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: Immobilized full-length GST-mDia2 and deletion mutant thereof (7), or GST as a negative control, served as baits to fish out an epitope-tagged version of the candidate under scrutiny in all pull-down experiments (Fig. 2A). CHMP5 and VTA-1 are subunits of the ESCRT-III complex, which regulates membrane budding, endosomal sorting, and multivesicular body biogenesis, receptor signaling, cytokinesis, autophagy, cell polarity and migration, and ribonucleic acid biology (30). CHMP5 and VTA-1 bound to mDia2, the former less efficiently than the latter (Fig. 2B). The ESCRT-III complex may contribute to the role of mDia2 in both cell motility and cytokinesis. Interestingly, microtubule plus-end-directed kinesin KIF20B also acts during cytokinesis (31) and turned out to be a genuine partner of mDia2. Tropomyosin-2, Tropomyosin-4, and Myosin-Vc are three actin-regulatory proteins belonging to the cell assembly and organization functional group unique for constitutively active mDia2. The interplay between mDia2 and Tropomyosins in stress fiber formation (32) raised our interest in testing Myosin-Vc, a nonconventional myosin involved in exocytosis (33). The interaction between Myosin-Vc (MYO5C) and mDia2 (Fig. 2B) suggest that they may cooperate in regulating actin dynamics in vesicle trafficking. The cyclin-dependent kinase CDK2 controlling cell cycle progression and centrosome duplication (34) specifically bound to full-length mDia2 (Fig. 2B) and may link the microtubule-stabilizing action of mDia2 to a microtubule-dependent process. The SKP1-Cullin 1-F-box-complex subunits SKP1 and FBXO3 (35) interacted with mDia2, providing a first-line validation for the link between mDia2 and the UPS. Specificity of the above interactions was further corroborated by the fact that none of the employed baits pulled down TRDMT1, a control nonrelated protein (Fig. 2B). The seven hits that we confirmed to be genuine mDia2-binding proteins interact with either full-length mDia2 only or both the full length and specific mDia2-deletion mutants, thereby suggesting the existence of multiple binding surfaces (Fig. 2B).
Affiliation: From the ‡Division of Molecular Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;