Abstract
Recent studies identified cyclase-associated proteins (CAPs) as important regulators of actin dynamics that control assembly and disassembly of actin filaments (F-actin). While these studies significantly advanced our knowledge of their molecular functions, the physiological relevance of CAPs largely remained elusive. Gene targeting in mice implicated CAP2 in heart physiology and skeletal muscle development. Heart defects in CAP2 mutant mice were associated with altered activity of serum response factor (SRF), a transcription factor involved in multiple biological processes including heart function, but also skeletal muscle development. By exploiting mouse embryonic fibroblasts (MEFs) from CAP2 mutant mice, we aimed at deciphering the CAP2-dependent mechanism relevant for SRF activity. Reporter assays and mRNA quantification by qPCR revealed reduced SRF-dependent gene expression in mutant MEFs. Reduced SRF activity in CAP2 mutant MEFs was associated with altered actin turnover, a shift in the actin equilibrium towards monomeric actin (G-actin) as well as and reduced nuclear levels of myocardin-related transcription factor A (MRTF-A), a transcriptional SRF coactivator that is shuttled out of the nucleus and, hence, inhibited upon G-actin binding. Moreover, pharmacological actin manipulation with jasplakinolide restored MRTF-A distribution in mutant MEFs. Our data are in line with a model in which CAP2 controls the MRTF-SRF pathway in an actin-dependent manner. While MRTF-A localization and SRF activity was impaired under basal conditions, serum stimulation induced nuclear MRTF-A translocation and SRF activity in mutant MEFs similar to controls. In summary, our data revealed that in MEFs CAP2 controls basal MRTF-A localization and SRF activity, while it was dispensable for serum-induced nuclear MRTF-A translocation and SRF stimulation.
Highlights
Cyclase-associated proteins (CAPs) have been recognized as actin-binding proteins (ABP) two decades a go[1,2,3,4], but significant progress in their molecular function has been achieved only r ecently[5,6,7,8,9,10,11]
We chose mouse embryonic fibroblasts (MEFs) as a cellular model system for our study, because in these cells CAP2 is expressed at substantial levels and serum response factor (SRF)-dependent gene regulation and upstream regulatory mechanisms have been intensively s tudied[21]
We found a shift in the actin equilibrium towards G-actin in CAP2 mutant MEFs, which was associated with a reduction in nuclear myocardin-related transcription factor A (MRTF-A), reduced SRF activity and decreased expression of established myocardin-related transcription factors (MRTFs)-SRF target genes
Summary
Cyclase-associated proteins (CAPs) have been recognized as actin-binding proteins (ABP) two decades a go[1,2,3,4], but significant progress in their molecular function has been achieved only r ecently[5,6,7,8,9,10,11]. CAPs emerged as important regulators of F-actin dynamics, the spatiotemporally controlled assembly and disassembly of F-actin[12] While these studies advanced our knowledge of their molecular functions, the physiological relevance of mammalian CAPs largely remained elusive, because appropriate animal models were lacking. Displayed a myopathy characterized by a large number of ring fibers associated with motor function d eficits[17] Together, these studies emphasized a pivotal role for CAP2 in striated muscles, in agreement with its abundant expression in heart and skeletal m uscle[14,17,18]. While our data were in line with a role for CAP2 in regulating SRF activity via the actin-MRTF-A pathway in non-stimulated MEFs, serum stimulation induced nuclear MRTF-A translocation and SRF activity in control and CAP2 mutant MEFs
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