FIG. 5.

Overexpression of kinase-dead S6K1 mutants inhibited S6K activity but failed to suppress the translational activation of L32-GFP mRNA. (a) 293 cells were transiently cotransfected with 1 μg of HA-p70S6K1 together with 1 μg of pEGFP-C1 and 16 μg of either a vector encoding the HA epitope (EMPTY) or a vector encoding HA-tagged p70S6K,K123M. Twenty-four hours later, highly fluorescent cells were sorted by fluorescence-activated cell sorter and reseeded. Forty-eight hours posttransfection cells were harvested, and cytoplasmic proteins were subjected to Western blot analysis using anti-rpS6 and anti-phospho-Ser240/244 antibodies. (b) 293 cells were transiently transfected with 1 μg of HA-S6K1, 1 μg of pEGFP-C1, and 16 μg of either a vector encoding HA (EMPTY) or a vector encoding HA-tagged p70Δ2-46/ΔCT104, K123M/T412E (Kinase-dead). Twenty-four hours later cells were starved for 2 h and then refed for 0.5 h and harvested. Cell extracts were subjected to immunoprecipitation with an anti-HA antibody, and a portion was assayed for S6 kinase activity. The reaction mixture was separated by SDS-PAGE and transferred onto a nitrocellulose membrane, and the radioactive signals of ³²P-S6 were autoradiographed (HA-S6K1 activity). Immunoblotting of the membrane with an anti-HA antibody enabled the detection of the wild-type S6K1 (HA-S6K1) and of the shorter kinase-dead (p70Δ2-46/ΔCT104, K123M/T412E) variant (Kinase-dead). (c) 293 cells were transfected with 2 μg of a vector encoding L32-GFP and 16 μg of a vector encoding the HA tag, p70S6K,K123M, and p70Δ2-46/ΔCT104, K123M/ T412E (Kinase-dead). Twenty-four to 36 h later cells were harvested untreated (Con) or after 2 h of amino acid starvation followed by 0.5 h of refeeding (aa refed). The translational efficiency of L32-GFP mRNA was analyzed and is presented as described in the legend to Fig. 2.