FIGURE 7.

RSK2 is an important H2BS32 kinase in the EGF-stimulated signal transduction pathway. A, H2BS32 phosphorylation occurs in cells treated with tumor promoters such as EGF. JB6 cells were seeded in 10-cm dishes and cultured overnight. The cells were subsequently cultured in 0.1% FBS-MEM for 24 h and then treated or not treated with EGF (10 ng/ml) and harvested at various times (5, 15, and 30 min). The proteins were extracted, and Western blotting was conducted using specific antibodies as indicated. Equal protein loading was confirmed using antibodies to detect total ERK1/2, RSK2, histone H2B, or H3. The asterisk indicates a nonspecific band signal B, RSK2 deficiency blocks EGF-induced H2BS32ph. RSK2 wild type (RSK2^+/+) and RSK2-deficient (RSK2^−/−) MEFs (1 × 10⁶) were each seeded in 10-cm dishes and grown to 90–95% confluence and subsequently cultured in 0.1% FBS-DMEM for 24 h. The cells were stimulated with EGF (10 ng/ml) and harvested 15 or 30 min later. Histone proteins were extracted, and Western blotting was conducted using specific antibodies as indicated. The RSK2 expression levels in RSK2 wild type (RSK2^+/+) and RSK2-deficient (RSK2^−/−) MEFs were also confirmed by Western blot (bottom). Signals of longer exposure time from the same blots are also shown for H3S10ph. C, knockdown of RSK2 in JB6 cells clearly attenuates the induction of H2BS32 phosphorylation by EGF. RSK2 expression levels in JB6 shMock and shRSK2 cells were first confirmed by Western blot (right). JB6 shMock and shRSK2 cells (1 × 10⁶) were seeded in 10-cm dishes and grown to 90–95% confluence and then cultured in 0.1% FBS-MEM 24 h. The cells were stimulated with EGF (10 ng/ml) and harvested 15 or 30 min later. The histone proteins were extracted and visualized by Western blot. Equal protein loading was confirmed using antibodies to detect β-actin, histone H2B, or H3. Phosphorylation of H3S10 served as a positive marker for EGF stimulation.