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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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FBXO3 is required for mDia2 to stimulate p53-mediated gene transcription in a conformation- and actin-nucleation-independent manner.A, Wild-type mDia2 increases the transcriptional activity of p53 more efficiently than its MA mutant. 293T cells were cotransfected with the reporter plasmid and the empty vector (−), Flag-tagged mDia2 WT (WT) or mDia2 MA (MA). Luciferase activity (arbitrary units, a.u.) was measured and plotted as described in the Experimental Procedures. Data represent mean ± S.D. (n = 12; One-way ANOVA). mDia2 expression was confirmed using anti-Flag antibodies and actin served as a loading control. B, Actin-nucleation-deficient mDia2 activates p53 as efficiently as wild-type mDia2. Cells were transfected and luciferase activity measured and plotted as in A (n = 9; One-way ANOVA). I704 was replaced by A in both wild-type and MA mDia2 to generate IA and IAMA, respectively. C, Generation of control and FBXO3 knockdown cells. 293T cells were infected with either control (shCtr) or FBXO3-targeting (shFBXO3) viruses. Total RNA and lysates were obtained as indicated in the Experimental Procedures. Bar graph: RT-qPCR shows the relative FBXO3 levels (Relative FBXO3 mRNA, arbitrary units (a. u.)) (n = 3; Repeated t Test). Blots: Total cell lysates (30 μg) were immunoblotted as indicated with actin providing a loading control. FBXO3 is reduced by 51.2 ± 3% (shFBXO3 #1) after normalization against actin. One of three experiments that were performed with similar results is shown. D, mDia2-mediated activation of p53 requires FBXO3. Control (shCtr) and FBXO3 (shFBXO3) knockdown cells were cotransfected with the reporter plasmid along with the empty vector (−) or Flag-tagged wild-type mDia2 (+) and luciferase activity measured and plotted as in A (n = 9; One-way ANOVA). E, mDia2 and FBXO3 jointly enhance p53 expression and activity. U2OS cells were cotransfected with the reporter plasmid, Flag-tagged wild-type mDia2 (+) or the empty vector (−), and either HA-tagged FBXO3 (+) or its corresponding empty vector (−). Luciferase activity was measured and plotted as in A (n = 15; One-way ANOVA). p53, mDia2 and FBXO3 expression was confirmed using anti-p53, anti-Flag and anti-HA antibodies, respectively.
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Figure 4: FBXO3 is required for mDia2 to stimulate p53-mediated gene transcription in a conformation- and actin-nucleation-independent manner.A, Wild-type mDia2 increases the transcriptional activity of p53 more efficiently than its MA mutant. 293T cells were cotransfected with the reporter plasmid and the empty vector (−), Flag-tagged mDia2 WT (WT) or mDia2 MA (MA). Luciferase activity (arbitrary units, a.u.) was measured and plotted as described in the Experimental Procedures. Data represent mean ± S.D. (n = 12; One-way ANOVA). mDia2 expression was confirmed using anti-Flag antibodies and actin served as a loading control. B, Actin-nucleation-deficient mDia2 activates p53 as efficiently as wild-type mDia2. Cells were transfected and luciferase activity measured and plotted as in A (n = 9; One-way ANOVA). I704 was replaced by A in both wild-type and MA mDia2 to generate IA and IAMA, respectively. C, Generation of control and FBXO3 knockdown cells. 293T cells were infected with either control (shCtr) or FBXO3-targeting (shFBXO3) viruses. Total RNA and lysates were obtained as indicated in the Experimental Procedures. Bar graph: RT-qPCR shows the relative FBXO3 levels (Relative FBXO3 mRNA, arbitrary units (a. u.)) (n = 3; Repeated t Test). Blots: Total cell lysates (30 μg) were immunoblotted as indicated with actin providing a loading control. FBXO3 is reduced by 51.2 ± 3% (shFBXO3 #1) after normalization against actin. One of three experiments that were performed with similar results is shown. D, mDia2-mediated activation of p53 requires FBXO3. Control (shCtr) and FBXO3 (shFBXO3) knockdown cells were cotransfected with the reporter plasmid along with the empty vector (−) or Flag-tagged wild-type mDia2 (+) and luciferase activity measured and plotted as in A (n = 9; One-way ANOVA). E, mDia2 and FBXO3 jointly enhance p53 expression and activity. U2OS cells were cotransfected with the reporter plasmid, Flag-tagged wild-type mDia2 (+) or the empty vector (−), and either HA-tagged FBXO3 (+) or its corresponding empty vector (−). Luciferase activity was measured and plotted as in A (n = 15; One-way ANOVA). p53, mDia2 and FBXO3 expression was confirmed using anti-p53, anti-Flag and anti-HA antibodies, respectively.

