PKA-dependent phosphorylation of serum response factor inhibits smooth muscle-specific gene expression.
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 SRF's involvement in smooth muscle cell (SMC)-specific and early response gene expression.


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.
Department of Pathology, University of North Carolina, Chapel Hill, NC 27599-7525, USA.
Alicia L
AL
Blaker
Joan M
JM
Taylor
Christopher P
CP
Mack
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026362:Serum Response Factor / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D015534:Trans-Activators / Q000235:genetics; D015533:Transcriptional Activation; D000099251:Myocardin
D017868:Cyclic AMP-Dependent Protein Kinases; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D026362:Serum Response Factor; D015534:Trans-Activators; D000099251:Myocardin