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.
Lin
Qiong
Q
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822, USA.
Wang
Jian
J
Childress
Chandra
C
Sudol
Marius
M
Carey
David J
DJ
Yang
Wannian
W
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D000075702:Nedd4 Ubiquitin Protein Ligases; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
D056827:Endosomal Sorting Complexes Required for Transport; D050505:Mutant Proteins; D004815:Epidermal Growth Factor; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; C403262:TNK2 protein, human; D046988:Proteasome Endopeptidase Complex