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J Proteome Res. 2016 Dec 2;15(12):4624-4637. Epub 2016 Nov 7.

Quantitative Proteomics Illuminates a Functional Interaction between mDia2 and the Proteasome.

Author information

1
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University , 3584 CH Utrecht, The Netherlands.
2
Netherlands Proteomics Centre and Cancer Genomics Centre, 3584 CH Utrecht, The Netherlands.

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.

KEYWORDS:

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

PMID:
27769112
DOI:
10.1021/acs.jproteome.6b00718
[Indexed for MEDLINE]

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