A7r5 rat aorta cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM high glucose (Invitrogen, Carlsbad, CA) with 10% fetal calf serum, 1% glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin. A7r5 cells were used as a model for smooth muscle that allows the expression of mutant proteins and knockdown by using small interfering RNA (siRNA). A7r5 cells proliferate as myoblasts and, after reaching the stationary phase, differentiate into a phenotype that resembles adult smooth muscle cells (Kimes and Brandt, 1976; Firulli et al., 1998). The cells express many smooth muscle specific marker proteins, including smooth muscle α actin, smooth muscle myosin, smooth muscle tropomyosin isoforms, h1 calponin, and SM22 α (Gimona et al., 2003). To ensure differentiation of the cells to the smooth muscle-like phenotype, cells were grown to confluence and serum starved for 24 h before all experiments.

For PLA using the Duolink in situ PLA kit (Olink, Uppsala, Sweden), coverslips were stained with the primary antibodies, washed, and further processed essentially according to the manufacturer's instructions. In brief, the coverslips were incubated with the secondary oligonucleotide-linked antibodies provided in the kit. The oligonucleotides bound to the antibodies were hybridized, ligated, amplified, and detected using a fluorescent probe. Dots were detected and counted using NIS Elements AR 2.30 software (Nikon, Melville, NY). Images were processed with Photoshop CS3 software (Adobe Systems, Mountain View, CA).

Cloning of the green fluorescent protein (GFP)-tagged and the Strep-tagged Par-4 cDNAs; the GFP-tagged ZIPK cDNA; and cloning of the constructs encoding the C-terminal regions of MYPT1, E2CCLZ (amino acids [aa] 892-1030), CC (aa 924-990), and LZ (aa 992-1030) has been described previously (Kögel et al., 1998; Vetterkind et al., 2005a,b; Lee et al., 2007). For the leucine zipper mutant Par-4 L3A, leucines 295, 316, and 330 were replaced by alanines, as described previously (Boosen et al., 2005). The phosphorylation site mutant Par-4 T155A, in which threonine-155 is replaced by an alanine, was generated by site-directed mutagenesis by using the primers 5′-AAG CGC CGC TCC GCT GGC GTG GTC AAC-3′ and 5′-GTT GAC CAC GCC AGC GGA GCG GCG CTT-3′.

LPA was purchased from Cayman Chemical (Ann Arbor, MI). Okadaic acid and calyculin A were purchased from Calbiochem (Gibbstown, NJ). Pharmalytes for isoelectric focusing were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom). General laboratory reagents were of analytical grade or better and were purchased from Sigma-Aldrich (St. Louis, MO) and Bio-Rad Laboratories (Hercules, CA). The following primary antibodies were used: rabbit polyclonal anti-PP1cδ antibody (1:250; Millipore, Billerica, MA), rabbit polyclonal anti-M130 antibody (1:1000; Covance Research Products, Princeton, NJ), rabbit polyclonal anti-pT696-MYPT1-antibody (1:500; Millipore), rabbit polyclonal anti-pSer19-LC20 antibody (1:500; Cell Signaling Technology, Danvers, MA), mouse monoclonal anti-LC20 antibody (1:3000; Sigma-Aldrich), mouse monoclonal anti-tubulin α antibody (Sigma-Aldrich), mouse monoclonal anti-GFP antibody (Clontech, Mountain View, CA), and mouse monoclonal anti-Par-4 antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA). For immunofluorescence experiments, goat anti-rabbit and goat anti-mouse Alexa Fluor 488 and Alexa Fluor 568 (1:1000; Invitrogen) were used as secondary antibodies. Goat Oregon Green 488- or Alexa Fluor 568-labeled anti-rabbit or anti-mouse immunoglobulin Gs were used as secondary antibodies in Western blot experiments (1:1000; LI-COR Biosciences, Lincoln, NE).

Transient transfection using the jetPEI transfection reagent (PolyPlus, New York, NY) was carried out according to the manufacturer's protocol. For Par-4 knockdown experiments, the following cyanine (Cy)3-labeled siRNA oligonucleotides (Dharmacon RNA Technologies, Lafayette, CO) were used: 5′-GAUGCUAUCACACAGCAGA-UU-3′ (antisense) and 3′-UU-CUACGAUAGUGUGUCGUCU-Cy3-5′ (sense) directed against rat Par-4 and 5′-GAUGCAAUUACACAACAGA-UU-3′ (antisense) and 3′-UU-CUACGUUAAUGUGUUGUCU-Cy3-5′ (sense) as mismatch control (directed against human Par-4; Kawai et al., 2003). Transfection with 40 nmol/l prehybridized siRNA molecules was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were processed for experiments 5 d after siRNA transfection.

