A novel PTB-PDZ domain interaction mediates isoform-specific ubiquitylation of mammalian Numb.
LNX was originally cloned as a Numb PTB-binding molecule, and it was subsequently found to act as a RING finger-type E3 ubiquitin ligase for the ubiquitylation and degradation of mNumb. Numb is a PTB domain-containing protein that functions as an intrinsic determinant of cell fate in asymmetric cell division. In mammals, four protein isoforms arise from alternative mRNA splicing. Here we report that while all four protein isoforms bind to LNX, only p72 and p66 Numb isoforms are ubiquitylated and degraded. The p72 and p66 Numb proteins differ from the other two isoforms by the presence of an 11-amino acid sequence insert in the PTB domain (PTBi). We demonstrate that the isoform-specific ubiquitylation of mNumb is due to a novel interaction between the first PDZ domain (PDZ1) of LNX and the PTBi variant. Deletion of LNX PDZ1 domain resulted in loss of ubiquitylation and subsequent degradation of the PTBi form of Numb. Interestingly efficient PTBi ubiquitylation not only depends on association with the LNX PDZ1 domain but also requires binding to the canonical PTB-binding motif NPAY in LNX. Thus two distinct modes of PTBi-mediated interaction with LNX work in concert to allow the effective and specific degradation of the p72 and p66 isoforms of mNumb.
The Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
Jing
J
Nie
Shawn S-C
SS
Li
C Jane
CJ
McGlade
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D038941:Polypyrimidine Tract-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014908:Triticum / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C113026:NUMB protein, human; C106293:Numb protein, mouse; D020033:Protein Isoforms; D011994:Recombinant Proteins; D025801:Ubiquitin; D038941:Polypyrimidine Tract-Binding Protein