EGF receptor and PKCδ kinase activate DNA damage-induced pro-survival and pro-apoptotic signaling via biphasic activation of ERK and MSK1 kinases

Abstract

DNA damage-mediated activation of extracellular signal-regulated kinase (ERK) can regulate both cell survival and cell death. We show here that ERK activation in this context is biphasic and that early and late activation events are mediated by distinct upstream signals that drive cell survival and apoptosis, respectively. We identified the nuclear kinase mitogen-sensitive kinase 1 (MSK1) as a downstream target of both early and late ERK activation. We also observed that activation of ERK→MSK1 up to 4 h after DNA damage depends on epidermal growth factor receptor (EGFR), as EGFR or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)/ERK inhibitors or short hairpin RNA-mediated MSK1 depletion enhanced cell death. This prosurvival response was partially mediated through enhanced DNA repair, as EGFR or MEK/ERK inhibitors delayed DNA damage resolution. In contrast, the second phase of ERK→MSK1 activation drove apoptosis and required protein kinase Cδ (PKCδ) but not EGFR. Genetic disruption of PKCδ reduced ERK activation in an in vivo irradiation model, as did short hairpin RNA-mediated depletion of PKCδ in vitro In both models, PKCδ inhibition preferentially suppressed late activation of ERK. We have shown previously that nuclear localization of PKCδ is necessary and sufficient for apoptosis. Here we identified a nuclear PKCδ→ERK→MSK1 signaling module that regulates apoptosis. We also show that expression of nuclear PKCδ activates ERK and MSK1, that ERK activation is required for MSK1 activation, and that both ERK and MSK1 activation are required for apoptosis. Our findings suggest that location-specific activation by distinct upstream regulators may enable distinct functional outputs from common signaling pathways.

Keywords: DNA damage; apoptosis; cell signaling; epidermal growth factor receptor (EGFR); extracellular signal–regulated kinase (ERK); kinase cascade; mitogen-activated protein kinase (MAPK); mitogen-sensitive kinase (MSK); protein kinase C (PKC); signal transduction.

Figure 1.

Biphasic activation of ERK drives survival and apoptosis. A, ParC5 cells were treated with 50 μm etoposide for the indicated times (hours). Cells were harvested and immunoblotted for pERK and stripped and reprobed for ERK and β-actin. B, quantitation by densitometry of three separate experiments similar to that shown in A. Data represent pERK/ERK/actin ratios ± S.E. Statistics represent one-way ANOVA and post hoc Tukey's multiple comparisons. **, p < 0.001; *, p < 0.05. C, ParC5 cells were pretreated with the DMSO control (black columns) or 10 μm PD98059 (gray columns) prior to addition of 50 μm etoposide for the indicated times (hours). D, ParC5 cells were pretreated with the DMSO control (black columns), 20 μm PD98059 (dark gray columns), or 20 μm U0126 (white columns) prior to addition of 0, 0.5, 5, or 50 μm etoposide for 18 h. E, ParC5 cells were pretreated with the DMSO control (black columns), 20 μm or 40 μm PD98059 (dark gray columns), or 20 μm or 40 μm U0126 (light gray and white columns) and then left untreated or treated with 10 Gy of IR. Cells were harvested 48 h after IR. Caspase-3 activity (C and E) and DNA fragmentation (D) were assayed as described under “Experimental procedures.” Data shown are representative experiments; statistics represent two-way ANOVA for comparison of time point or treatment with the corresponding control. **, p < 0.001; *, p < 0.05. Error bars represent S.E. from triplicate samples.

Figure 2.

EGFR activation of ERK promotes cell survival. A, ParC5 cells were pretreated with the DMSO control or 600 nm gefitinib prior to addition of 50 μm etoposide for the indicated times (hours). Cells were harvested and immunoblotted for the indicated proteins. B, ParC5 cells were pretreated with the DMSO control or 300 nm gefitinib prior to treatment with 10 Gy of IR. Cells were harvested at the indicated times (hours) post-IR and immunoblotted for the indicated proteins. C, ParC5 cells were pretreated with the DMSO control or 300 nm gefitinib or afatinib prior to addition of 50 μm etoposide. Cells were harvested for measurement of caspase-3 activity at the indicated times (hours). D, ParC5 cells were pretreated with DMSO, 300 nm gefitinib, or 20 μm PD98059 prior to irradiation with 10 Gy of IR. Following IR, cells were harvested at various time points (hours) and prepared using a neutral comet assay according to the Trevigen comet assay protocol. Data shown are representative experiments. Statistics represent two-way ANOVA for comparison of time point or treatment with the corresponding control. **, p < 0.001; *, p < 0.05.