Cells were fixed and stained as described previously (Vetterkind et al., 2005b). Transfected cells were fixed 48 h after transfection. For some experiments, cells were prepermeabilized with 0.25% Triton X-100 in cytoskeleton stabilization buffer [50 mmol/l NaCl, 3 mmol/l MgCl₂, 30 mmol/l sucrose, and 10 mmol/l piperazine-N,N′-bis(2-ethanesulfonic acid), pH 6.8] for 3 min at 37°C before fixation. Nuclei were stained with 4,6-diamidino-2-phenylindole (Sigma-Aldrich), filamentous actin was stained with Alexa Fluor 568 and Alexa Fluor 488 phalloidin (1:3000; Invitrogen). Cells were examined with an Eclipse TE2000-E fluorescence microscope (Nikon) equipped with a charge-coupled device camera and using filters optimized for double-label experiments.

For immunoprecipitation experiments, A7r5 cells were lysed in isotonic lysis buffer (50 mmol/l NaCl, 3 mmol/l MgCl₂, 1 mmol/l dithiothreitol, and 0.5% Nonidet-P40 in a 10 mmol/l sodium phosphate buffer, pH 8.0). To prevent protein degradation, lysis buffer was supplemented with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), and lysates were kept on ice or at 4°C at all times. Lysates were cleared by centrifugation, precleared with empty beads, and subjected to immunoprecipitation with the mouse monoclonal anti-Par-4 antibody (Santa Cruz Biotechnology), the rabbit polyclonal anti-PP1cδ antibody (Millipore) or the rabbit polyclonal anti-GFP antibody (Clontech) at 4°C overnight. The antigen–antibody complexes were adsorbed to protein G-Dynabeads (Invitrogen) and washed three times with lysis buffer. For pull down experiments, purified Par-4 (1 μg) was incubated with an Intein-tagged MYPT1 fragment (aa 850-1030) (Lee et al., 2007) immobilized on chitin beads (70 μl). Beads were washed three times with 200 μl of lysis buffer followed by one wash step with kinase buffer (50 mmol/l Tris, 100 mmol/l NaCl, 5 mmol/l MnCl₂, and 1 mmol/l dithiothreitol, pH 7.5). A sample of the beads (10 μl) was taken for analysis of Par-4 binding. The remaining beads were split into two samples of 30 μl each. Active ZIPK (0.25 μg; Cell Signaling Technology) was added, and samples were incubated either with or without 50 μM ATP at 37°C for 30 min, followed by three washes with lysis buffer. Samples were taken from all washing steps. Proteins in the samples were separated on 10% SDS polyacrylamide gels according to standard procedures. For Western blot analysis, proteins on SDS gels were transferred onto nitrocellulose membranes (Whatman, Florham Park, NJ). Bound proteins were detected with specific primary antibodies and appropriate secondary antibodies. Bands were visualized on an Odyssey infrared imaging system (LI-COR Biosciences). Densitometry analysis was performed with the Odyssey 2.1 software. Ponceau staining was used to verify equal protein loading and transfer.

Two-dimensional gel electrophoresis was performed as described previously (Kim et al., 2008) by using 0.4% Pharmalytes pH 3–10 and 1.6% Pharmalytes pH 4–6.5 (GE Healthcare). After isoelectric focusing at 400 V for 16 h, tube gels were transferred to 10% SDS polyacrylamide gels. LC20 phosphorylation was measured as an indicator for contractile potential. Glycerol-urea gel electrophoresis was used to separate nonphosphorylated, monophosphorylated, and diphosphorylated light chains as described previously (Kim et al., 2000). LC20 phosphorylation (in moles of phosphate per moles of LC20) was calculated from densitometric analyses as follows: [area (monophosphorylated) + 2× area (diphosphorylated)]/[area (unphosphorylated) + area (monophosphorylated) + area (diphosphorylated)].

