Figure 1.
PKC δ phosphorylates Ser-10 of histone H3 in vitro.
(A) and (B), Recombinant PKCδ was incubated with ATP and core histone (A) or histone H3 (B). After the in vitro kinase(IVK) assay, the samples were analyzed by immunoblotting (IB) with anti-PKCδ, anti-phospho histone H3 Thr 3 (pH3 T3), anti-phospho histone H3 Ser 10 (pH3 S10), anti-phospho histone H3 Thr 11 (pH3 T11), anti-phospho histone H2B Ser 14 (pH2B S14) or anti-histone H3 antibody. (C) Recombinant GST, GST-histone H3(1–15) or GST-histone H3(1–15, S10A) proteins were incubated with recombinant PKCδ. Immunoblottings were probed with anti-phospho histone H3 Ser 10 (pH3 S10) or anti-GST antibodies.
Figure 2.
Ectopic expression of PKCδ induces Ser-10 phosphorylation of histone H3.
(A) HEK 293A cells were transfected with EGFP vector, EGFP-PKCδ catalytic fragment (CF) and EGFP-PKCδ dominant negative form of catalytic fragment (CF DN), respectively.At 24 hours after transfection, cell lysates were subjected to immunoblot analysis with indicated antibodies. (B) Immunofluorescence staining of HEK293A cells transfected with PKCδ constructs. HEK293A cells were transfected with EGFP vector, EGFP-PKCδ CF and EGFP-PKCδ CF DN, respectively. Phosphorylation of histone H3 Ser 10 was monitored with specific antibody. (C) Kinase activity of PKCδ is required for histone H3 Ser 10 phosphorylation. Kinase-dead PKCδ CF DN was unable to phosphorylate histone H3 on Ser 10. Numbers of positive cells with anti-phospho histone H3 Ser 10 staining were calculated with means±SD value of three independent experiments. **, P<0.01.
Figure 3.
Phosphorylation of histone H3 on Ser10 in apoptotic cell death.
(A) and (B) Jurkat (A) or HeLa (B) cells were treated with 1 mM hydroxyurea for 24 hours and then treated with cisplatin (50 µM) for the incubated times. Whole cell lysates were analyzed by immunoblotting with indicated antibodies. (C) Jurkat cells transfected with control siRNA or PKCδ siRNA were untreated or treated with cisplatin (50 µM) for 12 hours. The samples were analyzed by immunoblotting with indicated antibodies. (D) Jurkat cells were transfected scramble control siRNA or PKCδ siRNA for 24 hours. Then cells were treated with cisplatin (50 µM) for 9 hours. Cell viability was measured by MTT assay. *, P<0.05. (E) HeLa cells were treated with 1 mM hydroxyurea for 24 hours and then treated with cisplatin (50 µM) for 12 hours, and anti-PKCδ or phospho-histone H3 Ser 10 staining cells were detected using immunofluorescence microscope. (F) Whole cell extracts from Jurkat cells treated with vehicle(DMSO) or cisplatin (50 µM) were immunoprecipitated with anti-PKCδ antibody, and the precipitates were immunoblotted with antibodies to PKCδ or histone H3.
Figure 4.
Phosphorylation of histone H3 on Ser-10 in apoptotic cells.
(A) and (B) HeLa cells were treated with 1 mM hydroxyurea for 24 hours and then treated with cisplatin (50 µM) for 12 hours, and TUNEL (A), cleaved caspase-3 (B) and phospho-histone H3 Ser 10 staining cells were detected using immunofluorescence microscope. Upper and lower panel images were taken from the same experimental set (A and B).
Figure 5.
PKCδ-mediated phosphorylation of histone H3 on Ser-10 contributes to apoptotic chromatin condensation.
HEK293A cells were transfected with EGFP vector, EGFP-PKCδ CF and EGFP-PKCδ CF DN, respectively. Chromatin condensation was analyzed with fluorescent microscope after Hoechst staining. The condensed nuclei were indicated with white arrows (middle panel).
Figure 6.
Two distinctive biological implications of the phosphorylation of histone H3 on Ser-10.
In response to mitogenic stimuli, histone H3 Ser-10 is phosphorylated by mitotic kinase such as Aurora B or VRK1 to promote the mitotic chromatin condensation (upper). On the other hand, in response to death stimuli, activated PKCδ phosphorylate the same position to facilitate the apoptotic chromatin condensation (lower).