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J. Biol. Chem., Vol. 275, Issue 33, 25723-25732, August 18, 2000

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Regulation of Human Endothelial Cell Focal Adhesion Sites and Migration by cGMP-dependent Protein Kinase I^*

Albert Smolenski, Wolfgang Poller, Ulrich Walter, and Suzanne M. Lohmann^§

From the Institut für Klinische Biochemie und Pathobiochemie, Medizinische Universitätsklinik, Josef-Schneider Strasse 2, D-97080 Würzburg, Germany

Received for publication, December 6, 1999, and in revised form, May 8, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cGMP-dependent protein kinase type I (cGK I), a major constituent of the atrial natriuretic peptide (ANP)/nitric oxide/cGMP signal transduction pathway, phosphorylates the vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins involved in regulation of the actin cytoskeleton. Here we demonstrate that stimulation of human umbilical vein endothelial cells (HUVECs) by both ANP and 8-(4-chlorophenylthio)guanosine 3':5'-monophosphate (8-pCPT-cGMP) activates transfected cGK I and causes detachment of VASP and its known binding partner (zyxin) from focal adhesions in >60% of cells after 30 min. The ANP effects, but not the 8-pCPT-cGMP effects, reversed after 3 h of treatment. In contrast, a catalytically inactive cGK I mutant (cGK I-K405A) was incapable of mediating these effects. VASP mutated (Ser/Thr to Ala) at all three of its established phosphorylation sites (vesicular stomatitis virus-tagged VASP-AAA mutant) was not phosphorylated by cGK I and was resistant to detaching from HUVEC focal adhesions in response to 8-pCPT-cGMP. Furthermore, activation of cGK I, but not of mutant cGK I-K405A, caused a 1.5-2-fold inhibition of HUVEC migration, a dynamic process highly dependent on focal adhesion formation and disassembly. These results indicate that cGK I phosphorylation of VASP results in loss of VASP and zyxin from focal adhesions, a response that could contribute to cGK alteration of cytoskeleton-regulated processes such as cell migration.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nitric oxide and atrial natriuretic peptide (ANP)¹ activate soluble or particulate guanylyl cyclases, respectively. The cGMP produced by these cyclases has multiple effectors within the cell, including cGMP-gated channels, cGMP-regulated phosphodiesterases, and cGMP-dependent protein kinases (cGKs). cGK I (including - and -splice variants) and cGK II have been identified in mammalian cells. The cGK I studied here is a major mediator of the nitric oxide/cGMP signal transduction pathway in several tissues (1); and recently, the functional role of cGK I in relaxation of smooth muscle, inhibition of Ca²⁺ transients, and inhibition of platelet adhesion and aggregation was consolidated by data from cGK I knockout mice (2, 3). Furthermore, cGK I is present in only certain endothelial cells, and only these respond to cGMP with a reduction in thrombin-induced calcium transients and paracellular permeability (4, 5).

The relationship between cGK I substrate phosphorylation and cellular function is still an emerging field and is the subject of this investigation. One extensively studied substrate of cGK I is the vasodilator-stimulated phosphoprotein (VASP), originally discovered in human platelets (6), but a substrate of cGK I in many cells, including endothelial cells (1, 5). In platelets, both cAMP- and cGMP-dependent phosphorylation of VASP closely correlate with inhibition of agonist-induced aggregation and in particular with inhibition of fibrinogen receptor (integrin _IIb3) activation (7). In knockout mice lacking endogenous VASP, cGMP-mediated inhibition of platelet aggregation was reduced, and agonist-induced platelet activation was enhanced (8, 9). Similarly, knockout mice lacking endogenous cGK I also displayed defective cGMP-mediated inhibition of platelet aggregation (3). cGMP-dependent phosphorylation of VASP (only Ser¹⁵⁷ was measurable at that time), but not cAMP-dependent phosphorylation of VASP, disappeared from the platelets of cGK I knockout mice (3) as well as from cGK I-deficient platelets from chronic myelocytic leukemia patients (10) and from endothelial cells that lost cGK I in culture (5), clear evidence that cGMP-dependent phosphorylation of VASP is mediated by cGK and not by activation of cAK. In contrast, cAMP-dependent phosphorylation of VASP is mediated by cAK.

In cells studied so far, VASP has been shown to be localized at highly dynamic membrane regions, focal adhesions, and cell-cell contacts and in a periodic pattern along stress fibers (11, 12). Recently, VASP-related proteins (now designated as members of the Ena/VASP protein family), which share some structural and functional characteristics with VASP, were identified (13, 14). Demonstration of VASP interaction with other proteins present at several intracellular sites suggested a role for VASP in cytoskeletal function. The N-terminal Ena/VASP homology 1 (EVH1) domain of the Ena/VASP protein family was shown to interact with a proline-rich FP₄-containing motif present in the focal adhesion proteins zyxin and vinculin (15) and also present in ActA, a surface protein of the intracellular pathogen Listeria monocytogenes to which VASP is recruited to facilitate F-actin assembly essential for Listeria motility (15-21). The central VASP domain containing four copies of a GP₅ motif binds profilin, an actin regulatory protein that has been shown to promote F-actin assembly (22). In addition, the VASP C-terminal EVH2 domain was shown to bind F-actin and to facilitate actin bundling (23, 24), to influence VASP localization to focal adhesions (25), and also to be responsible for tetramerization of VASP (23), which may stabilize interactions of the VASP EVH1 domain with the focal adhesion proteins vinculin and zyxin (26).

