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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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Phosphorylation of E2F4 on serines 244 and 384 promotes its transcriptional activity. A. and B. 293T cells were transfected with pCDNA3.1 empty vector (EV) or encoded for HA-tagged- human wild-type (WT) E2F4 or E2F4 mutants as indicated. After 48 h, cell lysates were analyzed for the expression of E2F4 proteins. C. 293T cells were transfected with EV or encoding for HA-E2F4 WT, HA-E2F4 S244A, HA-E2F4 S384A or HA-E2F4 S244A/S384A. 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 recombinant ERK1 for 5 min. Radiolabeled proteins were separated on SDS-PAGE and autoradiographed or analyzed for the expression of E2F4. D. 293T cells were co-transfected with DP2, thymidine kinase luciferase reporter with either empty vector, HA-wtE2F4, HA-E2F4 S244E, HA-E2F4 S384E or HA-E2F4 S244/S384E. pRL-SV40 Renilla luciferase reporter control vector was also co-transfected. Forty-eight hours following transfection, luciferase activity was quantified and normalized using the Renilla reporter, with the empty vector condition set at 1. A representative experiment of three experiments is shown. *Significantly different from control at p < 0.001, **Significantly different from WT at p < 0.002, ***Significantly different from WT at p < 0.001 (Student’s t test). E. HIEC grown on coverslips were transfected with either empty vector, HA-wtE2F4, HA-E2F4 S244E, HA-E2F4 S384E or HA-E2F4 S244/S384E. After 48 h, cells were analyzed by immunofluorescence for subcellular localization of HA-tagged E2F4 forms. Total cell number was determined using DAPI staining and cells exhibiting E2F4 forms into the nucleus or into the cytoplasm or in both compartments were counted. The percentage of cells exhibiting HA staining into the nucleus, into the cytoplasm or in both compartments was calculated and shown in the graph. Representative experiment is shown.
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Figure 3: Phosphorylation of E2F4 on serines 244 and 384 promotes its transcriptional activity. A. and B. 293T cells were transfected with pCDNA3.1 empty vector (EV) or encoded for HA-tagged- human wild-type (WT) E2F4 or E2F4 mutants as indicated. After 48 h, cell lysates were analyzed for the expression of E2F4 proteins. C. 293T cells were transfected with EV or encoding for HA-E2F4 WT, HA-E2F4 S244A, HA-E2F4 S384A or HA-E2F4 S244A/S384A. 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 recombinant ERK1 for 5 min. Radiolabeled proteins were separated on SDS-PAGE and autoradiographed or analyzed for the expression of E2F4. D. 293T cells were co-transfected with DP2, thymidine kinase luciferase reporter with either empty vector, HA-wtE2F4, HA-E2F4 S244E, HA-E2F4 S384E or HA-E2F4 S244/S384E. pRL-SV40 Renilla luciferase reporter control vector was also co-transfected. Forty-eight hours following transfection, luciferase activity was quantified and normalized using the Renilla reporter, with the empty vector condition set at 1. A representative experiment of three experiments is shown. *Significantly different from control at p < 0.001, **Significantly different from WT at p < 0.002, ***Significantly different from WT at p < 0.001 (Student’s t test). E. HIEC grown on coverslips were transfected with either empty vector, HA-wtE2F4, HA-E2F4 S244E, HA-E2F4 S384E or HA-E2F4 S244/S384E. After 48 h, cells were analyzed by immunofluorescence for subcellular localization of HA-tagged E2F4 forms. Total cell number was determined using DAPI staining and cells exhibiting E2F4 forms into the nucleus or into the cytoplasm or in both compartments were counted. The percentage of cells exhibiting HA staining into the nucleus, into the cytoplasm or in both compartments was calculated and shown in the graph. Representative experiment is shown.

Mentions: We identified seven putative ERK phosphorylation sites followed by a proline residue in E2F4 human sequence: T14, S202, S218, T224, S244, T248 and S384. Each of these specific serine/threonine residues was mutated into alanine. As shown in Figure 3A, mutation of serines 244 and 384 resulted in modification of the E2F4 electrophoretic profile in 293T cells, decreasing the amount of the slower migrating forms of E2F4. Of note, these slower migrating forms almost completely disappeared when both serines were mutated into alanine (Figure 3B). Accordingly, the S244A, S384A and the S244A/S384A mutants were much less phosphorylated by recombinant ERK1 in in vitro kinase assays (Figure 3C). Finally, the effect of E2F4 phosphorylation on E2F4 site-dependent transcription was measured on the thymidine kinase promoter, which represents the physiological E2F target gene [1,2]. Mutation of each of these serines into phosphomimetic sites, namely S244E, S384E and S244E/S384E, significantly increased the transcriptional activity of E2F4 (Figure 3D), confirming the involvement of the phosphorylation of these residues in the control of E2F4 transcriptional activity. To verify if mutations of serines 244 and 384 also alter the localization of E2F4, the S244A, S384A and the S244A/S384A E2F4 mutants were transiently expressed in HIEC and analyzed for their subcellular localization. As shown in Figure 3E, when overexpressed, wild-type E2F4 was mainly found into the cytoplasm but also in a minor proportion into the nucleus. By contrast, all the phosphomimetic mutants were consistently less expressed into the cytoplasm and more detected into the nucleus, correlating with their increased transcriptional activity (Figure 3D). Nevertheless, the observation of some cytoplasmic staining for the phospho-mimetic mutants indicate that other mechanisms than phosphorylation might also be involved in the control E2F4 nuclear translocation.


