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ERK-associated changes in E2F4 phosphorylation, localization and transcriptional activity during mitogenic stimulation in human intestinal epithelial crypt cells.

Paquin MC, Cagnol S, Carrier JC, Leblanc C, Rivard N - BMC Cell Biol. (2013)

Bottom Line: Stimulation of HIEC with epidermal growth factor (EGF) also led to the activation of ERK1/2 but, in contrast to serum or lysophosphatidic acid (LPA), EGF failed to induce E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition.The present results indicate that MEK/ERK activation and GSK3 inhibition are both required for E2F4 phosphorylation as well as its nuclear translocation and S phase entry in HIEC.This finding suggests that dysregulated E2F4 nuclear localization may be an instigating event leading to hyperproliferation and hence, of tumor initiation and promotion in the colon and rectum.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département d'Anatomie et Biologie Cellulaire, Cancer Research Pavillon, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3201, Jean-Mignault, Sherbrooke, J1E4K8, QC, Canada.

ABSTRACT

Background: The transcription factor E2F4 controls proliferation of normal and cancerous intestinal epithelial cells. E2F4 localization in normal human intestinal epithelial cells (HIEC) is cell cycle-dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, the intracellular signaling mechanisms regulating such E2F4 localization remain unknown.

Results: Treatment of quiescent HIEC with serum induced ERK1/2 activation, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition while inhibition of MEK/ERK signaling by U0126 prevented these events. Stimulation of HIEC with epidermal growth factor (EGF) also led to the activation of ERK1/2 but, in contrast to serum or lysophosphatidic acid (LPA), EGF failed to induce E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition. Furthermore, Akt and GSK3β phosphorylation levels were markedly enhanced in serum- or LPA-stimulated HIEC but not by EGF. Importantly, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition were all observed in response to EGF when GSK3 activity was concomitantly inhibited by SB216763. Finally, E2F4 was found to be overexpressed, phosphorylated and nuclear localized in epithelial cells from human colorectal adenomas exhibiting mutations in APC and KRAS or BRAF genes, known to deregulate GSK3/β-catenin and MEK/ERK signaling, respectively.

Conclusions: The present results indicate that MEK/ERK activation and GSK3 inhibition are both required for E2F4 phosphorylation as well as its nuclear translocation and S phase entry in HIEC. This finding suggests that dysregulated E2F4 nuclear localization may be an instigating event leading to hyperproliferation and hence, of tumor initiation and promotion in the colon and rectum.

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E2F4 is phosphorylated by ERK kinases. A. Subconfluent HIEC were serum-deprived for 36 h, treated or not (DMSO) during 10 min with 20 μM U0126 and then stimulated during 30 min with 5% FBS. Thereafter, cells were lysed and proteins were analyzed by SDS-PAGE (on 7.5% or 12% acrylamide gels) for Western blot analysis of the expression of phosphorylated ERK1/2, total ERK1/2, E2F4 and β-actin. B. E2F4 was immunoprecipitated from subconfluent serum-deprived and 30 min serum-stimulated HIEC. Beads containing E2F4 immune complexes were incubated with PP1 phosphatase for 30 min prior to Western blot analysis for E2F4 expression. C. E2F4 was immunoprecipitated from HIEC stimulated during 30 min with FBS in presence or absence of 20 μM U0126. E2F4 immune complexes were analyzed by SDS-PAGE for Western blot analysis with antibodies recognizing phosphorylated serine or E2F4. D. 293T cells were transfected with pCDNA3.1 containing or not HA-E2F4. After 48 h, cells were lysed and immunoprecipitated with anti-HA antibody. Kinase assays were performed by incubating beads containing HA-E2F4 immune complexes with active recombinant ERK1 for 5 min. Radiolabeled proteins were separated on SDS-PAGE and autoradiographed or analyzed by Western blot for the expression of E2F4.
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Figure 2: E2F4 is phosphorylated by ERK kinases. A. Subconfluent HIEC were serum-deprived for 36 h, treated or not (DMSO) during 10 min with 20 μM U0126 and then stimulated during 30 min with 5% FBS. Thereafter, cells were lysed and proteins were analyzed by SDS-PAGE (on 7.5% or 12% acrylamide gels) for Western blot analysis of the expression of phosphorylated ERK1/2, total ERK1/2, E2F4 and β-actin. B. E2F4 was immunoprecipitated from subconfluent serum-deprived and 30 min serum-stimulated HIEC. Beads containing E2F4 immune complexes were incubated with PP1 phosphatase for 30 min prior to Western blot analysis for E2F4 expression. C. E2F4 was immunoprecipitated from HIEC stimulated during 30 min with FBS in presence or absence of 20 μM U0126. E2F4 immune complexes were analyzed by SDS-PAGE for Western blot analysis with antibodies recognizing phosphorylated serine or E2F4. D. 293T cells were transfected with pCDNA3.1 containing or not HA-E2F4. After 48 h, cells were lysed and immunoprecipitated with anti-HA antibody. Kinase assays were performed by incubating beads containing HA-E2F4 immune complexes with active recombinant ERK1 for 5 min. Radiolabeled proteins were separated on SDS-PAGE and autoradiographed or analyzed by Western blot for the expression of E2F4.