Mentions: The interaction of mDia2 with FBXO3 and p53 prompted us to assess the effects of mDia2 on p53-dependent gene transcription. Wild-type and constitutively active mDia2, or an empty vector as a control, were transfected in 293T cells along with a plasmid having a minimal p53-responsive promoter located upstream of the luciferase gene (39). Strikingly, wild-type mDia2 did not only enhance the transcriptional activity of p53 but it also displayed a more prominent stimulatory action than the MA mutant (Fig. 4A). A point mutation abrogating actin nucleation (I704-to-A, hereafter referred to as IA) (3) made constitutively active mDia2 indistinguishable from the wild type (IAMA versus WT, Fig. 4B). In keeping with wild-type mDia2 attaining the closed conformation, the IA mutant activated p53 as efficiently as the wild type (IA versus WT, Fig. 4B). The notion that mDia2 plays a scaffolding role is further corroborated by the C-terminal region being required and sufficient for mDia2 to increase the transcriptional activity of p53 (supplemental Fig. S3A). The C-terminal region of mDia2 activated p53 also upon introduction of the MA mutation (supplemental Fig. S3A), thus ruling out that binding to endogenous mDia2 could account for these observations. Additionally, the inability of wild-type mDia1 to affect p53 activity (supplemental Fig. S3B) ruled out that the effects of mDia2 are caused by the sequestration of Rho GTPases or general Drf-binding proteins.


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)

FBXO3 is required for mDia2 to stimulate p53-mediated gene transcription in a conformation- and actin-nucleation-independent manner.A, Wild-type mDia2 increases the transcriptional activity of p53 more efficiently than its MA mutant. 293T cells were cotransfected with the reporter plasmid and the empty vector (−), Flag-tagged mDia2 WT (WT) or mDia2 MA (MA). Luciferase activity (arbitrary units, a.u.) was measured and plotted as described in the Experimental Procedures. Data represent mean ± S.D. (n = 12; One-way ANOVA). mDia2 expression was confirmed using anti-Flag antibodies and actin served as a loading control. B, Actin-nucleation-deficient mDia2 activates p53 as efficiently as wild-type mDia2. Cells were transfected and luciferase activity measured and plotted as in A (n = 9; One-way ANOVA). I704 was replaced by A in both wild-type and MA mDia2 to generate IA and IAMA, respectively. C, Generation of control and FBXO3 knockdown cells. 293T cells were infected with either control (shCtr) or FBXO3-targeting (shFBXO3) viruses. Total RNA and lysates were obtained as indicated in the Experimental Procedures. Bar graph: RT-qPCR shows the relative FBXO3 levels (Relative FBXO3 mRNA, arbitrary units (a. u.)) (n = 3; Repeated t Test). Blots: Total cell lysates (30 μg) were immunoblotted as indicated with actin providing a loading control. FBXO3 is reduced by 51.2 ± 3% (shFBXO3 #1) after normalization against actin. One of three experiments that were performed with similar results is shown. D, mDia2-mediated activation of p53 requires FBXO3. Control (shCtr) and FBXO3 (shFBXO3) knockdown cells were cotransfected with the reporter plasmid along with the empty vector (−) or Flag-tagged wild-type mDia2 (+) and luciferase activity measured and plotted as in A (n = 9; One-way ANOVA). E, mDia2 and FBXO3 jointly enhance p53 expression and activity. U2OS cells were cotransfected with the reporter plasmid, Flag-tagged wild-type mDia2 (+) or the empty vector (−), and either HA-tagged FBXO3 (+) or its corresponding empty vector (−). Luciferase activity was measured and plotted as in A (n = 15; One-way ANOVA). p53, mDia2 and FBXO3 expression was confirmed using anti-p53, anti-Flag and anti-HA antibodies, respectively.
