Quantitative Proteomics Illuminates A Functional Interaction Between Mouse Homolog Of Diaphanous 2 (mDia2) And The Proteasome
Formin mDia2 is a cytoskeleton-regulatory protein that switches reversibly between a closed, auto-inhibited and an open, active conformation. Although the open conformation of mDia2 induces actin assembly thereby controlling many cellular processes, mDia2 possesses also actin-independent and conformation-insensitive scaffolding roles related to microtubules and p53, respectively. Thus, we hypothesise that mDia2 may have other unappreciated functions and regulatory modes. Here we identify and validate proteasome and Ubiquitin as genuine mDia2-interacting partners using quantitative proteomics and a multi-disciplinary approach, respectively.
Sample Processing Protocol
After immunoprecipitation of SILAC-labeled wild-type and mutant mDia2 expressing cells, interactors of mDia2 were identified using MaxQuant and Perseus. Findings were further complemented by/corroborated with biochemical experiments. SILAC samples were run on Thermo.
Data Processing Protocol
For immunoprecipitation of WT and MA mDia2-transfected cells, SILAC light (no label) and heavy (Arg10/Lys8) labels were used. SDS-PAGE separation, follwed by in-gel digestion: trypsin gold (Promega) o/n.Data were run on an LTQ Orbitrap connected to an Agilent 1200 LC system, on a 20 mm 100 µm i.d. Reprosil C18 trap column and a 400 mm 50 µm i.d. Reprosil C18 analytical column, split-flow to 100nL/min. Data were processed in MaxQuant 220.127.116.11 and Perseus. M(ox) and SILAC H/L as variable modifications, carbamidomethylation (C) as fixed modification.
Isogai T, van der Kammen R, Bleijerveld OB, Goerdayal SS, Argenzio E, Altelaar AF, Innocenti M. Quantitative Proteomics Illuminates a Functional Interaction between mDia2 and the Proteasome. J Proteome Res. 2016 Dec 2;15(12):4624-4637 PubMed: 27769112