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  current news   Press   selected story    
     
  2nd March  
 

Down-regulation of active ACK1 is mediated by association with the E3 ubiquitin ligase Nedd4-2

 
 




Authors
Wing Chan1, Rui Tian1, Yeow-Fong Lee1, Soon Tuck Sit1, Louis Lim1, 2 and Ed Manser1*.

From GSK-IMCB Group1, Institute of Molecular and cell Biology, Singapore 138673 and Department of Molecular Science, Institute of Neurology2, University College London, London.

*Address correspondence to: Dr. Edward Manser, GSK-IMCB, Proteos, 61 Biopolis Drive, Singapore 138673. Phone: (65)-6586 9545. Fax: (65)-67740742; Email: mcbmansr@imcb.a-star.edu.sg

Abstract
ACK1 (activated Cdc42-associated kinase 1) is a cytoplasmic tyrosine kinase implicated in trafficking through binding to EGFR 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 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 co-expressed, 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. Since EGF stimulation activates ACK1 (3), our result suggest that EGFR-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 EGFR, impinge on cell receptor dynamics. These processes are particularly pertinent given the report of genomic amplification of the ACK1 locus in metastatic tumors.

 
 



 
 

Figure Legend:

  1. Domain structure of ACK1 (Activated Cdc42-associated Kinase1) with the key domains including the EGFR binding domain. Nedd4-2 binds to the PPaY motif in the proline-rich domain. UBA is involved in ubiquitin binding and promotes ACK1 ubiquitination.
  2. ACK1 turnover requires binding to Nedd4-2 and ligase activity of Nedd4-2. Flag-tagged full length ACK1 or an Nedd4-2 binding deficient ACK1 construct (ACKDPPAY) was cotransfected with full-length GST-tagged Nedd4-2 or ligase deficient Nedd4-2C/S. Forty hours after transfection, total ACK1 level was detected with anti-Flag antibody and Nedd4 level was detected with anti-Nedd4 antibody.
  3. Schematic diagram showing the inter-relationship of Nedd4-2, ACK1 and EGFR signaling and events involving ubiquitination pathways. Ligand binding to EGFR leads to ACK1 activation  through a mechanism that is not yet defined. This increases ACK1 association with the receptor and increases Nedd4-2 interaction with the activated ACK1, which in turn promotes ACK1 turnover via the proteasome. Clathrin-mediated endocytosis of both EGFR and ACK1 to endosomes and subsequent sorting within multi-vesicular bodies likely underlies some of these processes. The tumor suppressor WWox binds to ACK1 at the same site as Nedd4 and thus both proteins may compete for binding.

Published in J. Biol. Chem., Jan 2009; doi:10.1074/jbc.M806877200

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