FIGURE 6.

CK1 and PP1 regulate rpS6 recruitment to 7-methylguanosine cap complex. A, inhibition of rpS6 kinases reduces rpS6 binding to 7-methylguanosine beads. HEK 293T cells were treated with rapamycin (Rap) or D4476 for 3 h prior to stimulation with 5% fetal bovine serum for 1 h. Cells were then harvested, and 500 μg of protein was incubated with 50 μl of 7-methylguanosine beads for 2 h at 4 °C. Precipitates were washed in lysis buffer and analyzed along with whole cell lysates (WCL) by immunoblotting with α-rpS6 and α-eIF4E antibodies. DMSO, dimethyl sulfoxide. B, knockdown of PP1cs promotes rpS6 recruitment to 7-methylguanosine beads. HEK 293T cells were transfected with siRNA directed against GFP or PP1cs for 48 h. Cells were serum-starved 24 h prior to harvesting. Cell extracts were prepared, and cap binding assays were performed as described. C, binding of rpS6 mutants to 7-methylguanosine cap complex. HEK 293T cells were transfected with plasmids encoding HA-tagged rpS6^WT or the indicated phosphorylation site mutants. Cells were harvested 24 h later, cell extracts were prepared, and cap binding assays were performed as described. EV, empty vector. S235/236A, S235A/S236A; S240/244A, S240A/S244A; S235/236D, S235D/S236D; S240/244D, S240D/S244D. D, rpS6^S247A displays decreased cap binding. HEK 293T cells were transfected with plasmids encoding HA-tagged rpS6^WT or rpS6^S247A. Cells were harvested 24 h later, cell extracts were prepared, and cap binding assays were performed as described. (Bars depict mean ± S.E. * indicates p < 0.05; n = 4.) E, rpS6^S235D/S236D (S235/236D) cap binding is resistant to D4476. HEK 293T cells were transfected with plasmids encoding HA-tagged rpS6^WT or rpS6^S235D/S236D. Cells were harvested 24 h later, extracts were prepared, and cap binding assays were performed as described. Where indicated, cells were treated with D4476 for 3 h. These data are representative of at least three independent experiments.