Quantitative Proteomics Illuminates a Functional Interaction between mDia2 and the Proteasome

Tadamoto Isogai, Rob Van Der Kammen, Onno B. Bleijerveld, Soenita S. Goerdayal, Elisabetta Argenzio, A. F Maarten Altelaar*, Metello Innocenti*

*Corresponding author for this work

    Research output: Contribution to journalArticleAcademicpeer-review

    Abstract

    Formin mDia2 is a cytoskeleton-regulatory protein that switches reversibly between a closed, autoinhibited 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 hypothesize that mDia2 may have other unappreciated functions and regulatory modes. Here we identify and validate proteasome and Ubiquitin as mDia2-interacting partners using stable isotope labeling with amino acids in cell culture-based quantitative proteomics and biochemistry, respectively. Although mDia2 is ubiquitinated, binds ubiquitinated proteins and free Ubiquitin, it is not a proteasome substrate. Surprisingly, knockdown of mDia2 increases the activity of the proteasome in vitro, whereas mDia2 overexpression has opposite effects only when it adopts the open conformation and cannot induce actin assembly. Consistently, a combination of candidate and unbiased proteome-wide analyses indicates that mDia2 regulates the cellular levels of proteasome substrate β-catenin and a number of ubiquitinated actin-regulatory proteins. Hence, these findings add more complexity to the mDia2 activity cycle by showing that the open conformation may control actin dynamics also through actin-independent regulation of the proteasome.

    Original languageEnglish
    Pages (from-to)4624-4637
    Number of pages14
    JournalJournal of Proteome Research
    Volume15
    Issue number12
    DOIs
    Publication statusPublished - 2 Dec 2016

    Keywords

    • affinity purification
    • biochemistry
    • Formin
    • mass spectrometry
    • quantitative proteomics
    • SILAC

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