VASP is phosphorylated by cAMP- and cGMP-dependent protein kinases on Ser¹⁵⁷, located just distal to the EVH1 domain in the central proline-rich domain of VASP, and on Ser²³⁹ and Thr²⁷⁸, located in the VASP C-terminal EVH2 domain (23, 25, 27). So far, only phosphorylation of Ser¹⁵⁷ and Ser²³⁹ has been demonstrated to be of major significance in intact cells (27), and phosphorylation of these sites can be distinguished using specific antibodies (28). The phosphorylation site containing Ser¹⁵⁷ is preferred by cAK, whereas the site containing Ser²³⁹ is preferred by cGK in vitro and in intact cells; however, both kinases phosphorylate all three sites (27, 28). Information is, however, scarce concerning the influence of cGMP-mediated VASP phosphorylation on the described VASP interactions with other proteins or on cytoskeletal function. Furthermore, studies on many cell types, including endothelial cells, are hampered by the rapid decline in cGK levels in cell culturing and passaging (5, 29). Therefore, in this work, we present a system for studying the effect of VASP phosphorylation, using endothelial cells lacking endogenous cGK I, in the absence and presence of expressed cGK I. cGK I phosphorylation of VASP promoted detachment of VASP (but not a triple phosphorylation site mutant of VASP) and zyxin from focal adhesions, thus altering the composition and possible functional integrity of these structures. In support of this, cGK I also inhibited collagen-, fibronectin-, and fibrinogen-mediated endothelial cell migration, which is a highly dynamic process dependent on focal adhesion formation and destruction.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells-- Endothelial cells were isolated from human umbilical veins and cultured as described previously (30). HUVECs from only passages 1-3 were used for experiments. Primary human dermal fibroblasts (passages 7-10) and the human embryonic kidney cell line (HEK 293) were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Eggenstein, Germany) supplemented with 10% fetal calf serum and 1% antibiotic solution. All cells were kept at 37 °C with 5% CO₂.

Antibodies-- The following antibodies against VASP were used: rabbit antiserum M4 against VASP (6), monoclonal anti-VASP antibodies IE226 and IE273 (71), monoclonal anti-phospho-Ser²³⁹ antibody 16C2 raised against a VASP peptide phosphorylated on Ser²³⁹ (28), and monoclonal anti-phospho-Ser¹⁵⁷ antibody 5C6 (prepared by the same approach used for making the 16C2 antibody and to be described in more detail elsewhere).² Other antibodies used were as follows: rabbit antiserum A10 against cGK I (31); rabbit antiserum AS83-1 against zyxin (32); and monoclonal anti-vinculin antibody hVIN-1, monoclonal anti-VSV antibody P5D4, and fluorescein isothiocyanate-labeled anti-rabbit secondary antibody (Sigma, Deisenhofen, Germany). Cy2-labeled anti-mouse and Cy3-labeled anti-rabbit antibodies were from Rockland, Inc. (Gilbertsville, PA), and Texas Red-labeled anti-mouse antibody was from Molecular Probes, Inc. (Leiden, The Netherlands).

Vectors and Constructs-- The recombinant adenoviral vector Ad5-cGK I containing human cGK I cDNA (33, 34) was constructed as described previously (35). The catalytically inactive cGK I-K405A mutant was newly constructed by oligonucleotide-directed mutagenesis (Transformer site-directed mutagenesis kit, CLONTECH, Palo Alto, CA) of cGK I cDNA. The sequence of the mutagenic primer was 5'-GTT TCT TGA GAA TCg cCA TTG CAA ACG TTT TG-3', and the mutated region was checked by sequencing. The recombinant virus Ad5-cGK I-K405A was developed by cloning human mutant cGK I into the adenoviral transfer plasmid pZS2 using the multiple cloning site between the cytomegalovirus promotor and the bovine growth hormone 3'-untranslated region. The resulting recombinant plasmid pZS2/cGK I-K405A also contained 500 base pairs of 5'-terminal sequence of wild-type adenovirus type 5 upstream of cGK I. This plasmid was linearized with XbaI and ligated with the long XbaI fragment of RR5 DNA, an Ad5 mutant that carries a unique XbaI site and a deletion of the E1 region ranging from nucleotides 445 to 3333 of the wild-type adenovirus type 5 genome. Subsequently, the ligation product was transfected into E1a-transformed HEK 293 cells; and after 10-14 days, recombinant virus was recovered and plaque-purified as described previously (36).

Plasmid expression vectors were constructed using pcDNA3 (Invitrogen, DeSchelp, The Netherlands) and human VASP N-terminally tagged with an epitope of VSV glycoprotein G. The vectors for wild-type VASP and for three mutants carrying single amino acid substitutions at each phosphorylation site (S157A, S239A, and T278A) were described previously (28). Now, double (AAT) and triple (AAA) phosphorylation mutants of VSV-VASP were generated by combining one or more singly mutated regions using unique restriction sites.

