Figure 4

MSK1 activity was required for LMP1-induced phosphorylation of histone H3 at Ser10. (A) Histone H3 kinase activity in CNE1G and CNE1GL cells. Cell extracts (20 μg) were incubated with the pure histone H3 protein (1 μg), ATP (200 μM), and the presence or absence of H89 (10 μM). The expressions of phosphorylated and total histone H3 were detected by Western blot analysis. (B) MSK1 kinase activity in CNE1G and CNE1GL cells. Equal amounts of whole cell extracts (200 μg) were immunoprecipitated with anti-phosphorylated MSK1 antibody, and then incubated with the pure histone H3 protein (1 μg), ATP (200 μM). The expressions of phosphorylated and total histone H3 were detected by Western blot analysis. (C) CNE1G and CNE1GL cells were serum-starved for 36h and the expressions of phosphorylated and total ERK1/2 or MSK1 were detected by Western blot analysis. (D) CNE1 cells were transfected with pcDNA3.0 or pcDNA3.0-LMP1 vector. PD98059 or H89 was added to the culture medium at the concentration indicated every 12 h after transfection. Histone protein was extracted and the expressions of phosphorylated and total histone H3 were detected by Western blot analysis. (E) CNE1GL cells were transfected with si-mock or si-MSK1 vector. After 48 h of transfection, total protein and histone protein were extracted. The expressions of MSK1 and phosphorylated histone H3 were detected by Western blot analysis. β-actin and total histone H3 were used as loading controls.