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Nedd4 mediates ErbB4 JM‐a/CYT–1 ICD ubiquitination and degradation in MDCK II cells
Author(s) -
Zeng Fenghua,
Xu Jie,
Harris Raymond C.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.08-121947
Subject(s) - nedd4 , lactacystin , ubiquitin ligase , ubiquitin , chemistry , microbiology and biotechnology , proteasome , biology , biochemistry , proteasome inhibitor , gene
ABSTRACT ErbB4, a type I transmembrane receptor tyrosine kinase, is a member of the epidermal growth factor receptor family. Its cleavage releases an intracellular C‐terminal domain (ICD), which can be either degraded following ubiqitination or translocated to the nucleus and regulate gene expression. There are 2 ErbB4 ICD isoforms:CYT‐1 andCYT‐2. We and others have previously reported that following cleavage,CYT‐2 selectively translocates to the nucleus. In the current study we found that following cleavage, the intracellular levels ofCYT‐1 ICD decreased rapidly, while levels ofCYT‐2 ICD remained relatively stable.CYT‐1 ICD degradation could be prevented by administration of either the proteasome inhibitor lactacystin or the lysosome inhibitor chloroquine, indicating both proteasomal and lysosomal degradation. Further studies implicated Nedd4, an E3 ubiquitin ligase, as a mediator ofCYT‐1 ubiquitination and degradation. The interaction of Nedd4 withCYT‐1 was shown by coimmnunoprecipitation, an in vitro direct binding assay, and an in vitro ubiquitination assay. Three PPxY or PY motifs present in theCYT‐1 C terminus are necessary for binding by Nedd4 WW domains, because impaired interactions are seen in mutation of any of the PY motifs. Nedd4‐CYT‐1 binding was associated with increasedCYT‐1 ubiquitination following proteasome inhibitor treatment. Impaired Nedd4 binding toCYT‐1 by PY motif mutations led to increasedCYT‐1 ICD stability, whereas only one of the PY motif mutations (Y1056A), which disrupts the binding sites for both a WW domain and an SH2 domain of PI3 kinase, demonstrated enhanced nuclear translocation following HB‐EGF treatment. These studies indicate that Nedd4 mediates ErbB4CYT‐1 ICD ubiquitination and degradation, and the prevention of both WW binding and PI3 kinase activity are required for ErbB4 nuclear translocation.—Zeng, F.,Xu, J., Harris, R.C. Nedd4 mediates ErbB4JM‐a/CYT‐1 ICD ubiquitination and degradation in MDCK II cells. FASEB J. 23, 1935–1945 (2009)