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