Cell lysates were subjected to immunoprecipitation using the anti-PP1cδ antibody (Millipore). Immunoprecipitates were washed three times in lysis buffer and twice in PP1 assay buffer (20 mmol/l Tris-HCl, pH 7.0, 1% Triton X-100, 0.25 mol/l sucrose, 1 mmol/l MnCl₂, and 0.1% β-mercaptoethanol) and then split into two equal aliquots and resuspended in phosphatase assay buffer containing 10 mmol/l para-nitrophenyl phosphate (p-NPP; Sigma-Aldrich) as phosphatase substrate and either 5 nmol/l okadaic acid or 5 nmol/l calyculin A as PP1/PP2A inhibitors. PP1 is inhibited by calyculin A at 5 nmol/l but not okadaic acid at 5 nmol/l. Samples, including blank samples, were incubated at 30°C for 2 h. The reaction was stopped by adding 50 mmol/l EDTA. Production of the hydrolysis product para-nitrophenol was measured at 405 nm with a spectrophotometer (Shimadzu, Columbia, MD). To eliminate the activities of potentially copurified/contaminating phosphatases, the activity in the calyculin A sample was subtracted from the activity in the okadaic acid sample, and the remaining calyculin A-sensitive activity was considered as PP1 activity. Specific activity was determined by relating the PP1 activity to the total protein content of the same sample.

ZIPK assay reactions were performed in ZIPK assay buffer (50 mmol/l Tris-HCl, pH 7.5, 100 mmol/l NaCl, 5 mmol/l MnCl₂, and 1 mmol/l dithiothreitol) in a total volume of 20 μl containing 0.1 μg (1.27 pmol) of active purified recombinant ZIPK (Cell Signaling Technology) and 5 μg of LC20 peptide (BIOMOL Research Laboratories, Plymouth Meeting, PA) as substrate. Where indicated, purified recombinant Par-4 was added in a molar ratio of 1:1 (1.27 pmol) or 10:1 (12.7 pmol). Reactions were incubated for 30 min at 30°C and stopped by adding 10 mmol/l EGTA (end concentration). Then, 2 μl of each reaction was spotted on a nitrocellulose membrane. Bovine serum albumin and LC20 peptide were spotted as controls (each 0.5 μg in 2 μl of kinase assay buffer). The dot blots were blocked and analyzed with anti-Par-4, anti-ZIPK, and anti-pSer19-LC20 antibodies.

Strep-tagged wild-type Par-4 was expressed in the Escherichia coli strain BL21 (DE3) (Stratagene, La Jolla, CA). Bacteria were transformed with Par-4 expression vectors and grown in DYT medium (1.6% tryptone, 1.0% yeast extract, 0.5% NaCl) at 37°C. Protein expression was induced in late log phase with 1 mmol/l isopropyl β-d-thiogalactoside (Sigma-Aldrich). Bacteria were harvested 3 h after induction and solubilized in solubilization buffer (50 mmol/l Tris, pH 7.5, and 200 mmol/l MgCl₂) by ultrasonic disruption. Lysates were cleared from cell debris by centrifugation. Recombinant proteins were purified from the cell lysates using StrepTactin Sepharose (IBA, Göttingen, Germany) essentially according to the manufacturer's instructions. The MYPT1 peptides were purified as described previously (Lee et al., 2007).

All SPR experiments were carried out on a BIAcore 3000 (Biacore, Uppsala, Sweden) at 25°C. Sensor chips and reagents for immobilization [1-ethyl-3-(3-dimethylamino-propyl)carbodiimide, N-hydroxysuccinimide, and ethanolamine] were purchased from Biacore. Recombinant Par-4 protein was immobilized onto a CM5 sensor chip using the amine coupling method. Typically, 400-1000 resonance units of peptides were immobilized. Running buffer was HEPES-buffered saline containing 10 mmol/l HEPES and 150 mmol/l NaCl, pH 7.4, with 0.005% surfactant P20. Twofold serial dilutions of the MYPT1 peptides CCLZ (aa 925-1030), E2CCLZ (aa 892-1030), CC (aa 925-991), and LZ (aa 992-1030) starting at 348 to 593 μmol/l were used for the binding analysis. SPR data were analyzed using BIAevaluation 4.1 software (Biacore).

All values given in the text are mean ± SE. Differences between means were evaluated using a two-tailed Student's t test. Significant differences were taken at the p < 0.05 level. Sequence alignment was performed using the ClustalW server (Larkin et al., 2007), and prediction of coiled coil regions and leucine zipper motifs was performed using the 2ZIP server (Bornberg-Bauer et al., 1998).

