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. 2011 Oct 12;286(48):41171–41182. doi: 10.1074/jbc.M111.280982

FIGURE 1.

FIGURE 1.

ORF45/RSK axis induces phosphorylation of eIF4B and rpS6. A, ORF45 induces eIF4B and rpS6 phosphorylation. HEK293 cells were cotransfected with plasmid pKH3-RSK1 and plasmids expressing FLAG-tagged wild-type ORF45 or mutant constructs Δ(19–77) and F66A, both of which lost most of their ability to activate RSK. After serum starvation for 24 h, whole-cell lysates were immunoblotted with antibodies as indicated. B, ORF45 preferentially stimulates RSK rather than S6K kinase activity. The HA-tagged expression vector pKH3-RSK1 or pRK7-HA-S6K1 plasmids were cotransfected with pCR3.1-ORF45 or empty vector into HEK293 cells. After serum starvation for 24 h, the transfected cells were treated with TPA or vehicle (DMSO) for 10 min, and whole-cell lysates were then prepared. The HA-RSK1 and HA-S6K1 kinases were immunoprecipitated with anti-HA affinity beads and used in in vitro kinase assays with GST-S6 as substrate. Signals on the autoradiograph are phosphorylated GST-S6, autophosphorylated HA-RSK1, and ORF45 that was coimmunoprecipitated with RSK and phosphorylated by the associated kinases (17). The immunoprecipitated complexes were also analyzed by Western blot (WB) with antibodies as indicated. The right panel represents the relative kinase activities that were normalized to the input GST-S6 and HA-tagged kinases. Quantitative analysis of the band intensities in the left panel was performed with ImageJ software from NIH. C, RSK activity is required for the ORF45-induced eIF4B and rpS6 phosphorylation. HEK293 cells were cotransfected with ORF45 expression vector plus HA-tagged wild-type RSK2 (WT), RSK2-KD mutant (K100A/Y707A), or RSK2-CA mutant (Y707A) constructs in combinations as indicated. Beginning 48 h after transfection, the cells were serum-starved for an additional 24 h. Whole-cell lysates were analyzed by Western blot with antibodies as indicated. D, knockdown of RSK1 and RSK2 expression by siRNAs inhibits ORF45-induced eIF4B phosphorylation. RSK1/2 siRNA- or control siRNA-transduced HEK293 cells (17) were transfected with ORF45-expressing vectors or mock-transfected with control empty vectors. After serum starvation for 24 h, the cell lysates were analyzed by Western blots with antibodies as indicated.