Proteomic analyses uncover a new function and mode of action for mouse homolog of Diaphanous 2 (mDia2).
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.
Affiliation: From the ‡Division of Molecular Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;
- Microtubule-Associated Proteins/metabolism*
- NADPH Dehydrogenase/metabolism*
- Sequence Homology, Amino Acid*
- Chromatography, Affinity
- DNA Damage
- F-Box Proteins/metabolism
- Fetal Proteins/metabolism
- Gene Knockdown Techniques
- HeLa Cells
- Mass Spectrometry
- Microfilament Proteins/metabolism
- Nuclear Proteins/metabolism
- Protein Binding
- Protein Conformation
- Protein Interaction Maps
- Reproducibility of Results
- Tumor Suppressor Protein p53/metabolism
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Figure 3: mDia2 forms a complex with FBXO3 and p53.A, mDia2 and p53 coprecipitate. 293T cells were transfected with Flag-tagged p53 (1.5 μg) and myc-tagged mDia2 (8.5 μg). Total cell lysates (1 mg) were immunoprecipitated with anti-myc, anti-Flag and control (ctr) antibodies as described in the Experimental Procedures. Lysate (2%) and immunocomplexes (IP) were separated by SDS-PAGE and immunoblotted as indicated. B, FBXO3 and p53 coprecipitate. 293T cells were transfected with Flag-tagged p53 (1.5 μg) and HA-tagged FBXO3 (8.5 μg). Immunoprecipitations with anti-HA, anti-Flag and control (ctr) antibodies was performed as described in the Experimental Procedures. FBXO3 and p53 were detected as indicated. The lower bands in the IP lanes of the anti-p53 blot represent cross-reaction with the IgGs. C, Endogenous p53, FBXO3 and mDia2 form a complex. Total cell lysates (2 mg) were immunoprecipitated with anti-p53 and control (ctr) antibodies. Lysate (2%) and immunocomplexes (IP) were separated by SDS-PAGE and immunoblotted as indicated. D, Endogenous FBXO3 and p53 coprecipitate with myc-mDia2. 293T cells were transfected with either myc-tagged mDia2 (+) or the corresponding empty vector (−). Immunoprecipitation with anti-myc antibodies was performed as in A. Lysate (2%) and immunocomplexes (IP) were separated by SDS-PAGE and immunoblotted as indicated. Anti-myc and anti-mDia2 antibodies detected overexpressed and total mDia2, respectively. Selected short and long exposures of the anti-myc blot are illustrated (short exp. and long exp., respectively). In the long exposure, anti-myc cross-reacting bands are visible in the control IP lane. E, Endogenous mDia2 and p53 coprecipitate with HA-FBXO3. 293T cells were transfected with either HA-tagged FBXO3 (+) or the corresponding empty vector (−). Immunoprecipitation with anti-HA antibodies was performed as in B. Lysate (2%) and immunocomplexes (IP) were separated by SDS-PAGE and immunoblotted as indicated. Anti-HA and anti-FBXO3 antibodies detected overexpressed and total FBXO3, respectively. In the anti-mDia3 blot, some residual mDia2 signal is visible as a faint (and shifted) band in the IP lane. F, mDia2, FBXO3 and p53 form a complex. 293T cells were transfected with Flag-tagged p53 (1,5 μg) and HA-tagged FBXO3 (8,5 μg), with either myc-tagged mDia2 (10 μg) (+) or empty vector (−). Immunoprecipitations were performed as in A and B. mDia2, FBXO3 and p53 were detected as indicated. A–F: one of two experiments that were performed with similar results is shown.
The fact that FBXO3 regulates p53 activity through a mechanism that does not involve inhibition of p300 and HIPK2 ubiquitination (25) raised the possibility that mDia2 binds to FBXO3 and p53. As all available antibodies did not allow us to selectively immunoprecipitate either endogenous mDia2 or endogenous FBXO3, we tested this hypothesis as explained below. First, we demonstrated that myc-tagged mDia2 and HA-tagged FBXO3 coprecipitate with Flag-tagged p53 and vice versa, mDia2 less robustly than FBXO3 (Fig. 3A and 3B, respectively). In keeping with this, endogenous FBXO3 coprecipitated with endogenous p53 more efficiently than endogenous mDia2 and the absence of actin from the p53-based immunocomplexes demonstrated the specificity of these interactions (Fig. 3C). Second, we proved that endogenous FBXO3 and p53 bound to myc-tagged mDia2 (Fig. 3D). Third, we could also show that endogenous mDia2 and p53, but not mDia1 or mDia3, associated with mildly overexpressed HA-tagged FBXO3 (Fig. 3E). Fourth and to give insight into the topology of these interactions, we co-expressed Flag-tagged p53 and HA-tagged FBXO3 at levels comparable to and far below the endogenous counterparts, respectively, either alone or with heavily overexpressed myc-tagged mDia2 (supplemental Fig. S2D). Because the association between p53 and FBXO3 was not outcompeted by mDia2, which instead could be specifically detected in both anti-Flag and anti-HA immunocomplexes (Fig. 3F left and right, respectively), it is likely that FBXO3 holds mDia2 and p53 in a complex.