Abstract
The Ras-like small GTPase Rheb is an upstream activator of the mammalian target of rapamycin (mTOR). It has recently been shown that Rheb activates mTOR by binding to its endogenous inhibitor FKBP38 and preventing it from association with mTOR. The interaction of Rheb with FKBP38 is controlled by its guanine nucleotide binding states, which are responsive to growth factor and amino acid conditions. In this study, we show that Rheb interacts with FKBP38 through a section within its switch I region that is equivalent to the effector domain of other Ras-like small GTPases. We find that the ability for Rheb to interact with FKBP38 correlates with its activity for mTOR activation. Our findings suggest that FKBP38 is a bona fide effector of Rheb and that the ability to interact with FKBP38 is important for Rheb as an activator of mTOR.
Highlights
Rheb is a Ras-related small GTPase that functions as an upstream activator of the mammalian target of rapamycin2 [1, 2]
It binds strongly to FKBP38 in GTP-bound form and weakly in GDP bound form. This GTP-dependent binding suggests that FKBP38 is an effector of Rheb in the mammalian target of rapamycin (mTOR) pathway, which represents the first known effector of Rheb that is regulated by guanine nucleotide [12]
We have previously shown that Rheb physically interacts with FKBP38, which suggests a co-localization of both proteins
Summary
Thr46) were purchased from Cell Signaling Inc. Anti-HA Coimmunoprecipitation—HEK293 cells were co-transfected (12CA5) and c-Myc (9E10) antibodies were purchased from with vectors expressing FKBP38 and Rheb. Roche Applied Science, and anti-FLAG M2 antibody was from Dulbecco’s modified Eagle’s medium for 30 h, transfected cells. The pCMV-Myc-FKBP38 vector was constructed by were washed with cold PBS on ice and lysed in buffer containing 50. FKBP52 gene was amplified by PCR from the FKBP52 cDNA incubated with anti-Myc antibody (9E10)-linked Protein A-agarclone (ID 3542330) obtained from OpenBiosystems and cloned ose beads for 3 h at 4 °C with agitation. PcDNA3.1-Myc-FKBP52TM was created by inserting the Washed beads were resuspended in 60 l of 2ϫ SDS sample buffer. Cells were incubated overnight at 4 °C with anti-Rheb antibody (1:100 dilution), followed by staining with Alexa Fluor 488-conjugated anti-rabbit IgG (Molecular Probes) (1:1000).
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