A, HEK293E cells were serum-starved for 18 h and pretreated with U0126 (10 μM) and/or rapamycin (100 nM) for 30 min before stimulation with serum (10%) over a time course. Dual phosphorylation of rpS6 at Ser^235/236 and Ser^240/244 was monitored using phosphospecific antibodies. The cell lysates were also immunoblotted for total protein levels (rpS6, S6K1, ERK1/2), and phosphorylated S6K1 and ERK1/2. B and C, activation status of RSK2 and S6K1 was monitored in cells treated as indicated. Endogenous RSK2 and S6K1 were immunoprecipitated and assayed for their phosphotransferase activity against recombinant GST-rpS6 fusion protein in vitro. Relative ³²P incorporation is displayed in the histogram above the autoradiogram. These data are representative of greater than three independent experiments.

A, serum-starved HEK293E cells were pretreated with U0126 (10 μM) and/or rapamycin (100 nM) for 30 min before stimulation with PMA for 20 min. Phosphorylation of rpS6 at Ser^235/236 and Ser^240/244 was monitored using phosphospecific antibodies. The cell lysates were also immunoblotted for total protein levels (rpS6, ERK1/2), and phosphorylated RSK1/2 and ERK1/2. B and C, cells were transfected with control vector, oncogenic Ras (61L), dominant-negative Ras (17N), or activated MEK1 (DD), serum-starved for 16–18 h, and analyzed for rpS6 phosphorylation using phosphospecific antibodies. Total protein levels of rpS6, ERK1/2 were also determined, as well as levels of RSK1/2 and ERK1/2 phosphorylation. C, cells were treated with the MEK1/2 inhibitor U0126 for 30 min prior to harvesting cells for immunoblotting analysis.

A, requirement for S6K1, RSK1, and RSK2 in HEK293E cells was determined using RNA interference. Expression of S6K1, RSK1, and RSK2 was knocked down using specific siRNAs and cell lysates were assayed for rpS6 phosphorylation at Ser^235/236 and Ser^240/244. Total protein levels of rpS6, S6K1, RSK1, RSK2, and ERK1/2 were also determined, as well as levels of ERK1/2 phosphorylation. A scrambled siRNA was used as control. For sequences, see “Experimental Procedures.” B, HEK293E cells were transfected with scrambled control or a combination of RSK1 and RSK2 siRNAs. Cells were serum-starved for 18 h and stimulated with serum (10%) or EGF (25 ng/ml) for 15 min. Cell lysates were assayed for rpS6 phosphorylation at Ser^235/236 and Ser^240/244, ERK1/2 phosphorylation, and total levels of ERK1/2, rpS6, RSK1, and RSK2.

A, requirement for RSK activity during a time course of serum stimulation was tested in HeLa cells. Serum-starved cells were treated with 2 μM fmk for 30 min before stimulation with serum from 0– 60 min. Cell lysates were assayed for rpS6 phosphorylation at Ser^235/236 and Ser^240/244, RSK phosphorylation at Ser³⁸⁰, and ERK1/2 phosphorylation at Thr²⁰²/Tyr²⁰⁴. The protein levels of RSK1 and ERK1/2 were also determined by immunoblotting. B, requirement for RSK activity was tested in HEK293E cells by treating cells with 5 μM fmk for 30 min before stimulating with serum (10%), PMA (50 ng/ml), EGF (25 ng/ml), or insulin (50 nM). Cell lysates were assayed for rpS6 and RSK1/2 phosphorylation, and rpS6 protein levels by immunoblotting. Immunoprecipitates of endogenous RSK2 were subjected to in vitro kinase activity assays using GST-rpS6 as substrate. These data are representative of greater than three independent experiments.

