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. 2013 Aug 23;288(34):24972-83.
doi: 10.1074/jbc.M113.456244. Epub 2013 Jul 6.

Steroid-sensitive Gene 1 Is a Novel Cyclic GMP-dependent Protein Kinase I Substrate in Vascular Smooth Muscle Cells

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Free PMC article

Steroid-sensitive Gene 1 Is a Novel Cyclic GMP-dependent Protein Kinase I Substrate in Vascular Smooth Muscle Cells

Guang-rong Wang et al. J Biol Chem. .
Free PMC article

Abstract

NO, via its second messenger cGMP, activates protein kinase GI (PKGI) to induce vascular smooth muscle cell relaxation. The mechanisms by which PKGI kinase activity regulates cardiovascular function remain incompletely understood. Therefore, to identify novel protein kinase G substrates in vascular cells, a λ phage coronary artery smooth muscle cell library was constructed and screened for phosphorylation by PKGI. The screen identified steroid-sensitive gene 1 (SSG1), which harbors several predicted PKGI phosphorylation sites. We observed direct and cGMP-regulated interaction between PKGI and SSG1. In cultured vascular smooth muscle cells, both the NO donor S-nitrosocysteine and atrial natriuretic peptide induced SSG1 phosphorylation, and mutation of SSG1 at each of the two predicted PKGI phosphorylation sites completely abolished its basal phosphorylation by PKGI. We detected high SSG1 expression in cardiovascular tissues. Finally, we found that activation of PKGI with cGMP regulated SSG1 intracellular distribution.

Keywords: Cardiovascular Disease; Protein Kinase G (PKG); Signal Transduction; Steroid-sensitive Gene 1; Vascular Biology; Vascular Smooth Muscle Cells.