Expression Experiments and Western Blot Analysis-- HUVECs or human fibroblasts seeded on six-well plates were infected with Ad5 vectors at concentrations of 10⁹ to 10¹¹ particles/ml in serum-free medium for 2 h at 37 °C. Subsequently, the virus-containing medium was replaced by fresh serum-containing medium. Alternatively, plasmid transfections of HUVECs were performed using Superfect (QIAGEN Inc., Hilden, Germany) according to the instructions of the manufacturer. Later, 16-24 h after either infection or transfection, cells were washed once with serum-free medium and incubated for 5-30 min with 100 nM ANP (Sigma), 100 µM 8-pCPT-cGMP (BioLog, Bremen, Germany), or 3 µM forskolin (Sigma) in serum-free medium containing 0.1% bovine serum albumin at 37 °C. Subsequently, cells were briefly washed with PBS and either lysed in hot SDS sample buffer for later immunoblot analysis or fixed for immunofluorescence as described below. For immunoblotting, samples were separated by SDS-polyacrylamide gel electrophoresis using 9% gels and subsequently analyzed on Western nitrocellulose blots. Nitrocellulose sheets were blocked overnight in blocking medium (PBS, 0.3% Triton X-100, 0.05% Tween 20, 1% hemoglobin, and 0.01% NaN₃); incubated for 1 h at room temperature with anti-cGK I antiserum A10 (diluted 1:200), monoclonal anti-VASP antibody IE273 (diluted 1:1000), monoclonal anti-phospho-Ser²³⁹ VASP antibody 16C2 (1 µg/ml), or monoclonal anti-phospho-Ser¹⁵⁷ VASP antibody 5C6 (2 µg/ml); and then subsequently incubated for 1 h with 3.7 kBq/ml ¹²⁵I-labeled protein A or 7.4 kBq/ml ¹²⁵I-labeled sheep anti-mouse antibody (both from Amersham Pharmacia Biotech, Freiburg, Germany) followed by autoradiography (see Figs. 1A and 2) or with horseradish peroxidase-coupled anti-mouse or anti-rabbit secondary antibodies followed by ECL detection (Amersham Pharmacia Biotech) (see Figs. 1B, 4, and 10A). The amount of cGK expressed (Fig. 1A) (data not shown) was determined by cutting the radioactively labeled bands out of the nitrocellulose sheet and counting them in a scintillation counter. Various concentrations of purified recombinant cGK were used as a protein standard.

VASP Immunoprecipitation and in Vitro Phosphorylation-- Plasmid vectors for VSV-tagged VASP and VASP phosphorylation site mutants (Ser/Thr to Ala) were transfected into HEK 293 cells using calcium phosphate. Two days after transfection, cells were harvested and solubilized in buffer containing 20 mM Tris-HCl (pH 7.4), 75 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 10 mM EDTA, 10 mM sodium pyrophosphate, and 20 units/ml aprotinin. The expressed proteins were immunoprecipitated using an anti-VSV antibody and rat anti-mouse IgG1 coupled to magnetic beads (Dynal, Oslo, Norway). Immune complex phosphorylation was performed with the precipitates in buffer containing 10 mM HEPES (pH 7.4), 5 mM MgCl₂, 1 mM dithioerythritol, 0.2 mM EDTA, 4 µM ATP, 1 µCi of [-³²P]ATP, and either 50 ng of cAK catalytic subunit purified as described (37) or 50 ng of purified cGK I (38) activated with 20 µM cGMP. Samples were phosphorylated for 30 min at 30 °C and then stopped and analyzed using Western blotting as described above. Expressed VSV-VASP was detected using the monoclonal anti-VSV antibody, a horseradish peroxidase-coupled anti-mouse secondary antibody, and ECL analysis. After washing ECL labeling from the nitrocellulose, ³²P incorporation into VASP was subsequently detected by autoradiography.

Indirect Immunofluorescence-- HUVECs, grown on glass coverslips previously coated with 1% gelatin for 45 min and then fixed for 15 min with 0.5% glutaraldehyde and washed five times with M199 medium, were treated with cGMP-elevating agents as described below, washed twice with PBS, fixed with 4% paraformaldehyde for 15 min on ice, washed twice with PBS, permeabilized by 0.2% Triton X-100 in PBS for 10 min at room temperature, and then incubated with primary antibodies (see below) for 1 h at 37 °C and with secondary antibodies for 1 h at 37 °C. The coverslips were mounted in Moviol 4-88 (Hoechst, Frankfurt, Germany)-containing mounting medium with 1% N-propyl gallate, and cells were examined with a Leitz Aristoplan microscope. Photographs were taken with Tri-X-Pan 400 or TMAX 400 film (both from Eastman Kodak Co.).

