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The rapamycin and FKBP12 target (RAFT) displays phosphatidylinositol 4-kinase activity
D M Sabatini1, B A Pierchala, R K Barrow
1Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Abstract:
The immunosuppressant rapamycin prevents cell cycle progression in several mammalian cell lines and the yeast Saccharomyces cerevisiae. In mammalian cells, rapamycin binds to the small FK506-binding protein, FKBP12, allowing the drug-receptor complex to interact with the 289-kDa RAFT1/FRAP proteins. These proteins, along with their yeast homologs, TOR1/DRR1 and TOR2/DRR2, contain a C-terminal domain with amino acid homology to several phosphatidylinositol (PI) 4- and 3-kinases. However, no direct demonstration of kinase activity for this family of proteins has been reported. We now show that RAFT1, immunoprecipitated from rat brain and MG63 and HEK293 cells, contains PI 4-kinase activity and that rapamycin-FKBP12 has no effect on this activity. Thus, it is likely that, in vivo, rapamycin does not directly inhibit the PI 4-kinase activity and affects the RAFT1/FRAP protein through another mechanism.
Insights
The immunosuppressant drug rapamycin halts cell growth. Researchers found that rapamycin does not directly inhibit the PI 4-kinase activity of RAFT1/FRAP proteins, suggesting an indirect mechanism of action.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Rapamycin is an immunosuppressant that inhibits cell cycle progression in yeast and mammalian cells.
- Rapamycin forms a complex with FKBP12, which interacts with RAFT1/FRAP proteins.
- RAFT1/FRAP proteins share homology with phosphatidylinositol (PI) 4- and 3-kinases, but their kinase activity remains unproven.
Purpose of the Study:
- To investigate the direct kinase activity of RAFT1/FRAP proteins.
- To determine if rapamycin directly inhibits the PI 4-kinase activity of RAFT1/FRAP.
Main Methods:
- Immunoprecipitation of RAFT1 from rat brain and human cell lines (MG63, HEK293).
- Assay of PI 4-kinase activity in immunoprecipitated RAFT1.
- Assessment of the effect of the rapamycin-FKBP12 complex on RAFT1 PI 4-kinase activity.
Main Results:
- Immunoprecipitated RAFT1 protein exhibits PI 4-kinase activity.
- The rapamycin-FKBP12 complex did not affect the observed PI 4-kinase activity of RAFT1.
Conclusions:
- The RAFT1/FRAP protein family possesses intrinsic PI 4-kinase activity.
- Rapamycin likely inhibits RAFT1/FRAP function through an indirect mechanism, not by directly inhibiting its PI 4-kinase activity.