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Proteomic analyses uncover a new function and mode of action for mouse homolog of Diaphanous 2 (mDia2).

Isogai T, van der Kammen R, Goerdayal SS, Heck AJ, Altelaar AF, Innocenti M - Mol. Cell Proteomics (2015)

Bottom Line: Taking FBXO3 as a test case, we show that mDia2 binds FBXO3 and p53, and regulates p53 transcriptional activity in an actin-nucleation-independent and conformation-insensitive manner.Increased mDia2 and FBXO3 levels elevate p53 activity and expression thereby sensitizing cells to p53-dependent apoptosis, whereas their decrease produces opposite effects.Thus, we discover a new role of mDia2 in p53 regulation suggesting that the closed conformation is biologically active and an FBXO3-based mechanism to functionally specify mDia2's activity.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Division of Molecular Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;

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mDia2 and FMN1 cooperate in the regulation of p53.A, Generation of control, mDia2, FMN1 and mDia2-FMN1 knockdown cells. 293T cells were infected with either control (Control KD) or mDia2-targeting (mDia2 KD) viruses. These cells were further infected with either control (shCtr) or FMN1-targeting (shFMN1) viruses. Total cell lysates were immunoblotted as indicated. One of two similar experiments is shown. B, Formin-expression landscape in control (Control KD) and mDia2 knockdown (mDia2 KD) cells. RT-qPCR analyses were performed using the indicated Formin-specific primers to determine relative expression levels (Relative mRNA, arbitrary units (a. u.)) (n = 6; Two-way ANOVA). Cut-off red line is based on the fact that mDia1 silencing in mDia2 KD cells did not affect p53 activity (not shown). C, FMN1 silencing in mDia2 KD cells restores normal FMN1 expression. Cells were generated and total RNA obtained as in A and Fig. 4C, respectively. RT-qPCR shows the relative FMN1 levels (Relative FMN1 mRNA, arbitrary units (a. u.)) (n = 6; One-way ANOVA). FMN1 down-regulation in the Control KD cells is not evident because of its very low basal expression. D, mDia2 and FMN1 cooperate in regulating p53 activity. Cells were plated, transfected and the luciferase activity measured and plotted as in B (n = 6; One-way ANOVA). Similar results were obtained with a second FMN1-specific hairpin (not shown). E, Endogenous FBXO3 and p53 coprecipitate with EGFP-FMN1. 293T cells were transfected with either EGFP-tagged FMN1 (+) or the corresponding empty vector (−). Immunoprecipitation with GFP-trap beads was performed as described in the Experimental Procedures. Lysate (2%) and immunocomplexes (IP) were separated by SDS-PAGE and immunoblotted as indicated.
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Figure 5: mDia2 and FMN1 cooperate in the regulation of p53.A, Generation of control, mDia2, FMN1 and mDia2-FMN1 knockdown cells. 293T cells were infected with either control (Control KD) or mDia2-targeting (mDia2 KD) viruses. These cells were further infected with either control (shCtr) or FMN1-targeting (shFMN1) viruses. Total cell lysates were immunoblotted as indicated. One of two similar experiments is shown. B, Formin-expression landscape in control (Control KD) and mDia2 knockdown (mDia2 KD) cells. RT-qPCR analyses were performed using the indicated Formin-specific primers to determine relative expression levels (Relative mRNA, arbitrary units (a. u.)) (n = 6; Two-way ANOVA). Cut-off red line is based on the fact that mDia1 silencing in mDia2 KD cells did not affect p53 activity (not shown). C, FMN1 silencing in mDia2 KD cells restores normal FMN1 expression. Cells were generated and total RNA obtained as in A and Fig. 4C, respectively. RT-qPCR shows the relative FMN1 levels (Relative FMN1 mRNA, arbitrary units (a. u.)) (n = 6; One-way ANOVA). FMN1 down-regulation in the Control KD cells is not evident because of its very low basal expression. D, mDia2 and FMN1 cooperate in regulating p53 activity. Cells were plated, transfected and the luciferase activity measured and plotted as in B (n = 6; One-way ANOVA). Similar results were obtained with a second FMN1-specific hairpin (not shown). E, Endogenous FBXO3 and p53 coprecipitate with EGFP-FMN1. 293T cells were transfected with either EGFP-tagged FMN1 (+) or the corresponding empty vector (−). Immunoprecipitation with GFP-trap beads was performed as described in the Experimental Procedures. Lysate (2%) and immunocomplexes (IP) were separated by SDS-PAGE and immunoblotted as indicated.

Mentions: In order to measure the activity of p53 in cells devoid of mDia2, we took advantage of shRNA and lentiviral infection because all tested siRNAs resulted in partial (less than 50%) and very transient down-regulation of mDia2 (supplemental Fig. S3C and unpublished observations). We obtained mDia2 knockdown cells having mDia2 protein levels reduced by about 75% compared with the control knockdown population (Fig. 5A). Silencing of mDia2 did not alter either the expression of FBXO3, HIPK2, p300, and p53 (Fig. 5A). The activity of p53 in 293T cells did not change upon mDia2 knockdown (not shown and Fig. 5D), thus suggesting that other Formins might cooperate with mDia2 in regulating p53.


