Schematic representation of cPKC-dependent mSK1a regulation.

<p>(A) Phosphorylation-dependent regulation of mSK1a. (B) Schematic model for mSK1a regulation in response to extracellular stimuli. Abbreviations: mSK1a (mouse sphingosine kinase 1a), PIP₂ (phosphatidylinositol 4,5-bisphosphate), DAG (diacylglycerol), cPKC (conventional protein kinase C), PMA (phorbol 12-myristate 13-acetate), ERK (extracellular signal-regulated kinase), CaM (calmodulin), IP₃, (inositol trisphosphate), PLC-γ (phospholipase C-gamma), P (phosphate group).</p

S373 phosphorylation-dependent Inhibition of mSK1a.

<p>(A) The effect of mSK1a S373A mutation on PMA-induced inhibition of mSK1a activity. Wild-type (WT) mSK1a or the S373A mutant was transfected into COS-7 cells. One day after transfection, the cells were serum-deprived for 24 hr and treated with 100 nM PMA for the indicated periods of time. Protein extracts from COS-7 cells were assayed for SK activity <i>in vitro</i>. SK activity is expressed as the percentage of activity relative to the non-treated control. These data represent the means ± SEM. Inset: time-dependent phosphorylation of WT mSK1a and the S373A mutant (S373A). (B) Approximate stoichiometry of mSK1a phosphorylation. After PMA stimulation, WT mSK1a and its S373A mutant were immunoprecipitated and subjected to 2-D gel electrophoresis. mSK1a was detected using immunoblot analysis using α-FLAG Ab. S373-phosphorylated (P-mSK1a, red) and unphosphorylated (un-P-mSK1a, blue) proteins are indicated below the images. (C) Effect of mSK1a S373E mutation on PMA-induced inhibition of mSK1a activity. Wild-type mSK1a and the S373E mutant were purified from transfected COS-7 cells using α-FLAG Affi-Gel. The same amounts (5 ng) of purified WT mSK1a and S373E mutant were assayed for SK activity. The data are the means ± SEM (t- test, *p < 0.05, **p < 0.01). Inset: The amounts of purified mSK1a and S373E mutant were confirmed by SDS-PAGE followed by Coomassie Blue (CBB) Staining. (D) Calmodulin interaction of WT mSK1a and the S373 mutant. Identical concentrations of mSK1a and the S373E mutant were incubated with either calmodulin-agarose beads or control beads at two different Ca²⁺ concentrations, as indicated. mSK1a bound to beads were assessed by immunoblotting using α-FLAG antibody. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143695#pone.0143695.s001" target="_blank">S1 Fig</a> for additional data about the protein interactions of WT mSK1a and the S373E mutant.</p

Phosphorylation of mSK isoforms in response to extracellular stimuli.

<p>COS-7 cells were transfected with control vector, FLAG-tagged mSK1a, or FLAG-tagged mSK2 constructs. After serum deprivation and metabolic labeling with [³²P] inorganic phosphate, the cells were treated for 10 min with various agonists, as indicated: PMA (100 nM), EGF (100 ng/ml), or forskolin (20 μM). mSK isoforms were immunoprecipitated using α-FLAG Affi-Gel, transferred on nitrocellulose membrane, and exposed to X-ray photographic film (top panel) after Ponceau S staining (bottom). This result is representative of three independent experiments.</p

mSK1a S373 is the cPKC-dependent phosphorylation site.

<p>(A) PKC-dependent (spots a and b: arrowheads) and -independent (spot c: arrow) phosphopeptide(s) are indicated. Each peptide was eluted from the TLC plate and divided into two fractions, which were subjected to two-dimensional phosphoamino acid analysis or ³²P-release assay. (B) Phosphoamino acid analysis of three phosphopeptides. Acid-hydrolyzed peptides were subjected to 2-dimensional phosphoamino acid analysis. Dotted circles indicate the migration positions of phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) standards (left panel) and the image from autoradiography (right panel). (C) ³²P-release assay of three radiolabeled peptides. Radiolabeled peptides loaded onto the sequencing membrane (Millipore) were digested sequentially from the N-terminus by the Edman degradation method. Putative candidate sites for each phosphopeptide were predicted based on the relative position from the tryptic cleavage site, as shown on the right. (D) Site-directed mutagenesis of candidate sites. COS-7 cells were transfected with FLAG-tagged mSK1a constructs including wild-type (WT) and its mutant (S225A, S332A, S373A) and treated with PMA (100 nM). The relative phosphorylation level of each mSK1a form was assessed in duplicate. Data represent the means ± SE. (E) Two-dimensional phosphopeptide analysis of WT mSK1a and its S/A mutants (S225A, S332A, S373A). cPKC-dependent spots (a and b) and -independent spot (c) are indicated by arrowheads and arrow, respectively. (F) <i>In vitro</i> phosphorylation of WT mSK1a and its S/A mutants by PKCα. Wild-type mSK1a and S/A mutants (S373A, S225A) were expressed in COS-7 cells and purified using α-FLAG Affi-Gel. Each protein (100 ng) was incubated with PKCα (10 ng) in 1× phosphorylation buffer in the presence or absence of PMA and GF109204X (GFX). Incorporation of radioactivity was measured, and the transferred membrane was immunoblotted to monitor the input amount of each protein, as indicated.</p

PMA-induced phosphorylation and inhibition of mSK1a.

