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TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function
Stefanie S Schalm1, Diane C Fingar, David M Sabatini
1The Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Background:
The mammalian target of rapamycin, mTOR, is a serine/threonine kinase that controls cell growth and proliferation via the translation regulators eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). We recently identified a TOR signaling (TOS) motif in the N terminus of S6K1 and the C terminus of 4E-BP1 and demonstrated that in S6K1, the TOS motif is necessary to facilitate mTOR signaling to phosphorylate and activate S6K1. However, it is unclear how the TOS motif in S6K1 and 4E-BP1 mediates mTOR signaling.
Results:
Here, we show that a functional TOS motif is required for 4E-BP1 to bind to raptor (a recently identified mTOR-interacting protein), for 4E-BP1 to be efficiently phosphorylated in vitro by the mTOR/raptor complex, and for 4E-BP1 to be phosphorylated in vivo at all identified mTOR-regulated sites. mTOR/raptor-regulated phosphorylation is necessary for 4E-BP's efficient release from the translational initiation factor eIF4E. Consistently, overexpression of a mutant of 4E-BP1 containing a single amino acid change in the TOS motif (F114A) reduces cell size, demonstrating that mTOR-dependent regulation of cell growth by 4E-BP1 is dependent on a functional TOS motif.
Conclusions:
Our data demonstrate that the TOS motif functions as a docking site for the mTOR/raptor complex, which is required for multisite phosphorylation of 4E-BP1, eIF4E release from 4E-BP1, and cell growth.
Insights
The TOR signaling (TOS) motif in 4E-BP1 acts as a docking site for the mTOR/raptor complex, facilitating cell growth regulation. This motif is crucial for 4E-BP1 phosphorylation and its release from eIF4E, impacting cell size.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mammalian target of rapamycin (mTOR) regulates cell growth and proliferation.
- mTOR signaling involves translation regulators eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1).
- A TOR signaling (TOS) motif in 4E-BP1 and S6K1 was identified, but its mechanism in mediating mTOR signaling was unclear.
Purpose of the Study:
- To elucidate the role of the TOS motif in 4E-BP1 in mediating mTOR signaling.
- To investigate how the TOS motif facilitates mTOR-dependent regulation of cell growth.
Main Methods:
- Investigated 4E-BP1 binding to raptor, an mTOR-interacting protein.
- Assessed in vitro and in vivo phosphorylation of 4E-BP1 by the mTOR/raptor complex.
- Utilized overexpression of a mutant 4E-BP1 (F114A) with a modified TOS motif to study cell size regulation.
Main Results:
- A functional TOS motif in 4E-BP1 is essential for its binding to raptor.
- The TOS motif is required for efficient in vitro and in vivo phosphorylation of 4E-BP1 by the mTOR/raptor complex.
- mTOR/raptor-regulated phosphorylation of 4E-BP1 is necessary for its release from eIF4E, and a mutated TOS motif reduces cell size.
Conclusions:
- The TOS motif serves as a docking site for the mTOR/raptor complex.
- This interaction is critical for multisite phosphorylation of 4E-BP1.
- The TOS motif-mediated process regulates eIF4E release and is essential for mTOR-dependent cell growth.