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Published on: September 27, 2015
Dissociation of the eukaryotic initiation factor-4E/4E-BP1 complex involves phosphorylation of 4E-BP1 by an
1Department of Biochemistry, School of Medical Sciences, University Walk, Bristol, UK. k.heesom@bris.ac.uk
FEBS Letters
|September 3, 1999
Abstract:
mTOR immunoprecipitates contain two 4E-BP1 protein kinase activities. One appears to be due to mTOR itself and results in the phosphorylation of 4E-BP1 on residues T(36) and T(45), as shown previously by others. The other is a kinase which can be separated from mTOR and which phosphorylates 4E-BP1 within a peptide(s) containing residues S(64) and T(69). This phosphorylation, which occurs predominantly on S(64), results in the dissociation of 4E-BP1 from eIF-4E.
Insights
Researchers found two kinase activities in mTOR immunoprecipitates that phosphorylate 4E-BP1. One activity, from mTOR, targets T36/T45. A separate kinase targets S64, causing 4E-BP1 to detach from eIF-4E.
Area of Science:
- Molecular Biology
- Protein Kinase Research
- Cellular Signaling
Background:
- The mechanistic target of rapamycin (mTOR) pathway is crucial for cell growth and proliferation.
- 4E-binding proteins (4E-BPs) regulate protein synthesis by inhibiting the eukaryotic initiation factor 4E (eIF-4E).
- Phosphorylation of 4E-BPs by kinases controls their interaction with eIF-4E.
Purpose of the Study:
- To characterize the distinct protein kinase activities associated with mTOR that phosphorylate 4E-BP1.
- To determine the specific phosphorylation sites on 4E-BP1 targeted by these kinases.
- To elucidate how these phosphorylation events affect the interaction between 4E-BP1 and eIF-4E.
Main Methods:
- Immunoprecipitation of mTOR from cell lysates.
- In vitro kinase assays using purified mTOR immunoprecipitates and 4E-BP1.
- Peptide mapping and mass spectrometry to identify phosphorylation sites.
- Analysis of 4E-BP1 and eIF-4E interaction under different phosphorylation conditions.
Main Results:
- mTOR immunoprecipitates exhibited two distinct 4E-BP1 kinase activities.
- One activity, attributed to mTOR, phosphorylated 4E-BP1 at threonine residues T36 and T45.
- A second, separable kinase activity predominantly phosphorylated 4E-BP1 at serine S64.
- Phosphorylation at S64 led to the dissociation of 4E-BP1 from eIF-4E.
Conclusions:
- Two distinct kinase activities regulate 4E-BP1 phosphorylation within mTOR immunoprecipitates.
- mTOR directly phosphorylates 4E-BP1 at T36/T45.
- A separate kinase phosphorylates 4E-BP1 at S64, promoting eIF-4E release and translation initiation.
- This dual phosphorylation mechanism provides a nuanced control over protein synthesis regulation.
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