Characterization of ubiquilin 1, an mTOR-interacting protein.
The mTOR protein kinase is known to control cell cycle progression and cell growth through regulation of translation, transcription, membrane traffic and protein degradation. Known interactions of mTOR do not account for the multiple functions of this protein. Using a non-catalytic segment of mTOR (1-670) as bait in a yeast two-hybrid screen for interacting proteins, ubiquilin 1 (NM013438) was identified. Ubiquilin 1 is a member of a phylogenetically conserved gene family of unknown function, characterized by an N-terminal ubiquitin-like (Ubq) domain, a C-terminal ubiquitin associated (Uba) domain and a central region containing numerous NPXvar phi motifs (X, any; phi, hydrophobic amino acid). GST-ubiquilin 1 binds specifically to FLAG-mTOR (residues 1-670) in mammalian cells; residues 570-670 of mTOR and 226-323 of ubiquilin 1 are required for this interaction. Both mTOR and ubiquilin immunoreactivity appear as fine speckles throughout the cytoplasm; significant colocalization with cytoskeletal elements, early endosomes or proteasomes is not observed. As assessed by cell fractionation, mTOR is predominantly associated with low density membranes, along with 10% of ubiquilin 1. Ubiquilin 1 is a rapamycin-insensitive phosphoprotein. Overexpression of ubiquilin 1 does not alter the kinase activity of cotransfected mTOR or the phosphorylation of the mTOR target, p70 S6 kinase, in the presence or absence of rapamycin. Our data suggest that we have identified a novel mTOR interactor, ubiquilin 1. The biological significance of this, presumably membrane based, interaction, requires further study.
Wu
Shilan
S
Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02135, USA.
Mikhailov
Alexei
A
Kallo-Hosein
Heidi
H
Hara
Kenta
K
Yonezawa
Kazuyoshi
K
Avruch
Joseph
J
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D000071183:Autophagy-Related Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D018797:Cell Cycle Proteins; D002458:Cell Fractionation; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D058570:TOR Serine-Threonine Kinases; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; C107792:UBQLN1 protein, human; C575873:Ubqln1 protein, rat; D011494:Protein Kinases; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases