Proteomic analyses uncover a new function and mode of action for mouse homolog of Diaphanous 2 (mDia2).
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.
Affiliation: From the ‡Division of Molecular Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;Show MeSH
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Mentions: To further evaluate the role of mDia2 and FBXO3 in apoptotic cell death, U2OS were treated with etoposide, a topoisomerare II inhibitor that arrests cells in G2/M and induces apoptosis (42, 43). Time-course experiments indicated that addition of etoposide rapidly increased p53 expression before reducing FBXO3 expression (Fig. 7A). The correlation between down-regulation of mDia2 and FBXO3 and DNA fragmentation at both 24 and 48 h (not shown and Fig. 7B–7D, respectively) suggests that these proteins are no longer required when late-stage, irreversible apoptotic events have taken place. To assess the centrality of FBXO3 in this new pro-apoptotic pathway, we generated control, mDia2 knockdown and FBXO3 knockdown U2OS cells. As expected, etoposide caused control knockdown cells to accumulate in G2/M and increased the sub-G1 population (Fig. 7B and 7E, respectively). mDia2 knockdown cells responded as the control ones (Fig. 7C, 7E, and 7F), further suggesting that compensation by FMN proteins is at play (supplemental Fig. S4C). FBXO3 silencing resulted in a G2/M arrest, apoptosis and increased p21 expression without altering p53 levels (Fig. 7D–7F). However, etoposide did not further increase either apoptosis or p21 levels in FBXO3 knockdown cells (Fig. 7D–7F). These observations confirm that FBXO3 has a crucial role in the Formin-dependent pathway controlling the p53 apoptotic program and suggest that FBXO3 might affect apoptosis also independently of p53.
Affiliation: From the ‡Division of Molecular Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;