Mentions: Western blot analysis of E2F4 revealed that 30 min serum stimulation with or without U0126 did not affect the total expression levels of E2F4 (Figure 2A, upper first panel), even after 24 h stimulation (data not shown). However, when using higher-resolution gels, three major bands of approximately 60–63 kDa were detected in serum-deprived HIEC, whereas only one band with a lower electrophoretic mobility was observed in serum-stimulated cells after 30 min (Figure 2A, arrows). Of note, treatment with U0126 abolished ERK phosphorylation and markedly reduced the expression of this latter prominent band (Figure 2A). Similar results were obtained when we used the more specific and potent MEK inhibitor PD184352 (Additional file 2: Figure S2). We therefore investigated whether E2F4 phosphorylation could be responsible for this occurrence. E2F4 was immunoprecipitated from serum-deprived or serum-stimulated HIEC. Beads containing E2F4 immune complexes were then incubated with the serine/threonine phosphatase PP1 in order to dephosphorylate serine/threonine residues on E2F4. As shown in Figure 2B, immunoprecipitated E2F4 exhibited three bands in control HIEC, in contrast to one prominent band in serum-stimulated cells. Of interest, treatment of E2F4 immunoprecipitates from serum-stimulated cells with the PP1 phosphatase modified the electrophoretic profile of E2F4, reducing the amount of the slower migrating form of E2F4. Moreover, the use of antibodies recognizing phosphorylated serine revealed that E2F4 was phosphorylated on serine residue(s) upon serum stimulation (Figure 2C). Of note, the levels of phosphorylated serine residues in immunoprecipitated E2F4 were not completely reduced by U0126 treatment, suggesting that E2F4 could also be phosphorylated in absence of serum and ERK activation in quiescent HIEC as we previously observed [9]. Kinase assays with active recombinant ERK1 confirmed that ERK1 strongly phosphorylated immunoprecipitated HA-tagged E2F4 in vitro (Figure 2D). These results clearly indicate that E2F4 is phosphorylated on serine residue(s) in response to serum in a MEK-dependent manner. The data also suggest ERK1/2 as candidate kinases.


ERK-associated changes in E2F4 phosphorylation, localization and transcriptional activity during mitogenic stimulation in human intestinal epithelial crypt cells.

Paquin MC, Cagnol S, Carrier JC, Leblanc C, Rivard N - BMC Cell Biol. (2013)

E2F4 is phosphorylated by ERK kinases. A. Subconfluent HIEC were serum-deprived for 36 h, treated or not (DMSO) during 10 min with 20 μM U0126 and then stimulated during 30 min with 5% FBS. Thereafter, cells were lysed and proteins were analyzed by SDS-PAGE (on 7.5% or 12% acrylamide gels) for Western blot analysis of the expression of phosphorylated ERK1/2, total ERK1/2, E2F4 and β-actin. B. E2F4 was immunoprecipitated from subconfluent serum-deprived and 30 min serum-stimulated HIEC. Beads containing E2F4 immune complexes were incubated with PP1 phosphatase for 30 min prior to Western blot analysis for E2F4 expression. C. E2F4 was immunoprecipitated from HIEC stimulated during 30 min with FBS in presence or absence of 20 μM U0126. E2F4 immune complexes were analyzed by SDS-PAGE for Western blot analysis with antibodies recognizing phosphorylated serine or E2F4. D. 293T cells were transfected with pCDNA3.1 containing or not HA-E2F4. After 48 h, cells were lysed and immunoprecipitated with anti-HA antibody. Kinase assays were performed by incubating beads containing HA-E2F4 immune complexes with active recombinant ERK1 for 5 min. Radiolabeled proteins were separated on SDS-PAGE and autoradiographed or analyzed by Western blot for the expression of E2F4.
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Related In: Results  -  Collection