<p>(A) Time-dependent phosphorylation of mSK1a in response to PMA treatment. COS-7 cells were transfected with mSK1a cDNA. After metabolic labeling with [³²P] <i>ortho</i>-phosphate, the cells were treated with 100 nM PMA for indicated periods of time. mSK1a was immunoprecipitated using the FLAG epitope. The autoradiogram and immunoblot images using α-FLAG antibody are shown (top and middle panels), and relative radioactivity was quantitated in duplicate (bottom). Data represent the means ± SE. (B) Time course of PMA-induced suppression of mSK1a activity. COS-7 cells, transfected with mSK1a cDNA, were stimulated with 100 nM PMA for the indicated periods of time. Data are from triplicate determinations from independent cultures, and are expressed as percentages relative to untreated controls. Data represent the means ± SEM. (One-way ANOVA test, *p < 0.05, **p < 0.01). (C) PMA-induced suppression of [³H]S1P formation by mSK1a. Formation of [³H]S1P was determined in COS-7 cells that are transfected with either control vector or mSK1a cDNA, and further stimulated with 100 nM PMA for 10 min prior to [³H]SPH labeling. Data represent the means ± SEM. (t-test, *p < 0.05, **p < 0.01).</p

Involvement of ROS in MS-induced PDGFR phosphorylation.

<p>(A) Cells were stimulated by 10% MS for 10 min, and ROS was determined using DCF fluorescence. Quantitative data for the corresponding DCF fluorescence in the left are presented as means ± SEM (n = 4). **<i>p</i><0.01 <i>vs.</i> control (Con). (B) Cells were stimulated by 10% MS for 10 min in the presence or absence of NAC (0.5 mM), a ROS inhibitor, and the levels of phosphorylated and total PDGFR isoforms (PDGFR-α and PDGFR-β) were determined by immunoblotting. Quantitative data for the corresponding blots in the left panel are presented as means ± SEM (n = 5). **<i>p</i><0.01 <i>vs.</i> corresponding control (Con). #<i>p</i><0.05, ##<i>p</i><0.01 <i>vs.</i> corresponding vehicle (Veh).</p

The individual roles of PDGFR isoforms in Akt phosphorylation in VSMC.

<p>(A) Representative immunoblots for the phosphorylated (p-Akt) and total Akt in cells stimulated with a PDGFR-α ligand (PDGF-AA) and PDGFR-β ligands (PDGF-BB and –DD) for 10 min (n = 5). (B) Cells were transfected with the indicated doses of PDGFR-α siRNA or PDGFR-β siRNA, and then stimulated by 10% MS for 4 hrs. Quantitative data for the corresponding blots are presented as means ± SEM (n = 5). **<i>p</i><0.01 <i>vs.</i> control (Con). #<i>p</i><0.05, ##<i>p</i><0.01 <i>vs.</i> vehicle (Veh).</p

Role of Akt in MS-induced MMP-2 production in VSMC.

<p>(A) Time-course of MS-enhanced MMP-2 promoter activity. VSMC was transfected with MMP-2 promoter-luciferase construct (pGL-MMP-2) or empty luciferase vector (pGL3) for 24 hrs and then stimulated with 10% MS for the indicated time. MMP-2 promoter activity was represented as relative luciferase activity. (B) Time-course of MS-induced MMP-2 mRNA expression. Quantitative data are presented as the means ± SEM (n = 5–6). **p<0.01, <i>vs.</i> control (Con). (C and D) Cells were stimulated by10% MS for 12 hrs in the absence (Veh) or presence of various inhibitors including a PI3K inhibitor, LY294002 (LY, 10 μM), Akt inhibitor IV (AI, 10 μM), and MAPK inhibitors, PD98059 (PD, 10 μM), SP600125 (SP, 10 μM) and SB203580 (SB, 10 μM). (F and G) Cells were transfected with scrambled (NC, negative control) and AktsiRNA (100 pmol), and then stimulated by 10% MS for 12 hrs. MMP-2 activity and expression were measured using gelatin zymography and Western blot, respectively. Right panels represent quantitative data for left panels, and represent means ± SEM (n = 4). **<i>p</i><0.01 <i>vs.</i> corresponding control (Con). #<i>p</i><0.05, ##<i>p</i><0.01 <i>vs.</i> corresponding vehicle (Veh). (E and H) Cell viability in VSMC treated with Akt inhibitor or transfected with Akt siRNA are represented as % of control, and data are presented as the means ± SEM (n = 4).</p

Effects of MS on MMP-2 activity and expression in VSMC.

<p>Cells were stimulated by MS at indicated forces (A) or time (B), and then gelatinolytic activity (MMP-2 and -9) was determined using gelatin zymography. The force- and time-dependent increase in MMP-2 expression in VSMC exposed to MS was determined by Western blot (C and D) and immunocytochemical studies (E and F). Representative images are from 4–6 independent experiments.</p

The individual roles of PDGFR isoforms in MS-induced MMP-2 production in VSMC.

<p>Cells were transfected with the indicated doses of PDGFR-α siRNA or PDGFR-β siRNA, and then stimulated with 5 ng/ml PDGF-BB (A and B) as well as by 10% MS (C and D) for 12 hrs. MMP-2 activity and production were determined using gelatin zymography and Western blot, respectively. Quantitative data for the corresponding blots are presented as means ± SEM (n = 6). **<i>p</i><0.01 <i>vs.</i> corresponding control (Con). #<i>p</i><0.05, ##<i>p</i><0.01 <i>vs.</i> corresponding vehicle (Veh).</p