FIGURE 5.

PKARIα/RSK1 interactions regulate RSK1 activation and apoptosis. A, peptide (Wt-PS) corresponding to the pseudosubstrate region of PKARIα disrupts the interaction of PKARIα and RSK1 and increases RSK1 phosphorylation in unstimulated cells. B82L cells were serum-starved overnight and then were treated with 2 μm each of Wt-PS or Mut-PS (for sequences, see the legend to Fig. 2) for 10 min. RSK1 from the cell lysates was immunoprecipitated (IP) with anti-RSK1 antibody. The proteins in the immune complex were detected with Western analysis. The quantitative data using band intensities of the indicated proteins from three experiments are shown in the panels on the right. B, Wt-PS, but not Mut-PS, disrupts the association of RSK1 with PKARIα, increases the binding of endogenous PKAc to PKARIα, and decreases phosphorylation of BAD on Ser-155. Cell lysates were prepared as in A, and PKARIα was immunoprecipitated with anti-PKARIα antibody. The proteins in the immune complexes or whole cell lysates (WCL) were subjected to Western analysis. Quantification of data using band intensities of the indicated proteins (n ≥ 3 experiments) are shown in the panels on the right. C, Wt-PS, but not Mut-PS, increases basal RSK1 activity and decreases apoptosis. After overnight deprivation of serum, B82L cells were treated with PKARIα peptides Wt-PS or Mut-PS (2 μm each) for 10 min followed by stimulation with 50 nm EGF for 10 min. Cell lysates were examined for phosphorylation of Ser-380 on RSK1 and Ser-112 on BAD. The panels on the right represent quantification of band intensities of the indicated proteins from three experiments. For the apoptosis assay, after treatments with or without EGF for 10 min, cells were treated with TNF-α (20 ng/ml) plus cycloheximide (25 μg/ml) (TNF/CHX) for 1 h, and DNA fragmentation was monitored. Data are the mean ± S.E. of A₄₀₅ per μg of protein (n = 3). D, silencing of RSK1 abrogates the ability of Wt-PS to inhibit apoptosis. Procedures were as in C, except that cells were transfected with RSK1 siRNAs for 56 h before experimentation. The inset shows silencing of RSK1 by the two siRNAs. Data are the mean ± S.E. of A₄₀₅ per μg of protein (n = 3).