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Figure 2: E2F4 is phosphorylated by ERK kinases. A. Subconfluent HIEC were serum-deprived for 36 h, treated or not (DMSO) during 10 min with 20 μM U0126 and then stimulated during 30 min with 5% FBS. Thereafter, cells were lysed and proteins were analyzed by SDS-PAGE (on 7.5% or 12% acrylamide gels) for Western blot analysis of the expression of phosphorylated ERK1/2, total ERK1/2, E2F4 and β-actin. B. E2F4 was immunoprecipitated from subconfluent serum-deprived and 30 min serum-stimulated HIEC. Beads containing E2F4 immune complexes were incubated with PP1 phosphatase for 30 min prior to Western blot analysis for E2F4 expression. C. E2F4 was immunoprecipitated from HIEC stimulated during 30 min with FBS in presence or absence of 20 μM U0126. E2F4 immune complexes were analyzed by SDS-PAGE for Western blot analysis with antibodies recognizing phosphorylated serine or E2F4. D. 293T cells were transfected with pCDNA3.1 containing or not HA-E2F4. After 48 h, cells were lysed and immunoprecipitated with anti-HA antibody. Kinase assays were performed by incubating beads containing HA-E2F4 immune complexes with active recombinant ERK1 for 5 min. Radiolabeled proteins were separated on SDS-PAGE and autoradiographed or analyzed by Western blot for the expression of E2F4.
Mentions: Western blot analysis of E2F4 revealed that 30 min serum stimulation with or without U0126 did not affect the total expression levels of E2F4 (Figure 2A, upper first panel), even after 24 h stimulation (data not shown). However, when using higher-resolution gels, three major bands of approximately 60–63 kDa were detected in serum-deprived HIEC, whereas only one band with a lower electrophoretic mobility was observed in serum-stimulated cells after 30 min (Figure 2A, arrows). Of note, treatment with U0126 abolished ERK phosphorylation and markedly reduced the expression of this latter prominent band (Figure 2A). Similar results were obtained when we used the more specific and potent MEK inhibitor PD184352 (Additional file 2: Figure S2). We therefore investigated whether E2F4 phosphorylation could be responsible for this occurrence. E2F4 was immunoprecipitated from serum-deprived or serum-stimulated HIEC. Beads containing E2F4 immune complexes were then incubated with the serine/threonine phosphatase PP1 in order to dephosphorylate serine/threonine residues on E2F4. As shown in Figure 2B, immunoprecipitated E2F4 exhibited three bands in control HIEC, in contrast to one prominent band in serum-stimulated cells. Of interest, treatment of E2F4 immunoprecipitates from serum-stimulated cells with the PP1 phosphatase modified the electrophoretic profile of E2F4, reducing the amount of the slower migrating form of E2F4. Moreover, the use of antibodies recognizing phosphorylated serine revealed that E2F4 was phosphorylated on serine residue(s) upon serum stimulation (Figure 2C). Of note, the levels of phosphorylated serine residues in immunoprecipitated E2F4 were not completely reduced by U0126 treatment, suggesting that E2F4 could also be phosphorylated in absence of serum and ERK activation in quiescent HIEC as we previously observed [9]. Kinase assays with active recombinant ERK1 confirmed that ERK1 strongly phosphorylated immunoprecipitated HA-tagged E2F4 in vitro (Figure 2D). These results clearly indicate that E2F4 is phosphorylated on serine residue(s) in response to serum in a MEK-dependent manner. The data also suggest ERK1/2 as candidate kinases.

Bottom Line: Stimulation of HIEC with epidermal growth factor (EGF) also led to the activation of ERK1/2 but, in contrast to serum or lysophosphatidic acid (LPA), EGF failed to induce E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition.The present results indicate that MEK/ERK activation and GSK3 inhibition are both required for E2F4 phosphorylation as well as its nuclear translocation and S phase entry in HIEC.This finding suggests that dysregulated E2F4 nuclear localization may be an instigating event leading to hyperproliferation and hence, of tumor initiation and promotion in the colon and rectum.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département d'Anatomie et Biologie Cellulaire, Cancer Research Pavillon, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3201, Jean-Mignault, Sherbrooke, J1E4K8, QC, Canada.

ABSTRACT

Background: The transcription factor E2F4 controls proliferation of normal and cancerous intestinal epithelial cells. E2F4 localization in normal human intestinal epithelial cells (HIEC) is cell cycle-dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, the intracellular signaling mechanisms regulating such E2F4 localization remain unknown.

Results: Treatment of quiescent HIEC with serum induced ERK1/2 activation, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition while inhibition of MEK/ERK signaling by U0126 prevented these events. Stimulation of HIEC with epidermal growth factor (EGF) also led to the activation of ERK1/2 but, in contrast to serum or lysophosphatidic acid (LPA), EGF failed to induce E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition. Furthermore, Akt and GSK3β phosphorylation levels were markedly enhanced in serum- or LPA-stimulated HIEC but not by EGF. Importantly, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition were all observed in response to EGF when GSK3 activity was concomitantly inhibited by SB216763. Finally, E2F4 was found to be overexpressed, phosphorylated and nuclear localized in epithelial cells from human colorectal adenomas exhibiting mutations in APC and KRAS or BRAF genes, known to deregulate GSK3/β-catenin and MEK/ERK signaling, respectively.

Conclusions: The present results indicate that MEK/ERK activation and GSK3 inhibition are both required for E2F4 phosphorylation as well as its nuclear translocation and S phase entry in HIEC. This finding suggests that dysregulated E2F4 nuclear localization may be an instigating event leading to hyperproliferation and hence, of tumor initiation and promotion in the colon and rectum.

Show MeSH
Related in: MedlinePlus