Figure 3.

Nuclear PKCδ activates ERK to drive apoptosis. A, ParC5 cells stably expressing either PKCδ shRNA (δ561) or a scrambled control were treated with 50 μm etoposide (Etop) for the indicated times (hours). Lysates were immunoblotted for the indicated proteins. B, 6-week-old female PKCδ^+/+ and PKCδ^−/− mice were irradiated with 5 Gy of IR and harvested at the indicated times (hours) post-IR. Protein lysates were prepared from the parotid gland and immunoblotted for pERK, ERK, and PKCδ. The graph shows relative densitometry units of pERK normalized to total ERK bands, with gray and black columns indicating PKCδ^+/+ and PKCδ^−/−, respectively. C, ParC5 cells stably expressing either PKCδ shRNA (δ561) or a scrambled control were treated with 50 μm etoposide for the indicated times (hours). Lysates were assayed for caspase-3 activity. D, ParC5 cells were transfected with constructs expressing GFP, PKCδWT, or PKCδNLS for the indicated times (hours). E, ParC5 cells were transfected with constructs expressing GFP or GFP-PKCδCF for 24 h. For both D and E, cells were harvested and immunoblotted for GFP, pERK, ERK, or β-actin. F, ParC5 cells were transduced with either Ad-GFP or Ad-GFP-PKCδNLS and plated in the presence of 10 μm U0126 (gray columns) or 20 μm PD98059 (white columns) or treated with the DMSO control (black columns). Cells were harvested and assayed 48 h post-transduction for caspase-3 activity. Data shown are representative experiments. Statistics represent two-way ANOVA for comparison of time point or treatment with the corresponding control. **, p < 0.001; *, p < 0.05.

Figure 4.

ERK activates MSK1 in response to DNA damage. A, ParC5 cells stably expressing either control (SCR) or PKCδ shRNA (δ561 or δ844) were treated with 50 μm etoposide (Etop) for the indicated times (hours). Cells were harvested and immunoblotted for the indicated proteins. B, ParC5 cells were pretreated with the DMSO control or 600 nm gefitinib prior to addition of 50 μm etoposide for the indicated times (hours). Cells were harvested and immunoblotted for the indicated proteins. C and D, ParC5 cells stably expressing either SCR control or MSK1 shRNA were treated with 50 μm etoposide (C) or irradiated with 10 Gy of IR (D). Cells were harvested for caspase-3 activity at the indicated times. The blot in C demonstrates the magnitude of MSK1 depletion in the cells used in C and D. Data shown are representative experiments. Statistics represent two-way ANOVA for comparison of time point or treatment with the corresponding control. **, p < 0.001; *, p < 0.05.

Figure 5.

PKCδ drives DNA damage–induced apoptosis via ERK→MSK1. A, ParC5 cells were transduced with Ad-GFP or Ad-GFP-PKCδNLS (m.o.i. 200) for 24 h. Cells were harvested at the indicated times post-IR and immunoblotted for the indicated proteins. B, ParC5 cells were pretreated with 20 μm PD98059 (PD), 5 μm SB203580 (SB), or a combination of both concurrently with transduction of either Ad-GFP or Ad-GFP-PKCδNLS adenovirus (m.o.i. 200). Cells were then harvested and immunoblotted for the indicated proteins. C and D, ParC5 cells stably expressing either SCR control or MSK1 shRNA were either left untransfected or transfected with GFP, GFP-PKCδNLS (C), or GFP-PKCδCF (D) plasmid for 24 h. Cells were harvested and assayed for caspase-3 activity. Data shown are representative experiments. Statistics represent two-way ANOVA for comparison of time point or treatment with the corresponding control. **, p < 0.001; *, p < 0.05.

Figure 6.

Biphasic activation of ERK and MSK1 in response to DNA damage regulates survival and apoptosis. Our study defines a common ERK→MSK1 pathway that, when activated by EGFR in response to DNA damage, drives cell survival but when activated by nuclear PKCδ drives apoptosis.