Figures

FIGURE 1.
FIGURE 1.
Optimization of PKGI phosphorylation screen and confirmation of phosphorylation of known PKGI substrates. A, GST, the GST-thromboxane receptor C-terminal tail domain (GST-TXR-S), and the GST-MBS C-terminal domain (GST-MBS) were phosphorylated by PKGI in vitro and subjected to SDS-PAGE and autoradiography. The positions of GST-TXR-S and GST-MBS are indicated by dashes. B, phosphorylation by PKGI of TXR-S and MBS in λGEX5. TXR-S and MBS were packaged into bacteriophage λ particles, plated with the E. coli BB4 strain, and overlaid with nitrocellulose membrane filters that were subsequently phosphorylated with PKGI as described under “Experimental Procedures.” The phosphorimager analysis of the filters is shown. Results are representative of three separate experiments.
FIGURE 2.
FIGURE 2.
PKGI phosphorylation screen. The human coronary artery smooth muscle cell cDNA library was ligated into the Sfi1 sites downstream of a GST sequence and an ampicillin resistance (Ampr) sequence. This bacterial expression cassette was ligated into the Not1 sites of λ phage DNA. Phage plaques were transferred to nitrocellulose, incubated with purified PKGI, and subjected to autoradiography to identify potential substrates (as shown on the left). For putative clones, the bacterial expression cassette was excised with Not1, circularized by self-ligation, and then the GST fusion proteins were expressed and purified for subsequent phosphorylation reactions (as shown on the right). ColE1, colicin E1 carrying plasmid; Ori, origin of replication.
FIGURE 3.
FIGURE 3.
Phosphorylation of putative PKGI substrates expressed as GST fusion proteins in bacteria. A, autoradiogram of clones isolated from the PKGI phosphorylation screen. The phage clones were purified as described under “Experimental Procedures” and expressed as GST fusion protein in bacteria, followed by purification and phosphorylation by PKGI. The phosphorylated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to autoradiography. Ctl, GST alone. Clone C3-3 was selected for further analysis. B, schematic showing clone GST-C3-3 with the predicted PKGI phosphorylation sites at serine 49 and 74 as well as the amino acid sequence. The predicted PKGI phosphorylation sites are underlined and blue. Results are representative of three separate experiments.
FIGURE 4.
FIGURE 4.
Analysis of clones from the human aorta cDNA library using C3-3 as a probe. A, Southern blot analysis. The digested phage DNA prepared from the positive clones was hybridized with a 32P-labeled C3-3 DNA probe as described under “Experimental Procedures.” B, phagemid DNA was prepared from the positive phage clones, digested with EcoR1, and separated by a 1% agarose gel. The arrows indicate the 3.8-kb full-length hSSG1, as confirmed by DNA sequencing, and the vector DNA. Results are representative of three separate experiments. C, schematic representation of the H1-1/H10-1, H8-1, and H12-3 sequences and their positions within the hBAC clone. The hBAC sequence was from the Roswell Park Cancer Institute Human BAC library.
FIGURE 5.
FIGURE 5.
Nucleotide sequence and predicted amino acid sequence of human gene SSG1. The underlined sequence specifies the C3-3 portion of the protein. The putative cleavable signal peptide is shaded in red. The predicted PKGI phosphorylation sites are marked by red stars. The truncating site of the sequence is marked by an arrowhead. The stop codon is marked by an asterisk.
FIGURE 6.
FIGURE 6.
In vitro transcription/translation of the phagemid and cloned hSSG1 cDNA. A, Ctl(−), no added DNA negative control; Ctl(+), translated products of luciferase (Luc) cDNA used as a positive control. The H1-1, H8-1, H10-1, and H12-3 lanes show the translated products of the phagemid cDNA clones. B, the vehicle lane shows the translated product of vector DNA (pCMV-Tag2B). The H1-1 and H10-1 show the phagemid cDNA-translated products. The pCSSG1(1), pCSSG1(2), and pCSSG1(3) lanes show individually cloned hSSG1 cDNA-translated products. The molecular weight of luciferase is 61 kDa, and hSSG1 is 62.1 kDa.
FIGURE 7.
FIGURE 7.
Direct interaction of hSSG1 protein with PKGI in vitro. Purified PKGI was mixed with 35S-labeled hSSG1 and immunoprecipitated (IP) with anti-PKGI antibodies. Top panel, autoradiograph of 35S-labeled hSSG1 bound to PKGI. Center panel, immunoblot analysis of PKG1 bound to the anti-PKG1 antibody. Bottom panel, densitometric analysis of 35S-SSG1 bound to PKG1. *, p < 0.05. n = 3 experiments. NI, Non-immune antibody; Pos, purified PKGI-positive control.
FIGURE 8.
FIGURE 8.
Phosphorylation of SSG1 by PKGI in vitro (A) cGMP and PKGIα-dependent phosphorylation of SSG1 in vitro. SSG1 was immunoprecipitated from Ao184 cell lysates, followed by incubation at 30 ºC for 20 min with 2.7 or 5.4 μm purified PKGIα and 1 μm cGMP or vehicle. B, the bacterially expressed and purified GST, GST-C3-3, and GST-C3-3 mutants S49A, S74A, and S49/74A were phosphorylated by purified PKGI and then subjected to SDS-PAGE and autoradiography. The arrow indicates the position of phosphorylated GST-C3-3. C, Coomassie-stained SDS-PAGE of GST and GST fusion proteins from B. The arrows indicate the positions of GST and GST-C3-3. D, quantitation of phospho/total SSG1 C3-3 from B and C. *, p < 0.05.
FIGURE 9.
FIGURE 9.
Phosphorylation of SSG1 by PKGI-activating molecules in vascular smooth muscle cells. A, Western blot analysis for phosphoserine or total SSG1 in SSG1 immunoprecipitates from Ao184 cells pretreated with ANP at 37 ºC for 10 min. *, p < 0.01 versus all other groups; †, p < 0.01 versus vehicle. B, 32P incorporation detected in SSG1 immunoprecipitate from Ao184 cells pretreated for 10 min with vehicle or with 20 μm of the NO donor SNOC. n = 3. **, p = 0.05. V, vehicle-treated.
FIGURE 10.
FIGURE 10.
Tissue expression of hSSG1. A, Northern blot analysis of RNA from human tissues. The arrows indicate the sizes of the three transcripts identified. B, Northern blot analysis of RNA from human cardiovascular tissues. The membranes (Clontech) were probed with 32P-labeled C3-3 DNA followed by autoradiography. L, left; R, right. C, identification of hSSG1 protein in cultured cells. Immunoblot analyses of cell culture lysates using anti-C3-3 IgG. Peri. Blood Leuk., peripheral blood leukocyte.; HAEC, human aortic endothelial cell; HUVEC, human umbilical vein endothelial cell. Results are representative of three separate experiments.
FIGURE 11.
FIGURE 11.
Specificity of rabbit anti-C3-3 (hSSG1) antibody for C3-3. A, detection of C3-3 by Western blot analysis (IB) of cell lysates using anti-C3-3 antibody. HUVEC, human umbilical vein endothelial cell. B, Western blot analysis of identical lysates used in A but with coincubation of C3-3-immunizing peptide. Results are representative of three separate experiments.
FIGURE 12.
FIGURE 12.
Intracellular localization of hSSG1 in Co396 cells. A, immunofluorescence image of Co396 cells labeled with anti-C3-3 IgG antibody and Cy3-linked anti-rabbit secondary antibody. B, subcellular fractionation of Co396 cells by differential centrifugation. Immunoblot analysis of total cell lysate, cytosol, plasma membrane, and nuclear fractions with anti-C3-3 IgG. Results are representative of three separate experiments.
FIGURE 13.
FIGURE 13.
Localization of hSSG1 in Co396 cells treated with 8Br-cGMP. A, immunofluorescence labeling of Co396 cells with anti-C3-3 antibody in the absence and presence of 8Br-cGMP. The concentrations of 8Br-cGMP used are shown above each image. B, subcellular fractionation of SSG1 in Co396 cells without and with treatment with 8Br-cGMP. The immunoblot analysis for hSSG1, with anti-C3-3 antibody, and for lamin A/C are shown at the top. The graph shows the density of hSSG1 normalized to lamin A/C for each fraction and at each concentration of 8Br-cGMP. n = 3. *, p < 0.05.Ctl, control.

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