on September 24, 2009
Published online before print September 24, 2009, doi: 10.1161/ATVBAHA.109.197285
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Submitted on May 6, 2008
Accepted on ,
PKA-Dependent Phosphorylation of Serum Response Factor Inhibits Smooth Muscle–Specific Gene Expression
Alicia L. Blaker ;
From the Departments of Cell and Molecular Physiology (A.L.B.) and Pathology and Laboratory Medicine (J.M.T., C.P.M.), University of North Carolina, Chapel Hill.
* To whom correspondence should be addressed. E-mail: cmack{at}med.unc.edu.
Objective—Our goal was to identify phosphorylation sites that regulate serum response factor (SRF) activity to gain a better understanding of the signaling mechanisms that regulate SRFs involvement in smooth muscle cell (SMC)-specific and early response gene expression.
Methods and Results—By screening phosphorylation-deficient and mimetic mutations in SRF-/- embryonic stem cells, we identified T159 as a phosphorylation site that significantly inhibits SMC-specific gene expression in an embryonic stem cell model of SMC differentiation. This residue conforms to a highly conserved consensus cAMP-dependent protein kinase (PKA) site, and in vitro and in vivo labeling studies demonstrated that it was phosphorylated by PKA. Results from gel shift and chromatin immunoprecipitation assays demonstrated that T159 phosphorylation inhibited SRF binding to SMC-specific CArG elements. Interestingly, the myocardin factors could at least partially rescue the effects of the T159D mutation under some conditions, but this response was promoter specific. Finally, PKA signaling had much less of an effect on c-fos promoter activity and SRF binding to the c-fos CArG.
Conclusions—Our results indicate that phosphorylation of SRF by PKA inhibits SMC-specific transcription suggesting a novel signaling mechanism for the control of SMC phenotype.
Key words: smooth muscle • serum response factor • protein kinase A