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Plk1 Regulates the Repressor Function of FoxM1b by inhibiting its Interaction with the Retinoblastoma Protein

View Article: PubMed Central - PubMed

ABSTRACT

FoxM1b is a cell cycle-regulated transcription factor, whose over-expression is a marker for poor outcome in cancers. Its transcriptional activation function requires phosphorylation by Cdk1 or Cdk2 that primes FoxM1b for phosphorylation by Plk1, which triggers association with the co-activator CBP. FoxM1b also possesses transcriptional repression function. It represses the mammary differentiation gene GATA3 involving DNMT3b and Rb. We investigated what determines the two distinct functions of FoxM1b: activation and repression. We show that Rb binds to the C-terminal activation domain of FoxM1b. Analyses with phospho-defective and phospho-mimetic mutants of FoxM1b identified a critical role of the Plk1 phosphorylation sites in regulating the binding of FoxM1b to Rb and DNMT3b. That is opposite of what was seen for the interaction of FoxM1b with CBP. We show that, in addition to GATA3, FoxM1b also represses the mammary luminal differentiation marker FoxA1 by promoter-methylation, and that is regulated by the Plk1 phosphorylation sites in FoxM1b. Our results show that the Plk1 phosphorylation sites in FoxM1b serve as a regulator for its repressor function, and they provide insights into how FoxM1b inhibits differentiation genes and activates proliferation genes during cancer progression.

No MeSH data available.


Plk1 Phosphorylation determines the binding partners of FoxM1b.(A) Phosphorylation of FoxM1 inhibits the interaction with Rb. MDA-MB-453 cells were transiently transfected with T7 tagged FoxM1 wild type (WT), T7 tagged phosphodefective mutant (T596A), and T7 tagged phosphomimetic mutant of S715 and S724 (FoxM1-DD). Cell lysates (800 ug) from each transfection were immunoprecipitated with a monoclonal T7 antibody. Western analysis of the upper and lower portion of the same blot was performed with CBP and Rb antibody respectively (middle and upper panel of Fig. 4A). Similar immunoprecipitations were performed in parallel to identify the interactions of these constructs with DNMT3b using a monoclonal antibody. Expression of T7-tagged wild type and phosphomutant proteins was shown in right panel. (B) Interactions of wild type and mutant FoxM1 with Rb in MCF7 cells. We performed the similar experiment in MCF7 cells as described in Fig. 4A with GFP-WT-FoxM1 and GFP- FoxM1 mutants. Cell lysates were immunoprecipitated with GFP antibody and the immunoprecipitates were assayed for the presence of Rb, CBP, DNMT3b, and FoxM1 after western transfer. Expression of GFP constructs was shown in right panel. (C) Quantifications of the Rb and CBP binding by the wild type and FoxM1-mutants was done using image J (p-values represented as *≤0.05; **≤0.01; ***≤0.001).
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f4: Plk1 Phosphorylation determines the binding partners of FoxM1b.(A) Phosphorylation of FoxM1 inhibits the interaction with Rb. MDA-MB-453 cells were transiently transfected with T7 tagged FoxM1 wild type (WT), T7 tagged phosphodefective mutant (T596A), and T7 tagged phosphomimetic mutant of S715 and S724 (FoxM1-DD). Cell lysates (800 ug) from each transfection were immunoprecipitated with a monoclonal T7 antibody. Western analysis of the upper and lower portion of the same blot was performed with CBP and Rb antibody respectively (middle and upper panel of Fig. 4A). Similar immunoprecipitations were performed in parallel to identify the interactions of these constructs with DNMT3b using a monoclonal antibody. Expression of T7-tagged wild type and phosphomutant proteins was shown in right panel. (B) Interactions of wild type and mutant FoxM1 with Rb in MCF7 cells. We performed the similar experiment in MCF7 cells as described in Fig. 4A with GFP-WT-FoxM1 and GFP- FoxM1 mutants. Cell lysates were immunoprecipitated with GFP antibody and the immunoprecipitates were assayed for the presence of Rb, CBP, DNMT3b, and FoxM1 after western transfer. Expression of GFP constructs was shown in right panel. (C) Quantifications of the Rb and CBP binding by the wild type and FoxM1-mutants was done using image J (p-values represented as *≤0.05; **≤0.01; ***≤0.001).

