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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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Increased mDia2 and FBXO3 levels sensitize cells to p53-dependent apoptosis.A, Increased mDia2 and FBXO3 levels sensitize cells to apoptosis. U2OS cells were transfected with Flag-tagged wild-type mDia2 (mDia2) and HA-tagged FBXO3 (FBXO3), either alone or in combination, a control empty vector (EV), or wild-type Flag-tagged p53 (p53). Apoptotic index (a. u. = arbitrary units) versus time (h = hours) was obtained by measuring Caspase-3/7 activity as described in the supplemental Experimental Procedures. Data represent mean ± S.D. (n = 7; Two-way ANOVA), color-coded asterisks indicate statistical significance between the cells co-expressing mDia2 and FBXO3 and those expressing either mDia2 (red) or FBXO3 alone (green). All but p53-overexpressing cells exhibit a significantly increased apoptotic index compared with EV. B, Increased mDia2 and FBXO3 levels upregulate the expression of Bax. U2OS were transfected as in A and, 48 h later, total RNA was isolated. RT-qPCR analyses were performed using the indicated gene-specific primers. In each set, the relative mRNA levels of the EV served as a reference (n = 6; One-way ANOVA). C, Generation of control and p53 knockdown cells. U2OS cells were infected with either control (shCtr) or p53-targeting (shp53) lentiviruses to obtain stable populations. Total cell lysates were immunoblotted as indicated. Black lines mark removal of intervening lanes. One of two similar experiments is shown. D, mDia2 and FBXO3-induced apoptosis involves p53. Control and p53-knockdown U2OS cells were transfected with empty vector (−) or Flag-tagged wild-type mDia2 (mDia2) and HA-tagged FBXO3 (FBXO3) (+). The Apoptotic index was measured at the same time points corresponding to the peak in A. Data represent mean ± S.E.M. (n = 6; One-way ANOVA). The expression of mDia2 and FBXO3 was confirmed using anti-mDia2 and anti-HA antibodies, respectively. p53 levels were assessed with both short and long exposures (short exp. and long exp., respectively) and actin served as loading control.
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Figure 6: Increased mDia2 and FBXO3 levels sensitize cells to p53-dependent apoptosis.A, Increased mDia2 and FBXO3 levels sensitize cells to apoptosis. U2OS cells were transfected with Flag-tagged wild-type mDia2 (mDia2) and HA-tagged FBXO3 (FBXO3), either alone or in combination, a control empty vector (EV), or wild-type Flag-tagged p53 (p53). Apoptotic index (a. u. = arbitrary units) versus time (h = hours) was obtained by measuring Caspase-3/7 activity as described in the supplemental Experimental Procedures. Data represent mean ± S.D. (n = 7; Two-way ANOVA), color-coded asterisks indicate statistical significance between the cells co-expressing mDia2 and FBXO3 and those expressing either mDia2 (red) or FBXO3 alone (green). All but p53-overexpressing cells exhibit a significantly increased apoptotic index compared with EV. B, Increased mDia2 and FBXO3 levels upregulate the expression of Bax. U2OS were transfected as in A and, 48 h later, total RNA was isolated. RT-qPCR analyses were performed using the indicated gene-specific primers. In each set, the relative mRNA levels of the EV served as a reference (n = 6; One-way ANOVA). C, Generation of control and p53 knockdown cells. U2OS cells were infected with either control (shCtr) or p53-targeting (shp53) lentiviruses to obtain stable populations. Total cell lysates were immunoblotted as indicated. Black lines mark removal of intervening lanes. One of two similar experiments is shown. D, mDia2 and FBXO3-induced apoptosis involves p53. Control and p53-knockdown U2OS cells were transfected with empty vector (−) or Flag-tagged wild-type mDia2 (mDia2) and HA-tagged FBXO3 (FBXO3) (+). The Apoptotic index was measured at the same time points corresponding to the peak in A. Data represent mean ± S.E.M. (n = 6; One-way ANOVA). The expression of mDia2 and FBXO3 was confirmed using anti-mDia2 and anti-HA antibodies, respectively. p53 levels were assessed with both short and long exposures (short exp. and long exp., respectively) and actin served as loading control.

Mentions: p53 is a tumor suppressor with a crucial function in the cellular apoptotic programs (41) and the interactome of wild-type mDia2 associates with the cell death functional group (Fig. 1B). Therefore, we monitored apoptosis in U2OS cells transfected with mDia2 and FBXO3, either alone or in combination, and compared it to the control ones. These experiments revealed that the activity of Caspase-3/7 was significantly increased in the cells expressing either mDia2 or FBXO3 compared with the control ones (Fig. 6A). Most importantly, the highest apoptotic index was measured in cells co-expressing mDia2 and FBXO3 (Fig. 6A). As elevated p53 levels were insufficient to trigger apoptosis in U2OS cells (Fig. 6A), there appears that mDia2 and FBXO3 jointly promote p53 activation. Consistent with this notion, (1) mDia2 and FBXO3 were present in the nuclear compartment both in control and etoposide-treated cells (supplemental Fig. S5), (2) knockdown of FBXO3, FMN1, and FMN1-mDia2 resulted in decreased p53 levels (Fig. 4D and Fig. 5A), whereas (3) co-expression of mDia2 and FBXO3 increased them (Fig. 4E).


