THE DEVELOPMENT AND MOLECULAR EXPRESSION IN MAMMALIAN CELLS OF AN HA-TAGGED PLASMID ENCODING FOR THE TARGET OF RAPAMYCIN (mTOR)

by Dougherty, Kevin S.

The protein kinase mammalian Target of Rapamycin (mTOR) is a master controller of cell growth and proliferation due to its ability to integrate growth signals and regulate translation through 4E-BP1 and p70-S6K. We developed four phCMV2-HA-tagged plasmids to overexpress TOR in mammalian cells: Wild Type, D2357E (Kinase Dead), R2109A (PA Binding Deficient) and, S2035T (Rapamycin Resistant) each verified by restriction enzyme digestion and direct sequencing. Plasmid DNA was overexpressed in COS-7 cells and the 289 kDa protein detected in Western blots developed with anti-HA and anti-mTOR antibodies. Kinase activity of the overexpressed protein was detected by in vitro phosphorylation of 4E-BP1 and S6K. QRT-PCR detected a 200 fold increase in mTOR gene expression over mock transfected cells, but found no change in PLD2 gene expression. Immunoflourescence microscopy indicated that mTOR is perinuclear, concentrated in endosomal formations. This study provides the molecular biology tools for continuing investigation into the crosstalk between mTOR, S6K, PLD1 and PLD2.