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 gain insight into the protein interactions engaged by mDia2, we devised a strategy relying on affinity purification and mass spectrometry (AP-MS). 293T cells were transfected with either wild-type mDia2 or its constitutively active mutant, or with an empty vector as a control. mDia2-based complexes were affinity-purified by using anti-Flag antibodies, separated by one-dimensional SDS-PAGE, and then processed for MS (supplemental Fig. S1D). We analyzed three biological replicas and produced three independent datasets whose label-free quantitative information was exploited by SAINT to assign confidence values to individual protein–protein interactions (18). We ranked the interactions according to the computed confidence values and inventoried 46 and 67 proteins having an average probability higher than 0.24 for wild-type and constitutively active mDia2, respectively (supplemental Table S1). Among them were importin α (IMA8) and Profilin-2, two known mDia2-binding proteins (1, 2, 22). Interaction maps built using the STRING protein–protein interaction database (23) and Cytoscape (24) illustrated the connectivity of both interactomes (Fig. 1A and 1B). We obtained 75 nonredundant mDia2-binding proteins: 38 common to both wild-type and constitutively active mDia2, eight and 29 unique for the former and the latter, respectively (Fig. 1C). Many proteins could be clustered into the cell cycle, the cell growth and proliferation, the cell assembly and organization, and the cell development functional groups accounting for the known biological roles of mDia2 (Fig. 1D). Furthermore, the presence of the cell assembly and organization functional group exclusively in the interactome of the MA confirmed that the open conformation is associated with actin- and microtubule-based processes. Bioinformatics conveyed also new information: (1) the post-translational modification functional group unveiled the potential involvement of mDia2 in Ubiquitin biology, and (2) the presence of the cell death functional group only in the interactome of the wild type suggested a conformation-specific role of mDia2 in apoptosis (Fig. 1D). Noticeable was the enrichment for proteins implicated in ubiquitin biology: E3 ubiquitin ligases (FBXO3-Cullin 1-containing (25), Zhg-11-Cullin 2-containing (26), and CACYBP/SIP-containing (27) cullin-RING ubiquitin ligases), the ubiquitin-like protein and cullin-regulator NEDD8 (28), the de-ubiquitinating enzyme USP9X, several proteasome subunits and proteasome-associating proteins (NipSnap2, p62/SQSTM1 (29)) could be isolated as mDia2-binding proteins (supplemental Table S1). Consistently, the protein ubiquitination pathway was both significantly enriched and highly represented in the mDia2 interactomes (Fig. 1E).
Affiliation: From the ‡Division of Molecular Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;