HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK

ACK (activated Cdc42-associated tyrosine kinase) (also Tnk2) is an ubiquitin-binding protein and plays an important role in ligand-induced and ubiquitination-mediated degradation of epidermal growth factor receptor (EGFR). Here we report that ACK is ubiquitinated by HECT E3 ubiquitin ligase Nedd4-1 and degraded along with EGFR in response to EGF stimulation. ACK interacts with Nedd4-1 through a conserved PPXY WW-binding motif. The WW3 domain in Nedd4-1 is critical for binding to ACK. Although ACK binds to both Nedd4-1 and Nedd4-2 (also Nedd4L), Nedd4-1 is the E3 ubiquitin ligase for ubiquitination of ACK in cells. Interestingly, deletion of the sterile alpha motif (SAM) domain at the N terminus dramatically reduced the ubiquitination of ACK by Nedd4-1, while deletion of the Uba domain dramatically enhanced the ubiquitination. Use of proteasomal and lysosomal inhibitors demonstrated that EGF-induced ACK degradation is processed by lysosomes, not proteasomes. RNA interference (RNAi) knockdown of Nedd4-1, not Nedd4-2, inhibited degradation of both EGFR and ACK, and overexpression of ACK mutants that are deficient in either binding to or ubiquitination by Nedd4-1 blocked EGF-induced degradation of EGFR. Our findings suggest an essential role of Nedd4-1 in regulation of EGFR degradation through interaction with and ubiquitination of ACK.Activated Cdc42-associated tyrosine kinase (ACK) (also Tnk2) is a member of the type VIII tyrosine kinase family. Activation of ACK, including both ACK1 and ACK2, occurs in response to signaling of epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF) receptor, insulin receptor, Gas-6 receptor (Mer), M3 muscarinic receptor, integrins, or proteoglycan (3, 7, 11, 23, 26, 30, 44, 47). In Drosophila, D-ACK mediates the function of Cdc42 in dorsal closure during embryonic development (31). The ACK homologue, Ark-1, in Caenorhabditis elegans negatively regulates EGF signaling (15).A number of studies suggest a role for ACK in EGFR degradation. ACK1 and ACK2, two alternatively spliced isoforms, possess a highly conserved clathrin-binding motif and interact with clathrin (37, 45). Overexpression of ACK2 severely impairs transferrin receptor endocytosis, causes aberrant localization of AP-2, and induces changes in clathrin assembly. Furthermore, ACK2 interacts with sorting nexin 9 (SNX9, also named SH3PX1), a member of the sorting nexin family, via its proline-rich domain 1 and phosphorylates SNX9 to facilitate the degradation of EGF receptors (22). In C. elegans, Ark-1 genetically interacts with UNC101, the homologue of mammalian clathrin-associated protein AP47, and SLI-1, the homologue of mammalian Cbl that is an E3 ubiquitin ligase for ubiquitination of EGFR, and negatively regulates EGFR signaling (15).Our previous studies showed that ACK1 interacts with EGFR upon EGF stimulation via a region at the carboxyl terminus, designated the EGFR-binding domain (EBD), which is highly homologous to the EGFR/ErbB2-binding domain of Gene-33/Mig-6/RALT (32, 43). The interaction of ACK1 with EGFR is dependent on kinase activity and tyrosine phosphorylation of EGFR. Immunofluorescent staining using anti-EGFR and GFP-ACK1 indicates that ACK1 is colocalized with EGFR on large vacuolar structures upon EGF stimulation. Suppression of the expression of ACK1 by ACK-RNA interference (RNAi) inhibits ligand-induced degradation of EGFR, suggesting that ACK1 plays an important role in the regulation of EGFR degradation in cells. Furthermore, we identified ACK1 as an ubiquitin-binding protein. Through an ubiquitin association (Uba) domain at the carboxyl terminus, ACK1 is capable of interacting with both poly- and monoubiquitin. Overexpression of an Uba domain deletion mutant of ACK1 blocked the ligand-dependent degradation of EGFR, suggesting that ACK1 regulates EGFR degradation via its Uba domain. Thus, ACK1 senses EGF signaling and regulates degradation of EGFR.EGF-induced degradation of EGFR is mediated by ubiquitination (16). The ubiquitination of EGFR is activated upon EGF stimulation by recruiting the RING family E3 ubiquitin ligase Cbl to pY1045 (20, 21). This ubiquitination functions as a sorting signal for transporting EGFR to lysosomes for degradation (14). Nedd4, the HECT domain-containing E3 ubiquitin ligase, is also involved in the regulation of EGFR trafficking by ubiquitination of endocytic or vesicle sorting proteins (28). For example, it has been observed that Nedd4 ubiquitinates Cbl, Eps15, Tsg101, Hrs, and secretory carrier membrane proteins (SCAMPs) and participates in the processes of EGFR endocytosis and degradation (1, 18, 25, 42). However, exactly how Nedd4 engages in the EGFR degradation process in response to EGF stimulation is not known.In this report, we show that EGF stimulation induces ACK degradation. This degradation is associated with ubiquitination of ACK. Nedd4-1, but not Nedd4-2, is identified as the E3 ubiquitin ligase for ubiquitination of ACK. Furthermore, EGF-induced degradation of ACK is EGFR activation dependent and processed by lysosomes. RNAi knockdown and mutational analysis demonstrated that Nedd4-1 and Nedd4-1-catalyzed ubiquitination of ACK are required for EGF-induced degradation of EGFR and ACK. Our findings suggest a new mechanism in regulation of EGFR degradation.