A, alignment of the C-terminal tail of rpS6 proteins from human, mouse, frog, and fly. The identified phosphorylation sites are indicated in boldface character and annotated according to human numbering. B, the in vitro specificity of RSK1, RSK2, and S6K1 was tested by incubating endogenous immunoprecipitates of these kinases with bacterially derived recombinant GST-rpS6 fusion proteins. HEK293E cells were stimulated with PMA (50 ng/ml; RSK1, RSK2) or insulin (50 nM; S6K1), and increasing amounts of endogenous immunoprecipitates were assayed for kinase activity in vitro using phosphospecific antibodies against Ser^235/236 and Ser^240/244. A portion of each sample was assayed for kinase activity using [³²P]ATP and phosphotransferase activity is displayed as an autoradiogram with corresponding histogram. The level of GST-rpS6 is shown as a Coomassie Blue-stained gel. C, HEK293E cells were transfected with control vector, wt RSK1, Myristoylated RSK1 (Myr), or kinase inactive RSK1 (kd), and serum-starved for 16–18 h. Quiescent cells were left untreated or treated with rapamycin (100 nM) for 30 min. A subset of cells was also stimulated with insulin (50 nM) for 15 min before harvesting. Cell lysates were assayed for rpS6 phosphorylation at Ser^235/236 and Ser^240/244, and the levels of transfected RSK1 (avian isoform) and endogenous rpS6 were also determined. D, HEK293E cells were transfected as indicated, serum-starved for 16–18 h, and treated for 30 min with either U0126 (UO; 10 μM), wortmannin (100 nM), or rapamycin (100 nM). Cells lysates were assayed for rpS6 phosphorylation and protein levels (B).

A, HEK293E cells were transfected with HA-tagged rpS6 wt, S235/36A (S2A), and S235/36D (S2D) constructs. Cells were stimulated with PMA (50 ng/ml), and total cell lysates were assayed for rpS6 phosphorylation at Ser^235/236 and Ser^240/244, ERK1/2 phosphorylation, and HA-rpS6 levels. B and C, association of wt and mutant HA-tagged rpS6 proteins to the 7-methylguanosine cap complex was monitored in cells grown in the presence of serum or serum-starved for 16–18 h. Alternatively, cells were serum-starved and stimulated with PMA for 20 min. The level of associated rpS6 was determined using anti-HA immunoblotting in 7-methyl-guanosine precipitates. Equal levels between precipitates were monitored through analysis of eIF4E, and levels of transfected HA-rpS6 were determined in protein lysates.

A bicistronic reporter plasmid that directs cap-dependent translation of the Renilla luciferase (RL) gene and cap-independent HCV IRES-mediated translation of the firefly (FL) gene was used to determine the role of RSK in cap-dependent translation. A and B, transfected HEK293E cells were grown in the presence of serum or serum-starved for 24 h in the presence of PMA (50 ng/ml), rapamycin (100 nM), or increasing dose of fmk. Cells were harvested and RL/FL luminescence quantified using a luminometer. C, HEK293E cells were co-transfected with the reporter vector and different RSK1 mutants. Cells were grown in serum or serum-starved for 24 h in the presence of PMA (100 ng/ml) before harvesting for luminescence. D, cells were co-transfected with the reporter vector and different RSK isoforms and grown in the presence of serum with or without rapamycin (100 nM) for 24 h. Luminescence was determined and graphed as in B. For all panels, the RL/FL luminescence ratio is expressed as a histogram ± S.E. from three independent experiments. E, HEK293E cells were serum-starved for 24 h or grown in the presence of serum, and cellular extracts were size-fractionated by centrifugation through sucrose gradients (20–50%). The absorbance of polysomes and subpolysomal particles was continuously monitored at 260 nm, and the vertical dashed line separates the polysomal (P) and the subpolysomal fractions (S). Representative A₂₆₀ nm traces are shown (n = 3). F, cells were grown in the presence of serum and incubated with rapamycin (100 nM) or fmk (5 μM) for 24 h or 1 h before subjecting cellular extracts to sucrose gradients. The area under the curves was calculated using ImageJ (NIH), and the proportion of P/S has been referred to in the histogram as the percentage of ribosomes engaged in translation. The results are presented as a mean ± S.E. (n = 4).

The growth factor stimulated signaling pathways that converge on the translational machinery to regulate cap-dependent translation are depicted in the schematic representation.