Fig 2: The mTOR kinase activity regulates the nuclear accumulation of ribosomal proteins independent of rapamycin and rictor expression(A-B) The nuclear abundance of ribosomal proteins is dependent on the mTOR kinase activity. MDA-MB-435 cells were incubated with 500 nM pp242 (A) or 100 nM Torin 1 (B) for the indicated time points and then lysed with the following fractionation to cytoplasmic (Cyt) and nuclear fractions (SENF and RNF). The isolated fractions were analyzed by immunoblotting for the levels of indicated proteins. The mTOR-dependent phosphorylation of S6K1 on Thr-389 and Akt on Ser-473 sites has been examined in the cytosolic fraction by the phospho-specific antibodies. Detection of the Nup98 and S6K1 proteins is shown as the loading controls. (C) Immunostaining of a free ribosomal protein L7a (rpL7a) in MDA-MB-435 cells incubated with or without 500 nM pp242 for 1 hour. The rpL7a antibody (Cell Signaling Technology, #2415) shows an exclusive nucleolar localization of rpL7a where this protein is enriched prior its assembly to ribosomal subunit. (D) Acute inhibition of the mTOR kinase activity does not suppress translation of the ribosomal proteins rpL26 and rpS6. MDA-MB-435 cells were incubated with 500 nM pp242 and labeled with 35S-methione/cysteine for 1 hr and cells were lysed in RIPA buffer. The rpL26 and rpS6 immunoprecipitates were analyzed by autoradiography and immunoblotting for the levels of newly synthesized proteins and total rpL26 and rpS6. (E) The kinase activity of mTOR is required for association of RanBP2 and mTOR. The actively growing HEK 293T cells were treated by 500 nM pp242 for the indicated times and were lysed in a buffer optimized to extract nuclear pore components (the buffer E). RanBP2 immunoprecipitates were prepared from the total cellular lysates and analyzed by immunoblotting. Abundance of the analyzed proteins in the total cellular lysate is shown in a lower panel. (F) The nuclear abundance of ribosomal proteins is dependent on RanBP2 expression. Following expression of the control (luciferase) or two different RanBP2 shRNAs, the MDA-MB-435 cells were analyzed by a sub-cellular fractionation and were analyzed by immunoblotting for the levels of indicated proteins as in A. (G) Rapamycin does not regulate the nuclear accumulation of ribosomal proteins, but induces rpS6 and S6K1translocation to the nuclear (SENF) fraction. MDA-MB-435 cells were incubated with 50 nM rapamycin for the indicated times. The sub-cellular fractions were isolated and analyzed by immunoblotting for the levels of indicated proteins as in A. (H) The nuclear accumulation of ribosomal proteins is rictor independent. Rictor null and wild type mouse embryonic fibroblasts (MEFs) were analyzed by a sub-cellular fractionation to the cytoplasmic and nuclear fractions. The obtained fractions were analyzed by immunoblotting with the indicated antibodies as in A.

Fig 3: Nuclear import of the GFP-tagged ribosomal protein rpL7a is dependent on the mTOR kinase activity(A) The nuclear accumulation of ribosomal proteins by inhibition of the nuclear export is dependent on the mTOR kinase activity. To interfere with the nuclear export MDA-MB-435 cells were incubated with 200 nM leptomycin B for 2 hours. The leptomycin B treated cells were treated with or without pp242 at concentration 500 nM for 1 hour. The cytosolic and nuclear abundance of rpS3, rpS6, and rpL26 has been analyzed by immunoblotting of the nuclear fractions SENF and RNF. B-E, HeLa cells were transiently transfected with GFP-rpL7a, GFP-Lamin A, or GFP-tagged with the nuclear localization signal (GFP-NLS). Localization of the GFP-tagged proteins has been detected by fluorescent microscopy. The following images are presented: (B1-B2) after four hours of transfection, expression of GFP-L7a at the initial time point is not detectable, (C1) Control cells shows accumulation of GFP-RPL7a in nucleolus, (C2) Cells incubated with 1 μM of pp242 shows accumulation of GFP-L7a mostly in cytoplasm after 5 hours of treatment, (D1-D2 and E1-E2) The nuclear import of lamin A or GFP-NLS is not sensitive to the treatment of cells with 1 μM pp242. Both proteins show nuclear staining in cells with or without the drug treatement. (F) The nuclear import of GFP-rpL7a is dependent on the mTOR kinase activity. HeLa cells were transiently transfected with the expression plasmid encoding GFP-rpL7a protein and incubated with (the lower panel) or without (the upper panel) 1 μM of pp242. Following the photo-bleaching by a laser pulse, the recovery of rpL7a in nucleolus has been monitored by fluorescent microscopy. The images were taken within 6 min intervals.