Proteomic analyses uncover a new function and mode of action for mouse homolog of Diaphanous 2 (mDia2).

Isogai T, van der Kammen R, Goerdayal SS, Heck AJ, Altelaar AF, Innocenti M - Mol. Cell Proteomics (2015)

mDia2 and FMN1 cooperate in the regulation of p53.A, Generation of control, mDia2, FMN1 and mDia2-FMN1 knockdown cells. 293T cells were infected with either control (Control KD) or mDia2-targeting (mDia2 KD) viruses. These cells were further infected with either control (shCtr) or FMN1-targeting (shFMN1) viruses. Total cell lysates were immunoblotted as indicated. One of two similar experiments is shown. B, Formin-expression landscape in control (Control KD) and mDia2 knockdown (mDia2 KD) cells. RT-qPCR analyses were performed using the indicated Formin-specific primers to determine relative expression levels (Relative mRNA, arbitrary units (a. u.)) (n = 6; Two-way ANOVA). Cut-off red line is based on the fact that mDia1 silencing in mDia2 KD cells did not affect p53 activity (not shown). C, FMN1 silencing in mDia2 KD cells restores normal FMN1 expression. Cells were generated and total RNA obtained as in A and Fig. 4C, respectively. RT-qPCR shows the relative FMN1 levels (Relative FMN1 mRNA, arbitrary units (a. u.)) (n = 6; One-way ANOVA). FMN1 down-regulation in the Control KD cells is not evident because of its very low basal expression. D, mDia2 and FMN1 cooperate in regulating p53 activity. Cells were plated, transfected and the luciferase activity measured and plotted as in B (n = 6; One-way ANOVA). Similar results were obtained with a second FMN1-specific hairpin (not shown). E, Endogenous FBXO3 and p53 coprecipitate with EGFP-FMN1. 293T cells were transfected with either EGFP-tagged FMN1 (+) or the corresponding empty vector (−). Immunoprecipitation with GFP-trap beads was performed as described in the Experimental Procedures. Lysate (2%) and immunocomplexes (IP) were separated by SDS-PAGE and immunoblotted as indicated.
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Related In: Results  -  Collection

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Figure 5: mDia2 and FMN1 cooperate in the regulation of p53.A, Generation of control, mDia2, FMN1 and mDia2-FMN1 knockdown cells. 293T cells were infected with either control (Control KD) or mDia2-targeting (mDia2 KD) viruses. These cells were further infected with either control (shCtr) or FMN1-targeting (shFMN1) viruses. Total cell lysates were immunoblotted as indicated. One of two similar experiments is shown. B, Formin-expression landscape in control (Control KD) and mDia2 knockdown (mDia2 KD) cells. RT-qPCR analyses were performed using the indicated Formin-specific primers to determine relative expression levels (Relative mRNA, arbitrary units (a. u.)) (n = 6; Two-way ANOVA). Cut-off red line is based on the fact that mDia1 silencing in mDia2 KD cells did not affect p53 activity (not shown). C, FMN1 silencing in mDia2 KD cells restores normal FMN1 expression. Cells were generated and total RNA obtained as in A and Fig. 4C, respectively. RT-qPCR shows the relative FMN1 levels (Relative FMN1 mRNA, arbitrary units (a. u.)) (n = 6; One-way ANOVA). FMN1 down-regulation in the Control KD cells is not evident because of its very low basal expression. D, mDia2 and FMN1 cooperate in regulating p53 activity. Cells were plated, transfected and the luciferase activity measured and plotted as in B (n = 6; One-way ANOVA). Similar results were obtained with a second FMN1-specific hairpin (not shown). E, Endogenous FBXO3 and p53 coprecipitate with EGFP-FMN1. 293T cells were transfected with either EGFP-tagged FMN1 (+) or the corresponding empty vector (−). Immunoprecipitation with GFP-trap beads was performed as described in the Experimental Procedures. Lysate (2%) and immunocomplexes (IP) were separated by SDS-PAGE and immunoblotted as indicated.
Mentions: In order to measure the activity of p53 in cells devoid of mDia2, we took advantage of shRNA and lentiviral infection because all tested siRNAs resulted in partial (less than 50%) and very transient down-regulation of mDia2 (supplemental Fig. S3C and unpublished observations). We obtained mDia2 knockdown cells having mDia2 protein levels reduced by about 75% compared with the control knockdown population (Fig. 5A). Silencing of mDia2 did not alter either the expression of FBXO3, HIPK2, p300, and p53 (Fig. 5A). The activity of p53 in 293T cells did not change upon mDia2 knockdown (not shown and Fig. 5D), thus suggesting that other Formins might cooperate with mDia2 in regulating p53.

Bottom Line: Taking FBXO3 as a test case, we show that mDia2 binds FBXO3 and p53, and regulates p53 transcriptional activity in an actin-nucleation-independent and conformation-insensitive manner.Increased mDia2 and FBXO3 levels elevate p53 activity and expression thereby sensitizing cells to p53-dependent apoptosis, whereas their decrease produces opposite effects.Thus, we discover a new role of mDia2 in p53 regulation suggesting that the closed conformation is biologically active and an FBXO3-based mechanism to functionally specify mDia2's activity.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Division of Molecular Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;

Show MeSH
Related in: MedlinePlus