Quantitative Proteomics Illuminates a Functional Interaction between mDia2 and the Proteasome

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.