In immunofluorescence experiments with the rat aorta cell line A7r5, endogenous Par-4 colocalizes with endogenous PP1cδ and endogenous MYPT1 along the actin cables (Supplemental Figure S1). To find out whether Par-4 interacts with PP1cδ and/or MYPT1 in vivo, coimmunoprecipitation experiments using a monoclonal anti-Par-4 antibody were performed. As shown in Figure 1A, endogenous PP1cδ and MYPT1 were coprecipitated with endogenous Par-4 but not in control experiments. Moreover, after immunoprecipitation of endogenous PP1cδ with an anti-PP1cδ antibody, both MYPT1 and Par-4 bands could be detected. Because Par-4 does not contain a PP1 binding motif but harbors a C-terminal leucine zipper motif, we speculated that binding of Par-4 to MP could be mediated by leucine zipper-leucine zipper interaction between Par-4 and MYPT1. To test this hypothesis, we performed coimmunoprecipitation experiments with transfected A7r5 cells overexpressing GFP-tagged wild-type Par-4 or a Par-4 mutant in which three leucines of the leucine zipper have been replaced by alanines (L3A) (Boosen et al., 2005). Supplemental Figure S2 shows the sequence of the Par-4 leucine zipper and the position of the mutated leucines in L3A. In immunoprecipitation experiments with an anti-GFP antibody, endogenous MYPT1 coimmunoprecipitated with wild-type Par-4-GFP, whereas the amount of MYPT1 that was coimmunoprecipitated with the Par-4 leucine zipper mutant L3A-GFP was significantly reduced, indicating that the leucine zipper of Par-4 mediates binding to MYPT1 (Figure 1B). Coimmunoprecipitation of PP1cδ and the Par-4 interaction partner ZIPK with L3A-GFP were also reduced. Because the association of Par-4 with the MP complex depends on the presence of the Par-4 leucine zipper, it seems likely that binding to the MP complex is mediated by a leucine zipper-leucine zipper interaction between Par-4 and MYPT1.

Endogenous interaction was confirmed by the in situ PLA, an immunofluorescence-based method that generates a quantifiable signal indicative of proximity (30–50 nm) between two antigens (Fredriksson et al., 2002; Greenberg et al., 2008; Söderberg et al., 2008; Mellberg et al., 2009). Control experiments showed that staining with each antibody alone (instead of antibody pairs) resulted in virtually no signal in any case (data not shown). As is shown in Figure 1C, the Par-4 and MYPT1 antibody pair produced a significantly higher PLA signal than a negative control antibody pair (MYPT1 and tubulin), and the Par-4 and MYPT1 PLA signal was of similar magnitude to that of a positive control antibody pair (LC20 and pSer19-LC20), indicating that Par-4 and MYPT1 are in proximity to each other in the cell, probably in the same protein complex. Furthermore, in cells transfected with either wild-type Par-4-GFP or the leucine zipper mutant L3A-GFP, the number of dots was significantly higher in cells transfected with Par-4 than in cells transfected with the leucine zipper mutant (Figure 1C), confirming a role for the Par-4 leucine zipper in binding to MYPT1.

Direct binding of Par-4 and MYPT1 was assayed by SPR experiments. For this purpose, purified recombinant wild type Par-4 was immobilized on a CM 5 sensor chip and tested for binding of specific purified MYPT1 peptides E2CCLZ (aa 891-1030), CCLZ (aa 924-1030), CC (aa 924-990), and LZ (aa 991-1030) (Lee et al., 2007). The data in Figure 1D show that Par-4 binds to LZ but not CC; similar results as those for LZ were obtained for E2CCLZ and CCLZ (data not shown), indicating that Par-4 binds to LZ and to peptides that contain LZ. Thus, the leucine-zipper of Par-4 binds the leucine zipper domain of MYPT1. An apparent dissociation constant (k_d) of 84 μmol/l, a likely overestimate because it does not take into account homodimerization of Par-4, was determined for binding of Par-4 and CCLZ. However, because purified Par-4 was not compatible with the buffer requirements for SPR flow channel, we were not able to calculate the k_d value for Par-4 dimerization and therefore cannot provide an accurate k_d value for interaction of Par-4 and MYPT1.

