd5 through the clathrin-dependent pathway. Interestingly, CVAK104 selectively interacts with and induces accumulation of Fzd5 but not Fzd1 or Fzd4. In addition, we find that Fzd5 internalized in the presence of CVAK104 is subsequently degraded by a lysosomal pathway, suggesting a novel mechanism for regulating the turnover of a specific subclass of Fzd receptors.