TY - JOUR
T1 - Quantitative Proteomics Illuminates a Functional Interaction between mDia2 and the Proteasome
AU - Isogai, Tadamoto
AU - Van Der Kammen, Rob
AU - Bleijerveld, Onno B.
AU - Goerdayal, Soenita S.
AU - Argenzio, Elisabetta
AU - Altelaar, A. F Maarten
AU - Innocenti, Metello
PY - 2016/12/2
Y1 - 2016/12/2
N2 - 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.
AB - 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.
KW - affinity purification
KW - biochemistry
KW - Formin
KW - mass spectrometry
KW - quantitative proteomics
KW - SILAC
UR - http://www.scopus.com/inward/record.url?scp=85000869312&partnerID=8YFLogxK
U2 - 10.1021/acs.jproteome.6b00718
DO - 10.1021/acs.jproteome.6b00718
M3 - Article
AN - SCOPUS:85000869312
SN - 1535-3893
VL - 15
SP - 4624
EP - 4637
JO - Journal of Proteome Research
JF - Journal of Proteome Research
IS - 12
ER -