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)

Increased mDia2 and FBXO3 levels sensitize cells to p53-dependent apoptosis.A, Increased mDia2 and FBXO3 levels sensitize cells to apoptosis. U2OS cells were transfected with Flag-tagged wild-type mDia2 (mDia2) and HA-tagged FBXO3 (FBXO3), either alone or in combination, a control empty vector (EV), or wild-type Flag-tagged p53 (p53). Apoptotic index (a. u. = arbitrary units) versus time (h = hours) was obtained by measuring Caspase-3/7 activity as described in the supplemental Experimental Procedures. Data represent mean ± S.D. (n = 7; Two-way ANOVA), color-coded asterisks indicate statistical significance between the cells co-expressing mDia2 and FBXO3 and those expressing either mDia2 (red) or FBXO3 alone (green). All but p53-overexpressing cells exhibit a significantly increased apoptotic index compared with EV. B, Increased mDia2 and FBXO3 levels upregulate the expression of Bax. U2OS were transfected as in A and, 48 h later, total RNA was isolated. RT-qPCR analyses were performed using the indicated gene-specific primers. In each set, the relative mRNA levels of the EV served as a reference (n = 6; One-way ANOVA). C, Generation of control and p53 knockdown cells. U2OS cells were infected with either control (shCtr) or p53-targeting (shp53) lentiviruses to obtain stable populations. Total cell lysates were immunoblotted as indicated. Black lines mark removal of intervening lanes. One of two similar experiments is shown. D, mDia2 and FBXO3-induced apoptosis involves p53. Control and p53-knockdown U2OS cells were transfected with empty vector (−) or Flag-tagged wild-type mDia2 (mDia2) and HA-tagged FBXO3 (FBXO3) (+). The Apoptotic index was measured at the same time points corresponding to the peak in A. Data represent mean ± S.E.M. (n = 6; One-way ANOVA). The expression of mDia2 and FBXO3 was confirmed using anti-mDia2 and anti-HA antibodies, respectively. p53 levels were assessed with both short and long exposures (short exp. and long exp., respectively) and actin served as loading control.
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Figure 6: Increased mDia2 and FBXO3 levels sensitize cells to p53-dependent apoptosis.A, Increased mDia2 and FBXO3 levels sensitize cells to apoptosis. U2OS cells were transfected with Flag-tagged wild-type mDia2 (mDia2) and HA-tagged FBXO3 (FBXO3), either alone or in combination, a control empty vector (EV), or wild-type Flag-tagged p53 (p53). Apoptotic index (a. u. = arbitrary units) versus time (h = hours) was obtained by measuring Caspase-3/7 activity as described in the supplemental Experimental Procedures. Data represent mean ± S.D. (n = 7; Two-way ANOVA), color-coded asterisks indicate statistical significance between the cells co-expressing mDia2 and FBXO3 and those expressing either mDia2 (red) or FBXO3 alone (green). All but p53-overexpressing cells exhibit a significantly increased apoptotic index compared with EV. B, Increased mDia2 and FBXO3 levels upregulate the expression of Bax. U2OS were transfected as in A and, 48 h later, total RNA was isolated. RT-qPCR analyses were performed using the indicated gene-specific primers. In each set, the relative mRNA levels of the EV served as a reference (n = 6; One-way ANOVA). C, Generation of control and p53 knockdown cells. U2OS cells were infected with either control (shCtr) or p53-targeting (shp53) lentiviruses to obtain stable populations. Total cell lysates were immunoblotted as indicated. Black lines mark removal of intervening lanes. One of two similar experiments is shown. D, mDia2 and FBXO3-induced apoptosis involves p53. Control and p53-knockdown U2OS cells were transfected with empty vector (−) or Flag-tagged wild-type mDia2 (mDia2) and HA-tagged FBXO3 (FBXO3) (+). The Apoptotic index was measured at the same time points corresponding to the peak in A. Data represent mean ± S.E.M. (n = 6; One-way ANOVA). The expression of mDia2 and FBXO3 was confirmed using anti-mDia2 and anti-HA antibodies, respectively. p53 levels were assessed with both short and long exposures (short exp. and long exp., respectively) and actin served as loading control.
Mentions: p53 is a tumor suppressor with a crucial function in the cellular apoptotic programs (41) and the interactome of wild-type mDia2 associates with the cell death functional group (Fig. 1B). Therefore, we monitored apoptosis in U2OS cells transfected with mDia2 and FBXO3, either alone or in combination, and compared it to the control ones. These experiments revealed that the activity of Caspase-3/7 was significantly increased in the cells expressing either mDia2 or FBXO3 compared with the control ones (Fig. 6A). Most importantly, the highest apoptotic index was measured in cells co-expressing mDia2 and FBXO3 (Fig. 6A). As elevated p53 levels were insufficient to trigger apoptosis in U2OS cells (Fig. 6A), there appears that mDia2 and FBXO3 jointly promote p53 activation. Consistent with this notion, (1) mDia2 and FBXO3 were present in the nuclear compartment both in control and etoposide-treated cells (supplemental Fig. S5), (2) knockdown of FBXO3, FMN1, and FMN1-mDia2 resulted in decreased p53 levels (Fig. 4D and Fig. 5A), whereas (3) co-expression of mDia2 and FBXO3 increased them (Fig. 4E).

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