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Figure 4: FBXO3 is required for mDia2 to stimulate p53-mediated gene transcription in a conformation- and actin-nucleation-independent manner.A, Wild-type mDia2 increases the transcriptional activity of p53 more efficiently than its MA mutant. 293T cells were cotransfected with the reporter plasmid and the empty vector (−), Flag-tagged mDia2 WT (WT) or mDia2 MA (MA). Luciferase activity (arbitrary units, a.u.) was measured and plotted as described in the Experimental Procedures. Data represent mean ± S.D. (n = 12; One-way ANOVA). mDia2 expression was confirmed using anti-Flag antibodies and actin served as a loading control. B, Actin-nucleation-deficient mDia2 activates p53 as efficiently as wild-type mDia2. Cells were transfected and luciferase activity measured and plotted as in A (n = 9; One-way ANOVA). I704 was replaced by A in both wild-type and MA mDia2 to generate IA and IAMA, respectively. C, Generation of control and FBXO3 knockdown cells. 293T cells were infected with either control (shCtr) or FBXO3-targeting (shFBXO3) viruses. Total RNA and lysates were obtained as indicated in the Experimental Procedures. Bar graph: RT-qPCR shows the relative FBXO3 levels (Relative FBXO3 mRNA, arbitrary units (a. u.)) (n = 3; Repeated t Test). Blots: Total cell lysates (30 μg) were immunoblotted as indicated with actin providing a loading control. FBXO3 is reduced by 51.2 ± 3% (shFBXO3 #1) after normalization against actin. One of three experiments that were performed with similar results is shown. D, mDia2-mediated activation of p53 requires FBXO3. Control (shCtr) and FBXO3 (shFBXO3) knockdown cells were cotransfected with the reporter plasmid along with the empty vector (−) or Flag-tagged wild-type mDia2 (+) and luciferase activity measured and plotted as in A (n = 9; One-way ANOVA). E, mDia2 and FBXO3 jointly enhance p53 expression and activity. U2OS cells were cotransfected with the reporter plasmid, Flag-tagged wild-type mDia2 (+) or the empty vector (−), and either HA-tagged FBXO3 (+) or its corresponding empty vector (−). Luciferase activity was measured and plotted as in A (n = 15; One-way ANOVA). p53, mDia2 and FBXO3 expression was confirmed using anti-p53, anti-Flag and anti-HA antibodies, respectively.
Mentions: The interaction of mDia2 with FBXO3 and p53 prompted us to assess the effects of mDia2 on p53-dependent gene transcription. Wild-type and constitutively active mDia2, or an empty vector as a control, were transfected in 293T cells along with a plasmid having a minimal p53-responsive promoter located upstream of the luciferase gene (39). Strikingly, wild-type mDia2 did not only enhance the transcriptional activity of p53 but it also displayed a more prominent stimulatory action than the MA mutant (Fig. 4A). A point mutation abrogating actin nucleation (I704-to-A, hereafter referred to as IA) (3) made constitutively active mDia2 indistinguishable from the wild type (IAMA versus WT, Fig. 4B). In keeping with wild-type mDia2 attaining the closed conformation, the IA mutant activated p53 as efficiently as the wild type (IA versus WT, Fig. 4B). The notion that mDia2 plays a scaffolding role is further corroborated by the C-terminal region being required and sufficient for mDia2 to increase the transcriptional activity of p53 (supplemental Fig. S3A). The C-terminal region of mDia2 activated p53 also upon introduction of the MA mutation (supplemental Fig. S3A), thus ruling out that binding to endogenous mDia2 could account for these observations. Additionally, the inability of wild-type mDia1 to affect p53 activity (supplemental Fig. S3B) ruled out that the effects of mDia2 are caused by the sequestration of Rho GTPases or general Drf-binding proteins.

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