Mentions: To further investigate the impact of Plk1 phosphorylation of FoxM1, we employed phospho-defective and phospho-mimetic mutants of FoxM1b. Mutation of T596 to alanine (T596A) was shown to inhibit Plk1 phosphorylation at S715 and S72443. Also, we generated S715A, S724A (AA) as Plk1 sites phospho-defective mutant, and S715D, S724D (DD) as Plk1 sites phospho-mimetic mutant. Epitope-tagged FoxM1b expression vectors were used to distinguish from the interactions of the endogenous FoxM1. GFP-tagged FoxM1b-mutants or T7-tagged mutants were analyzed for binding to Rb, CBP and DNMT3b in MDA-MB-453 (Fig. 4A) and MCF7 cells (Fig. 4B). Consistent with the experiments in Fig. 3, we observed that the phospho-defective mutants of FoxM1 maintain interactions with Rb (Fig. 4A,B). Quantifications of the binding data in MCF7 cells from 3 independent experiments (Figs 4B and S3D) are shown in Fig. 4C. Clearly, the phospho-mimetic mutant of FoxM1 is impaired in binding to Rb (Fig. 4). The opposite is true for interaction with CBP. The phospho-defective mutants are deficient in binding to CBP, but the phospho-mimetic mutant bound CBP as efficiently as the wild type, if not better (Fig. 4). The Rb-family protein p130 exhibited similar binding pattern with the mutant FoxM1 as Rb, whereas p107 appeared to retain some binding with the phospho-mimetic mutant of FoxM1 (Fig. S4). These observations on differential interactions of FoxM1 with Rb and CBP as a function of Plk1 phosphorylation suggest that Plk1 phosphorylation of FoxM1 functions as a switch that inhibits interaction with Rb or Rb-family proteins while promotes interaction with CBP. Interestingly, we observed that Plk1 phospho-mimetic mutant is deficient also in binding to DNMT3b (Figs 4 and S3B). That is somewhat surprising given that the DNMT3b binds to the N-terminal region in FoxM1. However, there is evidence for interactions between the N- and C-terminal domains of FoxM1b that is regulated by phosphorylation5051. Therefore, in the context of the full-length protein, it is not unexpected that phosphorylation of FoxM1b at the Plk1-sites would regulate its interaction with DNMT3b.