Par-4 is a phosphoprotein that is known to be regulated by upstream kinases such as ZIPK, PKA, and PKB/Akt (Page et al., 1999; Kawai et al., 2003; Goswami et al., 2005; Gurumurthy et al., 2005). To find out whether Par-4 phosphorylation is required for Par-4 binding to MP, we analyzed the phosphorylation state of Par-4 by 2-D gel electrophoresis before (Figure 2A, black bars) and after (Figure 2A, white bars) coimmunoprecipitation with PP1cδ. That the anti-PP1cδ antibody is able to coimmunoprecipitate MYPT1 is shown in Figure 1A. Par-4 spots were detected with a specific anti-Par-4 antibody. In pilot studies, phosphorylated and unphosphorylated Par-4 spots were identified by analyzing samples that were subjected to phosphatase and phosphatase inhibitor treatments (Supplemental Figure S3A). As shown in Figure 2A, densitometric analysis of 2-D gels indicates that in unstimulated cells, ∼30% of Par-4 is phosphorylated. After stimulation with LPA to induce Rho/Rho kinase (ROCK) and ZIPK activation, Par-4 phosphorylation levels were significantly increased to 45%. However, after immunoprecipitation of MP with an anti-PP1cδ antibody, the phosphorylation levels of coprecipitated Par-4 (white bars in Figure 2A) were indistinguishable between LPA stimulated and unstimulated cell extracts (26 and 29%, respectively). Control experiments showed that the phosphorylation state of Par-4 was preserved throughout the experiment (Supplemental Figure S3B). These results suggest that the majority of Par-4 bound to MP is unphosphorylated and that Par-4 phosphorylation is not required for binding to MP. To test whether phosphorylation of Par-4 might weaken the interaction with MYPT1, we have performed in vitro experiments with purified Par-4, an immobilized purified C-terminal MYPT1 fragment, and purified active ZIPK. We show in Figure 2B that Par-4 binds to the immobilized MYPT1 fragment (Figure 2B, input) and that it is released from the beads into the wash buffer upon addition of active ZIPK in the presence, but not in the absence, of ATP (Figure 2B). In addition, we show in Figure 2C that MYPT1 is coprecipitated with both wild type Par-4 and a phosphorylation site mutant (T155A) in untreated cells, whereas after LPA stimulation, coimmunoprecipitation of MYPT1 could only be detected for the phosphorylation site mutant. These results demonstrate that Par-4 binds to MP preferentially when it is unphosphorylated, whereas Par-4 phosphorylation, because it occurs after stimulation of Rho/ROCK and ZIPK by LPA, leads to its detachment from the MP complex.

To find out whether interaction with Par-4 affects MP activity, we assayed the activity of immunoprecipitated PP1 holoenzymes after overexpression of Par-4 or the leucine zipper mutant of Par-4 L3A. Proteins were overexpressed threefold to fivefold over endogenous Par-4. Interestingly, phosphatase activity was increased approximately threefold in Par-4–transfected cells compared with untransfected or GFP-transfected cells (Figure 3A). In L3A-expressing cells, PP1 activity was significantly lower than in Par-4–transfected cells and remained at the level of background PP1 activity (Figure 3A). To further test the hypothesis that Par-4 increases MP activity, we applied a knockdown approach. Endogenous Par-4 protein expression was down-regulated in the rat smooth muscle cell line A7r5 by using siRNA directed against rat Par-4. As a mismatch control, we used siRNA directed against human Par-4 (which differs in sequence by three nucleotides). In Par-4 siRNA-transfected cells, the Par-4 expression was significantly reduced to an average of 37% of that in the mismatch transfected cells, whereas expression of MYPT1, ZIPK, and PP1cδ was unaffected by the knockdown (Supplemental Figures S4, A and B). In the same experiments, PP1 activity was significantly reduced in the Par-4 siRNA-treated cells compared with the mismatch treated cells (0.015 ± 0.0029 vs. 0.041 ± 0.0057 U; Figure 3A).

