PKA-dependent phosphorylation of serum response factor inhibits smooth muscle-specific gene expression

Abstract

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.

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.

Figure 1. The phosphomimetic T159D mutation negatively regulated SRF-dependent SMC-specific transcription

A) Schematic of SRF functional domains and phosphorylation sites tested in these studies. B) SRF was transfected into SRF −/− ES cells along with SM22, SM α-actin, or c-fos promoter/luciferase constructs. Luciferase activity was measured 24h post-transfection and expressed relative to that measured in the presence of an equal amount of empty expression vector. C) The indicated SRF variants and promoter-reporter constructs were co-transfected into ES cells. Luciferase activity after 24h was measured and is expressed relative to empty expression vector set to 1. *p<0.05 versus Wt SRF D) Western blot showing expression levels of the SRF variants in ES cells 24h post-transfection.

Figure 2. PKA phosphorylated SRF T159 in vitro and in vivo

A) SRF sequence conservation near the consensus PKA/PKG phosphorylation site at T159 B) GST-SRF fusions (AA 1–203) containing Wt or T159A protein sequence were incubated with γ³²P-ATP in the presence of active PKA or PKG for 15 min. Following removal of unincorporated label, samples were separated on an SDS-Page gel and exposed to film. C) Cos-7 cells expressing flag-tagged Wt and T159A SRF were incubated with 1mCi ortho ³²P for 2h and then stimulated with 20μM forskolin or 100μM 8-pCPT-cGMP for 2h. SRF was then immunoprecipitated from RIPA lysates using anti-flag agarose. Following washing immunoprecipitants were run on an SDS-Page gel, transferred to nitrocellulose, and exposed to film. D) Endogenous SRF was immunoprecipitated from primary SMCs labeled with ortho ³²P and then treated with FSK for the indicated times. Immunoprecipitants were run on an SDS-Page gel, transferred to nitrocellulose, and exposed to film.

Figure 3. Myocardin factor over-expression rescued the inhibitory effects of the T159 mutation in a promoter-specific fashion

SM α-actin (A) and SM22 (B) promoter luciferase constructs were co-transfected into SRF −/− ES cells along with the indicated SRF variant and myocardin factor. Luciferase activity was measured at 24h. *p<0.05 versus Wt SRF.

Figure 4. Phosphorylation of T159 inhibited SRF binding to the SMC-specific CArGs

A) Radiolabeled oligonucleotide probes containing either the SM α-actin intronic CArG (left panel) or the SM22 far CArG (right panel) were incubated with the indicated SRF variant in the absence or presence of myocardin or ΔNMRTF-A. All proteins were in vitro translated and the total amount of TnT protein in each reaction was maintained by the addition of unprogrammed lysate. After 30 min binding reactions were run on a 5% non-denaturing gel, transferred to filter paper, and visualized by autoradiography. For supershifts (lane 1), 1μL of anti-flag antibody was added after 20 minutes of incubation. B) 10T1/2 cells were transfected with the Wt, T159A, or T159D SRF variant. After 48h cells were processed for ChIP assays by standard protocols (see methods for more details). PCR reactions were performed on immunoprecipitants using primers spanning the SM α-actin intronic CArG, the SM22 far CArG, a SM MHC 5′ CArG, and the c-fos CArG. Quantification of 3 separate ChIp assays is shown in the right panel. C) SMCs grown in 10% FBS were treated with 20μM forskolin for the indicated time before processing for ChIP assays. * p<0.05 versus vehicle treated. D) 10T1/2 cells expressing the T159 SRF variant were treated with forskolin for 1h before processing for ChIP assays. E) Western blot of SRF variant expression in 10T1/2 cells.

Figure 5. The T159D mutation inhibited endogenous SMC marker gene expression in an ES cell model of SMC differentiation

A) SRF −/− ES cells were transfected with Wt SRF. After 24 h cells were placed in differentiation media (minus LIF, plus 500nM retinoic acid) for 0, 3, and 6 days. SM α-actin and SM22 message levels were measured by quantitative PCR. * p<0.05 versus plus empty expression vector. B) SRF −/− ES cells expressing the indicated SRF variants were subjected to the SMC differentiation protocol. SM α-actin and SM22 mRNA levels measured at 0, 3, and 6 d are expressed relative to Wt SRF set to 1. * p<0.05 versus Wt SRF

Figure 6. PKA activation inhibited SM α-actin and SM22 promoter activity in primary SMC

A) Primary SMCs were transfected with SM α-actin, SM22, or c-fos promoter luciferase constructs. Luciferase activity was measured after treatment with 20μM forskolin for 9h. * p<0.05 versus vehicle. B) Primary SMC cultures were co-transfected with SM α-actin-luciferase and increasing concentrations of Wt or T159D SRF. * p<0.05 versus same concentration of Wt SRF.