HUVECs infected with 5 × 10⁹ particles of adenoviral cGK I vectors/ml (>90% transfection efficiency) were incubated with either 100 nM ANP or 100 µM 8-pCPT-cGMP for 30 min (in some cases, also 3 h) for determining the localization of VASP, zyxin, or vinculin. cGK staining was visualized using polyclonal anti-cGK I antibody A10 diluted 1:1000 and fluorescein isothiocyanate-labeled anti-rabbit secondary antibody diluted 1:30. For cGK I-VASP colocalization studies, VASP was visualized using undiluted monoclonal anti-VASP antibody IE226 and Texas Red-labeled anti-mouse secondary antibody diluted 1:50. Vinculin was stained using monoclonal anti-vinculin antibody hVIN-1 diluted 1:50 and Cy2-labeled anti-mouse secondary antibody diluted 1:150. For vinculin-VASP colocalization studies, VASP was stained using affinity-purified polyclonal antibody M4 diluted 1:100 and Cy3-labeled anti-rabbit secondary antibody diluted 1:300. For vinculin-zyxin colocalization studies, zyxin was stained using polyclonal antibody AS83-1 diluted 1:200 and Cy3-labeled anti-rabbit secondary antibody diluted 1:300. The percentage of cells containing VASP, zyxin, or vinculin at focal adhesions was determined in double-labeling experiments, in each of which 100 cells were examined.

In HUVECs transfected with plasmid vectors for cGK I and VSV-tagged wild-type or mutant VASP, cGK I was stained using polyclonal anti-cGK I antibody A10 diluted 1:1000 and fluorescein isothiocyanate-labeled anti-rabbit secondary antibody diluted 1:30; expressed VSV-VASP was stained using monoclonal anti-VSV antibody P5D4 diluted 1:10,000 and Texas Red-labeled anti-mouse secondary antibody diluted 1:50. The percentage of cGK I-expressing HUVECs (50-100 cells analyzed per experiment) staining positive for VSV-VASP (or the VSV-tagged triple phospho-VASP mutant) at focal adhesions after cGK I activation was determined.

Migration Assay-- Haptotactic cell migration assays were performed using modified Boyden chambers (Transwell filters, 8-µm pore size, Costar Corp., Cambridge, MA). The lower surface of the filter membranes was coated with 3 µg/ml human fibronectin, 10 µg/ml collagen I, or 10 µg/ml fibrinogen (all from Sigma). The upper surface of the membrane and the remaining free binding sites on the lower surface were blocked with 0.5% bovine serum albumin. Subconfluent HUVECs were trypsinized and resuspended in M199 medium containing 25 mM HEPES, 1 mM MgCl₂, and 0.5% bovine serum albumin, and then 30,000 cells were seeded per filter. After 5 h of incubation at 37 °C with or without 100 µM 8-pCPT-cGMP or 500 nM ANP, cells on the upper surface of the membrane were removed with a cotton swab. Migrated cells on the lower membrane surface were stained with 1% (w/v) crystal violet (Sigma) in 2% (v/v) ethanol, washed with H₂O, and dried. The stain was eluted with 10% (v/v) acetic acid, and its absorbance was measured at 600 nm.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Functional Expression of cGK I in HUVECs-- A replication-deficient adenoviral vector containing the cDNA of human cGK I was constructed and used to evaluate the function of cGK I in HUVECs lacking endogenous cGK I. Infection of HUVECs with different amounts of vector (10⁹ to 10¹¹ particles/ml) resulted in the expression of 0.2-2.0 µg of cGK I/mg of cell protein as assessed by immunoblotting (Fig. 1A) (data not shown), which is in the range of the endogenous cGK I concentration in other types of human endothelial cells (5). In subsequent experiments, adenoviral vectors were usually used at concentrations of 10¹⁰ particles/ml or less. At 10¹⁰ particles/ml (Fig. 1A), a well known cGK I degradation product (65 kDa) was observed; and at higher levels of expression, additional smaller (most likely breakdown) products were detected. Expressed cGK I was demonstrated to be functionally active by treatment of HUVECs with a cell membrane-permeant, hydrolysis-resistant, cGK-selective cGMP analog, 8-pCPT-cGMP, which activates the kinase. In response to 8-pCPT-cGMP, cGK I phosphorylated its well characterized substrate, VASP, as demonstrated by Western blot analysis (Fig. 1B). An antibody against total VASP (IE273) detected a shift