In combination with various regulatory subunits, PP1cδ is active against a wide array of different substrates (for review, see Ceulemans and Bollen, 2004). To specifically analyze the effect of Par-4 on PP1cδ as part of the MP holoenzyme, we monitored LC20 phosphorylation after overexpression of Par-4. For this purpose, we applied the proximity ligation assay using a mouse monoclonal anti-LC20 antibody and a rabbit polyclonal pSer19-LC20 antibody as primary antibodies, so that a signal is generated when LC20 is phosphorylated at serine 19, but not when it is unphosphorylated. This cell-based assay, in contrast to a traditional gel-based assay, has the advantage of allowing selected analysis of the subpopulation of transfected cells. Consistent with the activation of PP1 described above, overexpression of Par-4 led to reduced LC20 phosphorylation levels (Figure 3B). In contrast, expression of L3A, which is impaired in binding to MYPT1, had no effect on LC20 phosphorylation, indicating that Par-4 activates MP via binding to MYPT1 (Figure 3B). Overexpression of wild-type Par-4 with ZIPK had no effect over that of ZIPK alone. However, a Par-4 phosphorylation site mutant, T155A, which would prevent the displacement of Par-4 from MYPT1 and lead to an increased MP activity, decreased the phosphorylation level of LC20 compared with ZIPK alone (Figure 3B and model in Figure 4C). Competitive peptide blocking experiments were performed to test the specificity of the pSer19-LC20-antibody in the context of the proximity ligation assay. As shown in Figure 3C (inverted immunofluorescence images and graph), preincubation of the anti-pSer19-LC20 antibody with an unphosphorylated LC20 peptide containing the Ser19 phosphorylation site did not affect the number of signal dots. However, preincubation of the phospho-antibody with the same peptide after it had been phosphorylated by ZIPK, led to a significantly reduced signal in the PLA assay. The proximity ligation assay with the anti-LC20/anti-pSer19-LC20 antibody pair was also sensitive enough to detect changes in LC20 phosphorylation levels in A7r5 cells after treatment with the MLCK inhibitor ML-9 or with the PP1 and PP2B inhibitor calyculin A (graph in Figure 3C). Specificity of the pSer19-LC20 antibody is further shown in Figure 3C in a glycerol-urea gel analysis of A7r5 lysate.

In a previous publication, we have reported that knockdown of Par-4 leads to a reduced response of smooth muscle strips to agonist stimulation, indicating a role for Par-4 in inhibition of MP (Vetterkind and Morgan, 2009). Here, our findings show that Par-4 activates MP in resting cultured smooth muscle cells. To test whether Par-4 might have a dual function in the regulation of MP, we have performed siRNA knockdown of Par-4 in A7r5 cells, followed by Western blot to analyze MYPT1 phosphorylation at threonine 696, the phosphorylation site preferred by ZIPK (MacDonald et al., 2001; Borman et al., 2002). In smooth muscle, MYPT1 phosphorylation at threonine 696 can be detected upon contractile stimulation and seems to be involved in calcium sensitization and steady-state contraction rather than initial contraction (Shin et al., 2002; Mizuno et al., 2008; Ding et al., 2009; Khromov et al., 2009). LC20 phosphorylation was analyzed by glycerol-urea gel electrophoresis. Cells were stimulated with LPA, or left untreated, before quick freezing in trichloroacetic acid/acetone. As shown in Figure 3D, in Par-4 knockdown cells MYPT1 inhibitory phosphorylation at threonine 696 is elevated in unstimulated cells but reduced in LPA-treated cells, compared with mismatch siRNA-transfected cells. Consistently, LC20 phosphorylation is increased after Par-4 knockdown in untreated cells, but significantly reduced in LPA stimulated cells (see graph in Figure 3D for statistical analysis). These results are in agreement with our previous findings that in vascular smooth muscle, Par-4 acts as an inhibitor of MP during agonist stimulation. At the same time, these findings are consistent with the other findings presented here, identifying Par-4 as an activator of MP in unstimulated cells. Furthermore, when the data that were generated with differentiated smooth muscle in the previously published work (Vetterkind and Morgan, 2009) were analyzed retrospectively for basal tone, it was revealed that antisense knockdown of Par-4 not only reduces PGF-2α-induced contraction (Vetterkind and Morgan, 2009) but also increases basal tone (Supplemental Figure S4C), consistent with the finding that Par-4 supports MP activity in unstimulated cells. These results indicate that Par-4 has dual functions: 1) as an activator of MP in the absence of agonists and 2) as an inhibitor of MP in the presence of an agonist. Because ZIPK can phosphorylate LC20 directly (Kögel et al., 1998; Murata-Hori et al., 1999), we tested for direct regulation of ZIPK activity by Par-4 in a kinase assay using an LC20 peptide as substrate. As shown in Figure 3E, phosphorylation of the LC20 peptide by ZIPK was not affected by the presence of Par-4, thus ruling out direct regulation of ZIPK by Par-4.