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Over‐expression of Mutant Rheb Proteins and mTOR Signaling
Author(s) -
Teran Daniel,
Parmar Nitika
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1046.6
Subject(s) - rheb , mutant , phosphorylation , pi3k/akt/mtor pathway , mutagenesis , biology , phenotype , microbiology and biotechnology , wild type , hek 293 cells , signal transduction , gene , genetics , mtorc1
Activation of mTOR is regulated by the small G‐protein, Rheb. In mammalian systems there are two Rheb genes‐Rheb1 and Rheb2 (RhebL1). Based on a scan for putative residues subject to phosphorylation, we created several mutants for both Rheb1 and Rheb2 using a site‐directed mutagenesis protocol. These residues include Ser130, Lys45, Thr20, Ser44, and Gln130. Wild type and mutant Rheb proteins were over‐expressed in HEK293 cells and impact on mTOR signaling was assessed via monitoring the phosphorylation of the S6 protein. Experiments were also conducted under serum starved conditions to determine if over‐expression of wild type and/or mutant Rheb proteins offered a growth advantage to the cells. Phospho‐S6 was analyzed via immunoblotting in cells grown in enriched, starved and starved/stimulated conditions. Our preliminary results indicate that over‐expression of both wild‐type Rheb1 and Rheb2 is able to rescue the starved phenotype observed, i.e. decrease in S6 phosphorylation, however, the mutants S44A and K45A were unable to rescue this phenotype. Variations in phospho‐S6 observed for other mutants suggest that they may allow the cells to continue growth under starved conditions, similar to wild‐type. Our results so far are unique and provide evidence that the residues S44 and K45 may play a critical role in regulating Rheb's activity, thus warranting a deeper investigation of Rheb's impact on mTOR signaling.