in the electrophoretically determined size of VASP, from an apparent size of 46 kDa in unstimulated cells to 50 kDa in cGMP-stimulated, cGK I-expressing cells (Fig. 1B, first panel, sixth lane), a shift resulting from phosphorylation of VASP Ser¹⁵⁷. This was confirmed using a newly developed antibody (5C6) specific for Ser¹⁵⁷ in its phosphorylated state (Fig. 1B, second panel). VASP phosphorylation on Ser¹⁵⁷ was also observed with uninfected HUVECs stimulated with 3 µM forskolin, an activator of adenylate cyclase and thus cAK (Fig. 1B, first two panels, second lanes). A higher concentration of forskolin (10 µM) also caused Ser²³⁹ phosphorylation (data not shown). Furthermore, antibody 16C2, made against the VASP phosphorylation site containing Ser²³⁹, detected cGMP-stimulated phosphorylation of VASP on Ser²³⁹ (Fig. 1B, third panel) either without (46-kDa band) or with (shift to 50-kDa band) concomitant phosphorylation of Ser¹⁵⁷ in cGK I-expressing cells. Note that in the absence of added 8-pCPT-cGMP (fifth lane), the expressed cGK I has some basal activity. More important, no cGMP-mediated VASP phosphorylation was observed in HUVECs infected either with a newly developed, catalytically inactive cGK I mutant (cGK I-K405A) (Fig. 1B, third and fourth lanes; and see Fig. 4, first through third lanes) or with a luciferase control vector or in uninfected HUVECs (data not shown). These results clearly demonstrate that cGMP-dependent phosphorylation of VASP does not occur in the absence of cGK and therefore is not mediated by cAK. Similar results were obtained from in vitro kinase assays performed with lysates from cells treated as shown in Fig. 1B, i.e. cGMP-activable kinase activity was present only in cGK I-infected cells (data not shown). Polymerase chain reaction analysis also detected no cGK I mRNA in pure HUVECs uncontaminated by fibroblasts, which do contain cGK I (data not shown).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Adenovirus-mediated expression of cGK I and demonstration of cGK activity, cGMP-dependent phosphorylation of endogenous VASP, in intact HUVECs. A, HUVECs were infected with increasing concentrations (109 to 1011 particles/ml) of an adenoviral vector containing cGK I (Ad5-cGK I). One day later, cells were harvested, and homogenates (20 µg of protein each) were analyzed on Western blots using a specific antibody against cGK I as described under "Experimental Procedures." The first lane contained 3 ng of purified cGK I standard (St). B, subconfluent HUVECs were either uninfected or infected with equal amounts (5 × 109 particles/ml) of adenoviral vectors for either wild-type (wt) cGK I or a catalytically inactive mutant of cGK I (cGK I-K405A). After 1 day of infection, cells were incubated in the absence and presence of either 3 µM forskolin for 10 min or 100 µM 8-pCPT-cGMP for 5 min. Cell homogenates (20 µg of protein each) were analyzed on Western blots labeled with specific antibodies recognizing total VASP and VASP phosphorylation of Ser157 as a shift in VASP apparent molecular mass from 46 to 50 kDa (IE273 antibody; first panel), VASP phosphorylated on Ser157 (5C6 antibody; second panel), VASP phosphorylated on Ser239 (16C2 antibody; third panel), or cGK I (fourth panel). Uninfected HUVECs contained no detectable endogenous cGK I (fourth panel, first and second lanes), and VASP phosphorylation on Ser157 was detected in these cells only in response to forskolin (second lanes), but not 8-pCPT-cGMP (data not shown). In adenoviral vector infection studies of HUVECs, the control of catalytically inactive cGK I-K405A (third and fourth lanes) gave no VASP phosphorylation of Ser157 (first two panels) or Ser239 (third panel) in response to 8-pCPT-cGMP (fourth lanes). In contrast, expressed wild-type cGK I showed some basal activity (fifth lanes) that was further stimulated by 8-pCPT-cGMP (sixth lanes) with regard to both Ser157 (first two panels) and Ser239 (third panel) phosphorylation. Phosphorylation of VASP on Ser239, either without (46-kDa band) or with (50-kDa band) concomitant phosphorylation of Ser157, was detected by the 16C2 antibody. Shown are examples of experiments performed at least three times.

Mutant cGK I-K405A was not only itself catalytically inactive, but was also capable of inhibiting (Fig. 2B, third through sixth lanes) wild-type cGK I activity (second lane), as demonstrated in passage 8 human dermal fibroblasts containing endogenous cGK I (cGK I observed upon longer film exposure than that shown in Fig. 2A). VASP phosphorylation by 8-pCPT-cGMP-stimulated endogenous cGK I was completely inhibited by 5 × 10¹⁰ particles of mutant cGK I-containing adenoviral vector/ml, which produced a severalfold overexpression of the mutant kinase compared with the endogenous kinase. The mutated Lys⁴⁰⁵ is the homolog of the essential Lys⁷² in the ATP-binding site of cAK that is conserved in all protein kinases (39). The mutant's inhibitory effect on wild-type cGK I function could be abolished by raising the concentration of 8-pCPT-cGMP from 100 to 500 µM (data not shown), suggesting that the inactive mutant competed with wild-type cGK I for the cGMP analog. In contrast, VASP phosphorylation in fibroblasts induced by 10 µM prostaglandin E₁, an agonist of endogenous cAK, was not inhibited by mutant cGK I (data not shown).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Catalytically inactive cGK I-K405A mutant inhibits endogenous cGK I phosphorylation of VASP in human dermal fibroblasts. Fibroblasts were infected with increasing concentrations (1-50 × 109 particles/ml) of adenoviral vector for mutant cGK I-K405A and 1 day later were incubated either with 100 µM 8-pCPT-cGMP (+) or with vehicle alone (). Cell homogenates (20 µg of protein) were analyzed on Western blots labeled with specific antibodies for either cGK I (A) or VASP phosphorylated on Ser239 (16C2 antibody) (B). Uninfected fibroblasts (first two lanes) contain endogenous cGK I (not visible on the cGK I blot shown because of short film exposure), which phosphorylates endogenous VASP after stimulation by 8-pCPT-cGMP (B, second lane). Infection of fibroblasts with increasing amounts of adenoviral vector for mutant cGK I-K405A led to a dose-dependent increase in mutant cGK I expression (A, third through sixth lanes) and a concurrent decrease in VASP phosphorylation by endogenous wild-type cGK I (B). Examples of experiments performed three times are shown.

Early Detachment of VASP from Focal Adhesions after cGK I Activation in HUVECs-- In cultured HUVECs, VASP is localized at focal adhesions, microfilaments, and cell-cell contacts (Fig. 3, A, C, and E) (5). In our present studies, VASP localization was studied by indirect immunofluorescence in HUVECs infected with the adenoviral vector for cGK I (Fig. 3, C-F). Cells were infected with 10⁹ particles of viral vector for cGK I/ml and 24 h later were treated with 100 µM 8-pCPT-cGMP for 30 min to activate the kinase, the procedure that produced maximal VASP phosphorylation (blot in Fig. 4). Subsequently, cells were immediately fixed for immunofluorescence. Double labeling with antibodies against cGK I and VASP demonstrated that VASP was no longer detectable at focal adhesions (Fig. 3C) in any cGK I-expressing HUVECs (bright cells in Fig. 3D) in nonconfluent monolayers. Instead, a diffuse VASP signal was observed in the cytosol, and some VASP staining was still detected along stress fibers, although it did not resemble the normal periodic pattern. Whereas complete loss of VASP from focal adhesions occurred at 30 min, a reduction in VASP at focal adhesions was observed already after 5 min of 30 µM 8-pCPT-cGMP treatment, the earliest time point and lowest cGMP analog concentration at which maximal phosphorylation of VASP was detectable by immunoblotting (data not shown). This early loss appeared as a marked general reduction in focal adhesion labeling intensity, which progressed at later times to complete loss of labeling. The specificity of the effect of cGK I expression and activation on VASP localization was demonstrated by the lack of any such response to 8-pCPT-cGMP in uninfected cells (Fig. 3A), in cells at the periphery in Fig. 3C corresponding to cells not expressing detectable cGK I (Fig. 3D), or in cells infected with a control (luciferase) adenoviral vector (data not shown) or with an adenoviral vector for the catalytically inactive cGK I-K405A mutant (see Fig. 5A and 6A). Although cGK I activation caused a loss of VASP from focal adhesions in nonconfluent monolayers of HUVECs, it did not alter the predominant VASP localization at cell-cell contacts in confluent monolayers (Fig. 3E).

View larger version (84K): [in this window] [in a new window]   Fig. 3.   Detachment of endogenous VASP from focal adhesions in 8-pCPT-cGMP-treated HUVECs expressing cGK I. One day after Ad5-cGK I infection (109 particles/ml), cells were stimulated with 100 µM 8-pCPT-cGMP for 30 min and fixed for immunofluorescence. In the double-labeling experiments shown, a monoclonal antibody (IE226) was used to visualize VASP (A, C, and E) in cells either expressing (bright cells) or not expressing (dark cells) cGK I (B, D, and F) detected with polyclonal antiserum A10. A and B, uninfected cells, C-F, Ad5-cGK I-infected cells. A-D, nonconfluent cells; E and F, confluent cells. The VASP staining of focal adhesions observed in uninfected cells (A) was absent (C) in cGK I-expressing cells (bright cells in D) activated for 30 min in nonconfluent HUVEC cultures. However, at the same time in confluent HUVEC cultures, VASP remained in cell-cell contacts (E) of stimulated cGK I-expressing cells (F). Representative data from at least 10 experiments are shown. Bar = 20 µm.

Loss of Zyxin and Vinculin from Focal Adhesions after cGK I Activation in HUVECs-- To investigate cGK-mediated rearrangement of focal adhesion proteins in more detail, we analyzed the localization of VASP as well as that of the established VASP-binding proteins zyxin and vinculin at different times of cGK activation. cGK was activated either directly with 8-pCPT-cGMP or indirectly with ANP, which elevates intracellular cGMP levels via the ANP receptor guanylyl cyclase. The phosphorylation of VASP in HUVECs expressing cGK I, incubated for 5 min to 3 h with 100 nM ANP or 100 µM 8-pCPT-cGMP, is shown in Fig. 4. More important, no cGMP-dependent VASP phosphorylation was observed in HUVECs expressing the catalytically inactive cGK I-K405A mutant. In contrast to 8-pCPT-cGMP (hydrolysis-resistant) induction of constant maximal VASP phosphorylation for 3 h, ANP-induced VASP phosphorylation declined at 3 h. In parallel experiments, HUVECs were double-labeled using antibodies against either VASP and vinculin or zyxin and vinculin for immunofluorescence analysis. The transfection efficiency was adjusted so that nearly all cells expressed cGK. After 30 min of cGK activation by ANP or 8-pCPT-cGMP, VASP (Figs. 5E and 6E) as well as zyxin (Figs. 7E and 8E) were detached from focal adhesions (quantitation in Fig. 9). Vinculin staining per focal adhesion was diminished after 30 min of cGK activation by either 8-pCPT-cGMP or ANP (Figs. 5F, 6F, 7F, and 8F), i.e. the size of vinculin-containing focal adhesions often appeared reduced, although the total number of cells exhibiting vinculin staining was not greatly changed at 30 min (Fig. 9). However, 3 h of 8-pCPT-cGMP treatment caused depletion of all three proteins (VASP, zyxin, and vinculin) from focal adhesions (Figs. 6, G-H; 8, G-H; and 9) and, at the same time, a general loss of fluorescein isothiocyanate-phalloidin staining of the microfilament system (data not shown). In contrast, 3 h of ANP treatment of HUVECs resulted in some VASP and zyxin relocalization to focal adhesions, and the size of vinculin-staining focal adhesions increased again (Figs. 5, G-H; 7, G-H; and 9). This recovery corresponds to the decline in ANP-stimulated cGK-dependent VASP phosphorylation (Fig. 4, upper panel). Thus, ANP caused a time-dependent and reversible depletion of primarily VASP and zyxin from focal adhesions. The effects of both 8-pCPT-cGMP and ANP were dependent on cGK since HUVECs transfected with the inactive cGK I-K405A mutant did not display any change in VASP phosphorylation (Figs. 1B and 4) or in VASP, zyxin, or vinculin localization in focal adhesions (Figs. 5-8, A and B).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   cGK I mediation of ANP-dependent endogenous VASP phosphorylation in HUVECs. Subconfluent HUVECs infected with equal amounts (5 × 109 particles/ml) of either the catalytically inactive cGK I-K405A mutant or wild-type (wt) cGK I were incubated 1 day after infection for the indicated times with either 100 nM ANP or 100 µM 8-pCPT-cGMP (cGMP). Cell homogenates (20 µg of protein) were analyzed on Western blots using a monoclonal antibody (16C2) specific for VASP phosphorylated on Ser239 (upper panel) and, subsequently after stripping, were analyzed with a polyclonal antibody specific for cGK I (lower panel). Similar amounts of cGK were expressed in all samples. Weak phosphorylation of VASP on Ser239 was detected already in untreated cells expressing cGK I (upper panel, fourth lane). Treatment with either ANP or 8-pCPT-cGMP strongly enhanced VASP Ser239 phosphorylation and additionally Ser157 phosphorylation (VASP shift to 50 kDa) in cells expressing wild-type cGK I, but not catalytically inactive cGK I (K405A mutant). Data like that shown were obtained in three experiments.