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)

Phosphorylation of E2F4 on serines 244 and 384 promotes its transcriptional activity. A. and B. 293T cells were transfected with pCDNA3.1 empty vector (EV) or encoded for HA-tagged- human wild-type (WT) E2F4 or E2F4 mutants as indicated. After 48 h, cell lysates were analyzed for the expression of E2F4 proteins. C. 293T cells were transfected with EV or encoding for HA-E2F4 WT, HA-E2F4 S244A, HA-E2F4 S384A or HA-E2F4 S244A/S384A. 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 recombinant ERK1 for 5 min. Radiolabeled proteins were separated on SDS-PAGE and autoradiographed or analyzed for the expression of E2F4. D. 293T cells were co-transfected with DP2, thymidine kinase luciferase reporter with either empty vector, HA-wtE2F4, HA-E2F4 S244E, HA-E2F4 S384E or HA-E2F4 S244/S384E. pRL-SV40 Renilla luciferase reporter control vector was also co-transfected. Forty-eight hours following transfection, luciferase activity was quantified and normalized using the Renilla reporter, with the empty vector condition set at 1. A representative experiment of three experiments is shown. *Significantly different from control at p < 0.001, **Significantly different from WT at p < 0.002, ***Significantly different from WT at p < 0.001 (Student’s t test). E. HIEC grown on coverslips were transfected with either empty vector, HA-wtE2F4, HA-E2F4 S244E, HA-E2F4 S384E or HA-E2F4 S244/S384E. After 48 h, cells were analyzed by immunofluorescence for subcellular localization of HA-tagged E2F4 forms. Total cell number was determined using DAPI staining and cells exhibiting E2F4 forms into the nucleus or into the cytoplasm or in both compartments were counted. The percentage of cells exhibiting HA staining into the nucleus, into the cytoplasm or in both compartments was calculated and shown in the graph. Representative experiment is shown.
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Figure 3: Phosphorylation of E2F4 on serines 244 and 384 promotes its transcriptional activity. A. and B. 293T cells were transfected with pCDNA3.1 empty vector (EV) or encoded for HA-tagged- human wild-type (WT) E2F4 or E2F4 mutants as indicated. After 48 h, cell lysates were analyzed for the expression of E2F4 proteins. C. 293T cells were transfected with EV or encoding for HA-E2F4 WT, HA-E2F4 S244A, HA-E2F4 S384A or HA-E2F4 S244A/S384A. 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 recombinant ERK1 for 5 min. Radiolabeled proteins were separated on SDS-PAGE and autoradiographed or analyzed for the expression of E2F4. D. 293T cells were co-transfected with DP2, thymidine kinase luciferase reporter with either empty vector, HA-wtE2F4, HA-E2F4 S244E, HA-E2F4 S384E or HA-E2F4 S244/S384E. pRL-SV40 Renilla luciferase reporter control vector was also co-transfected. Forty-eight hours following transfection, luciferase activity was quantified and normalized using the Renilla reporter, with the empty vector condition set at 1. A representative experiment of three experiments is shown. *Significantly different from control at p < 0.001, **Significantly different from WT at p < 0.002, ***Significantly different from WT at p < 0.001 (Student’s t test). E. HIEC grown on coverslips were transfected with either empty vector, HA-wtE2F4, HA-E2F4 S244E, HA-E2F4 S384E or HA-E2F4 S244/S384E. After 48 h, cells were analyzed by immunofluorescence for subcellular localization of HA-tagged E2F4 forms. Total cell number was determined using DAPI staining and cells exhibiting E2F4 forms into the nucleus or into the cytoplasm or in both compartments were counted. The percentage of cells exhibiting HA staining into the nucleus, into the cytoplasm or in both compartments was calculated and shown in the graph. Representative experiment is shown.
Mentions: We identified seven putative ERK phosphorylation sites followed by a proline residue in E2F4 human sequence: T14, S202, S218, T224, S244, T248 and S384. Each of these specific serine/threonine residues was mutated into alanine. As shown in Figure 3A, mutation of serines 244 and 384 resulted in modification of the E2F4 electrophoretic profile in 293T cells, decreasing the amount of the slower migrating forms of E2F4. Of note, these slower migrating forms almost completely disappeared when both serines were mutated into alanine (Figure 3B). Accordingly, the S244A, S384A and the S244A/S384A mutants were much less phosphorylated by recombinant ERK1 in in vitro kinase assays (Figure 3C). Finally, the effect of E2F4 phosphorylation on E2F4 site-dependent transcription was measured on the thymidine kinase promoter, which represents the physiological E2F target gene [1,2]. Mutation of each of these serines into phosphomimetic sites, namely S244E, S384E and S244E/S384E, significantly increased the transcriptional activity of E2F4 (Figure 3D), confirming the involvement of the phosphorylation of these residues in the control of E2F4 transcriptional activity. To verify if mutations of serines 244 and 384 also alter the localization of E2F4, the S244A, S384A and the S244A/S384A E2F4 mutants were transiently expressed in HIEC and analyzed for their subcellular localization. As shown in Figure 3E, when overexpressed, wild-type E2F4 was mainly found into the cytoplasm but also in a minor proportion into the nucleus. By contrast, all the phosphomimetic mutants were consistently less expressed into the cytoplasm and more detected into the nucleus, correlating with their increased transcriptional activity (Figure 3D). Nevertheless, the observation of some cytoplasmic staining for the phospho-mimetic mutants indicate that other mechanisms than phosphorylation might also be involved in the control E2F4 nuclear translocation.

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