Plk1 Regulates the Repressor Function of FoxM1b by inhibiting its Interaction with the Retinoblastoma Protein
Plk1 Phosphorylation determines the binding partners of FoxM1b.(A) Phosphorylation of FoxM1 inhibits the interaction with Rb. MDA-MB-453 cells were transiently transfected with T7 tagged FoxM1 wild type (WT), T7 tagged phosphodefective mutant (T596A), and T7 tagged phosphomimetic mutant of S715 and S724 (FoxM1-DD). Cell lysates (800 ug) from each transfection were immunoprecipitated with a monoclonal T7 antibody. Western analysis of the upper and lower portion of the same blot was performed with CBP and Rb antibody respectively (middle and upper panel of Fig. 4A). Similar immunoprecipitations were performed in parallel to identify the interactions of these constructs with DNMT3b using a monoclonal antibody. Expression of T7-tagged wild type and phosphomutant proteins was shown in right panel. (B) Interactions of wild type and mutant FoxM1 with Rb in MCF7 cells. We performed the similar experiment in MCF7 cells as described in Fig. 4A with GFP-WT-FoxM1 and GFP- FoxM1 mutants. Cell lysates were immunoprecipitated with GFP antibody and the immunoprecipitates were assayed for the presence of Rb, CBP, DNMT3b, and FoxM1 after western transfer. Expression of GFP constructs was shown in right panel. (C) Quantifications of the Rb and CBP binding by the wild type and FoxM1-mutants was done using image J (p-values represented as *≤0.05; **≤0.01; ***≤0.001).
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f4: Plk1 Phosphorylation determines the binding partners of FoxM1b.(A) Phosphorylation of FoxM1 inhibits the interaction with Rb. MDA-MB-453 cells were transiently transfected with T7 tagged FoxM1 wild type (WT), T7 tagged phosphodefective mutant (T596A), and T7 tagged phosphomimetic mutant of S715 and S724 (FoxM1-DD). Cell lysates (800 ug) from each transfection were immunoprecipitated with a monoclonal T7 antibody. Western analysis of the upper and lower portion of the same blot was performed with CBP and Rb antibody respectively (middle and upper panel of Fig. 4A). Similar immunoprecipitations were performed in parallel to identify the interactions of these constructs with DNMT3b using a monoclonal antibody. Expression of T7-tagged wild type and phosphomutant proteins was shown in right panel. (B) Interactions of wild type and mutant FoxM1 with Rb in MCF7 cells. We performed the similar experiment in MCF7 cells as described in Fig. 4A with GFP-WT-FoxM1 and GFP- FoxM1 mutants. Cell lysates were immunoprecipitated with GFP antibody and the immunoprecipitates were assayed for the presence of Rb, CBP, DNMT3b, and FoxM1 after western transfer. Expression of GFP constructs was shown in right panel. (C) Quantifications of the Rb and CBP binding by the wild type and FoxM1-mutants was done using image J (p-values represented as *≤0.05; **≤0.01; ***≤0.001).
Mentions: To further investigate the impact of Plk1 phosphorylation of FoxM1, we employed phospho-defective and phospho-mimetic mutants of FoxM1b. Mutation of T596 to alanine (T596A) was shown to inhibit Plk1 phosphorylation at S715 and S72443. Also, we generated S715A, S724A (AA) as Plk1 sites phospho-defective mutant, and S715D, S724D (DD) as Plk1 sites phospho-mimetic mutant. Epitope-tagged FoxM1b expression vectors were used to distinguish from the interactions of the endogenous FoxM1. GFP-tagged FoxM1b-mutants or T7-tagged mutants were analyzed for binding to Rb, CBP and DNMT3b in MDA-MB-453 (Fig. 4A) and MCF7 cells (Fig. 4B). Consistent with the experiments in Fig. 3, we observed that the phospho-defective mutants of FoxM1 maintain interactions with Rb (Fig. 4A,B). Quantifications of the binding data in MCF7 cells from 3 independent experiments (Figs 4B and S3D) are shown in Fig. 4C. Clearly, the phospho-mimetic mutant of FoxM1 is impaired in binding to Rb (Fig. 4). The opposite is true for interaction with CBP. The phospho-defective mutants are deficient in binding to CBP, but the phospho-mimetic mutant bound CBP as efficiently as the wild type, if not better (Fig. 4). The Rb-family protein p130 exhibited similar binding pattern with the mutant FoxM1 as Rb, whereas p107 appeared to retain some binding with the phospho-mimetic mutant of FoxM1 (Fig. S4). These observations on differential interactions of FoxM1 with Rb and CBP as a function of Plk1 phosphorylation suggest that Plk1 phosphorylation of FoxM1 functions as a switch that inhibits interaction with Rb or Rb-family proteins while promotes interaction with CBP. Interestingly, we observed that Plk1 phospho-mimetic mutant is deficient also in binding to DNMT3b (Figs 4 and S3B). That is somewhat surprising given that the DNMT3b binds to the N-terminal region in FoxM1. However, there is evidence for interactions between the N- and C-terminal domains of FoxM1b that is regulated by phosphorylation5051. Therefore, in the context of the full-length protein, it is not unexpected that phosphorylation of FoxM1b at the Plk1-sites would regulate its interaction with DNMT3b.

View Article: PubMed Central - PubMed

ABSTRACT

FoxM1b is a cell cycle-regulated transcription factor, whose over-expression is a marker for poor outcome in cancers. Its transcriptional activation function requires phosphorylation by Cdk1 or Cdk2 that primes FoxM1b for phosphorylation by Plk1, which triggers association with the co-activator CBP. FoxM1b also possesses transcriptional repression function. It represses the mammary differentiation gene GATA3 involving DNMT3b and Rb. We investigated what determines the two distinct functions of FoxM1b: activation and repression. We show that Rb binds to the C-terminal activation domain of FoxM1b. Analyses with phospho-defective and phospho-mimetic mutants of FoxM1b identified a critical role of the Plk1 phosphorylation sites in regulating the binding of FoxM1b to Rb and DNMT3b. That is opposite of what was seen for the interaction of FoxM1b with CBP. We show that, in addition to GATA3, FoxM1b also represses the mammary luminal differentiation marker FoxA1 by promoter-methylation, and that is regulated by the Plk1 phosphorylation sites in FoxM1b. Our results show that the Plk1 phosphorylation sites in FoxM1b serve as a regulator for its repressor function, and they provide insights into how FoxM1b inhibits differentiation genes and activates proliferation genes during cancer progression.

No MeSH data available.