Down-regulation of active ACK1 is mediated by association with the E3 ubiquitin ligase Nedd4-2.
ACK1 (activated Cdc42-associated kinase 1) is a cytoplasmic tyrosine kinase implicated in trafficking through binding to epidermal growth factor (EGF) receptor and clathrin. Here, we have identified a new ACK1-binding partner, the E3 ubiquitin ligase Nedd4-2, which binds ACK1 via a conserved PPXY-containing region. We show that this motif also binds Nedd4-related proteins and several other WW domain-containing proteins, including the tumor suppressor oxidoreductase Wwox. In HeLa cells ACK1 colocalizes with Nedd4-2 in clathrin-rich vesicles, requiring this PPXY motif. Nedd4-2 strongly down-regulates ACK1 levels when coexpressed, and this process can be blocked by proteasome inhibitor MG132. ACK1 degradation via Nedd4 requires their mutual interaction and a functional E3 ligase; it is also driven by ACK1 activity. ACK1 is polyubiquitinated in vivo, and dominant inhibitory Nedd4 blocks endogenous ACK1 turnover in response to acute EGF treatment. Because EGF stimulation activates ACK1 ( Galisteo, M., Y., Y., Urena, J., and Schlessinger, J. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 9796-9801 ), our result suggest that EGF receptor-mediated ACK1 activation allows Nedd4-2 to drive kinase degradation. Thus the interplay between Nedd4-2-related E3 ligases that regulate ACK1 levels and Cbl that modifies EGF receptor impinges on cell receptor dynamics. These processes are particularly pertinent given the report of genomic amplification of the ACK1 locus in metastatic tumors.
Chan
Wing
W
GSK, Institute of Molecular and Cell Biology, Singapore 138673.
Tian
Rui
R
Lee
Yeow-Fong
YF
Sit
Soon Tuck
ST
Lim
Louis
L
Manser
Ed
E
D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D000075702:Nedd4 Ubiquitin Protein Ligases; D009362:Neoplasm Metastasis; D009369:Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D061988:Proteasome Inhibitors; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics; D050721:Proto-Oncogene Proteins c-cbl / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects / Q000502:physiology; D000074584:WW Domain-Containing Oxidoreductase
D002966:Clathrin; D015853:Cysteine Proteinase Inhibitors; D056827:Endosomal Sorting Complexes Required for Transport; D007976:Leupeptins; D061988:Proteasome Inhibitors; D025521:Tumor Suppressor Proteins; D004815:Epidermal Growth Factor; D010088:Oxidoreductases; D000074584:WW Domain-Containing Oxidoreductase; C486071:WWOX protein, human; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C512478:EGFR protein, human; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; C403262:TNK2 protein, human; D046988:Proteasome Endopeptidase Complex; C067419:CBL protein, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde