Related Experiment Videos
Regulation of eIF-4E BP1 phosphorylation by mTOR
K Hara1, K Yonezawa, M T Kozlowski
1Diabetes Unit and Medical Services, Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts 02129, USA.
Abstract:
The proteins eIF-4E BP1 and p70 S6 kinase each undergo an insulin/mitogen-stimulated phosphorylation in situ that is partially inhibited by rapamycin. Previous work has established that the protein known as mTOR/RAFT-1/FRAP is the target through which the rapamycin.FKBP12 complex acts to dephosphorylate/deactivate the p70 S6 kinase; thus, some mTOR mutants that have lost the ability to bind to the rapamycin.FKBP12 complex in vitro can protect the p70 S6 kinase against rapamycin-induced dephosphorylation/deactivation in situ. We show herein that such mTOR mutants also protect eIF-4E BP1 against rapamycin-induced dephosphorylation, and for both p70 S6 kinase and eIF-4E BP1, such protection requires that the rapamycin-resistant mTOR variant retains an active catalytic domain. In contrast, mutants of p70 S6 kinase rendered intrinsically resistant to inhibition by rapamycin in situ are not able to protect coexpressed eIF-4E BP1 from rapamycin-induced dephosphorylation. We conclude that mTOR is an upstream regulator of eIF-4E BP1 as well as the p70 S6 kinase; moreover, these two mTOR targets are regulated in a parallel rather than sequential manner.
Insights
The mechanistic target of rapamycin (mTOR) pathway regulates protein synthesis. This study shows mTOR directly controls both eIF-4E binding protein 1 (4E-BP1) and p70 S6 kinase in parallel, independent pathways.
Area of Science:
- Cellular signaling
- Molecular biology
- Biochemistry
Background:
- Insulin and mitogens stimulate protein synthesis via phosphorylation of key regulators.
- Rapamycin inhibits these processes by targeting the mTOR/RAFT-1/FRAP protein complex.
- mTOR's role in regulating p70 S6 kinase phosphorylation is established, but its effect on eIF-4E BP1 is less clear.
Purpose of the Study:
- To investigate the role of mTOR in the regulation of eIF-4E BP1 phosphorylation.
- To determine if mTOR acts upstream of both p70 S6 kinase and eIF-4E BP1.
- To elucidate the relationship between mTOR, p70 S6 kinase, and eIF-4E BP1 regulation.
Main Methods:
- Utilized rapamycin-resistant mTOR mutants to assess in situ phosphorylation.
- Examined the effect of mTOR mutants on both p70 S6 kinase and eIF-4E BP1.
- Investigated the requirement of mTOR's catalytic activity for its regulatory function.
Main Results:
- Rapamycin-resistant mTOR mutants protected eIF-4E BP1 from dephosphorylation, similar to p70 S6 kinase.
- Protection of both targets required an active catalytic domain in the mTOR mutant.
- Mutants of p70 S6 kinase resistant to rapamycin did not protect eIF-4E BP1.
Conclusions:
- mTOR acts as an upstream regulator of both eIF-4E BP1 and p70 S6 kinase.
- These two substrates are regulated by mTOR in parallel, rather than sequentially.
- The findings clarify the signaling network downstream of mTOR in response to growth factors.