View larger version (79K): [in this window] [in a new window]   Fig. 5.   ANP- and cGK I-dependent detachment of endogenous VASP and vinculin from focal adhesions in HUVECs. Subconfluent HUVECs infected with equal amounts (5 × 109 particles/ml) of either catalytically inactive cGK I-K405A mutant (A and B) or wild-type (wt) cGK I (C-H) were treated 1 day after infection without (C and D) or with 100 nM ANP for either 30 min (A, B, E, and F) or 3 h (G and H). In double-labeling experiments, VASP (polyclonal antibody M4; A, C, E, and G) was stained together with vinculin (monoclonal antibody hVIN-1; B, D, F, and H). ANP-stimulated cells expressing the catalytically inactive cGK I-K405A mutant (A and B) and unstimulated cells expressing wild-type cGK I (C and D) displayed essentially the same VASP localization observed in untransfected cells (compare with Fig. 3A). In contrast, 30 min of ANP stimulation of cGK I-expressing cells caused loss of VASP from focal adhesions and reduced the size of vinculin-containing focal adhesions (but not the percent of vinculin-expressing cells) (E and F), which was reversible at 3 h (G and H). See quantitation of data from three experiments in Fig. 9. Bar = 20 µm.

View larger version (80K): [in this window] [in a new window]   Fig. 6.   8-pCPT-cGMP stimulation of cGK I-dependent detachment of endogenous VASP and vinculin from focal adhesions in HUVECs. HUVECs were infected as described in the legend to Fig. 5; and 1 day after infection, cells were incubated without (C and D) or with 100 µM 8-pCPT-cGMP (cGMP) for either 30 min (A, B, E, and F) or 3 h (G and H). In double-labeling experiments, VASP (polyclonal antibody M4; A, C, E, and G) was stained together with vinculin (monoclonal antibody hVIN-1; B, D, F, and H). 8-pCPT-cGMP-stimulated cells expressing the catalytically inactive cGK I-K405A mutant (A and B) and unstimulated cells expressing wild-type (wt) cGK I (C and D) displayed essentially the same VASP localization observed in untransfected cells (compare with Fig. 3A). As observed for ANP in Fig. 5, 8-pCPT-cGMP stimulation of cGK I for 30 min caused loss of VASP from focal adhesions, but additionally reduced the percent of cells expressing vinculin in focal adhesions (E and F). However, a complete loss of both VASP and vinculin labeling was observed after 3 h (G and H). See quantitation of data from three experiments in Fig. 9. Bar = 20 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 7.   ANP- and cGK I-dependent detachment of endogenous zyxin and vinculin from focal adhesions in HUVECs. HUVECs infected and treated with 100 nM ANP as described in the legend to Fig. 5 were double-labeled for zyxin (polyclonal antibody AS83-1; A, C, E, and G) and vinculin (monoclonal antibody hVIN-1; B, D, F, and H). ANP-stimulated cells expressing the catalytically inactive cGK I-K405A mutant (A and B) or unstimulated cells expressing wild-type (wt) cGK I (C and D) demonstrated no changes in the localization of zyxin and vinculin in comparison with uninfected cells (not shown). ANP stimulation (30 min) of cGK I-expressing cells (E and F) nearly abolished zyxin labeling at focal adhesions and reduced the size of vinculin-containing focal adhesions (but not the percent of vinculin-expressing cells). These cellular changes reversed after 3 h of ANP treatment (G and H). See quantitation of data from three experiments in Fig. 9. Bar = 20 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 8.   8-pCPT-cGMP stimulation of cGK I-dependent detachment of endogenous zyxin and vinculin from focal adhesions in HUVECs. HUVECs were infected and treated with 100 µM 8-pCPT-cGMP (cGMP) as described in the legend to Fig. 6. In double-labeling experiments, zyxin (polyclonal antibody AS83-1; A, C, E, and G) was stained together with vinculin (monoclonal antibody hVIN-1; B, D, F, and H). 8-pCPT-cGMP-stimulated cells expressing the catalytically inactive cGK I-K405A mutant (A and B) or unstimulated cells expressing wild-type (wt) cGK I (C and D) displayed no change in the localization of zyxin and vinculin in comparison with uninfected cells (not shown). After 30 min of 8-pCPT-cGMP stimulation of cells expressing wild-type cGK I (E and F), zyxin labeling at focal adhesions was nearly abolished, and that of vinculin was reduced. In contrast to the results obtained with ANP (see Fig. 7), a complete loss of zyxin and vinculin labeling of focal adhesions was observed after 3 h of 8-pCPT-cGMP treatment (G and H). See quantitation of data from three experiments in Fig. 9. Bar = 20 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 9.   Quantitative evaluation of the effects of ANP and 8-pCPT-cGMP on cGK I-dependent detachment of endogenous VASP, zyxin, and vinculin from focal adhesions in HUVECs. Subconfluent HUVECs infected with Ad5-cGK I or the catalytically inactive Ad5-cGK I-K405A mutant were either untreated () or treated with 100 nM ANP or 100 µM 8-pCPT-cGMP (cGMP) for 30 min or 3 h and subsequently analyzed by immunofluorescence as described in the legends to Figs. 5-8. Shown is the percent of cells demonstrating VASP, zyxin, or vinculin at focal adhesions. In cGK I-expressing cells, VASP (upper panel) and zyxin (middle panel) demonstrated a similar time-dependent pattern of detachment from focal adhesions, i.e. notable detachment in >60% of cGK I-expressing cells after 30 min of ANP or 8-pCPT-cGMP treatment, with evidence of reversal of the ANP effect, but enhancement (90% detachment) of the 8-pCPT-cGMP effect after 3 h. In contrast, ANP treatment did not decrease the percent of cells showing vinculin labeling (lower panel) at focal adhesions, whereas 8-pCPT-cGMP did, although with a delayed time course (major decrease at 3 h) in comparison with VASP and zyxin labeling (major decrease at 30 min). No reduction in VASP, zyxin, or vinculin labeling of focal adhesions resulted from stimulation of cells expressing the cGK I-K405A mutant. Data represent means ± S.E. of three independent experiments, in each of which 100 cells were analyzed. wt, wild-type.

Dependence of VASP Intracellular Redistribution on VASP Phosphorylation by cGK I-- The role of cGK I-dependent VASP phosphorylation in the observed redistribution of VASP was examined using VASP phosphorylation mutants in which Ser¹⁵⁷, Ser²³⁹, and Thr²⁷⁸ were replaced by Ala, either singly or by double mutation of both serines (AAT) or by triple mutation of all three phosphorylation sites (AAA). To distinguish both transfected wild-type and mutant VASPs from endogenous VASP present in HUVECs, the former were expressed as VSV epitope-tagged fusion proteins and detected using an antibody directed against VSV in both Western blotting and immunofluorescence.

Efficacious VASP phosphorylation site mutation was demonstrated by expressing wild-type or mutant VASP in HEK 293 cells, immunoprecipitating them, and determining their ability to be phosphorylated by purified cGK I or cAK in vitro using [-³²P]ATP. The anti-VSV antibody detected essentially equal amounts of all VSV-VASP forms on Western blots of the phosphorylated samples (Fig. 10A); however, phosphate incorporation (Fig. 10B) into the double mutant (AAT, Ser¹⁵⁷ and Ser²³⁹ both changed to Ala) was severely impaired, and no significant phosphate incorporation into the triple mutant protein (AAA) was observed.

View larger version (22K): [in this window] [in a new window]   Fig. 10.   VSV-tagged VASP phosphorylation site mutants (Ser/Thr to Ala) expressed in and immunoprecipitated from HEK 293 cells demonstrate defective phosphorylation by cAMP- and cGMP-dependent protein kinases in vitro. Plasmid vectors (1 µg) for wild-type VASP (wt), for VASP mutated at phosphorylation sites Ser157 and Ser239 (AAT), and for VASP mutated at all three phosphorylation sites including Thr278 (AAA) were transfected as VSV-tagged constructs into HEK 293 cells. Two days later, VASP proteins were immunoprecipitated and incubated for 30 min either with 50 ng of purified cGK I (G) plus 20 µM cGMP or with 50 ng of purified cAK catalytic subunit (A) in the presence of [-32P]ATP. A, samples were then analyzed for VASP expression on Western blots using anti-VSV antibodies and ECL detection. B, subsequently, the nitrocellulose blot was stripped of ECL labeling and re-exposed to film for detection of [32P]phosphate incorporation. A standard of non-phosphorylated VSV-tagged wild-type VASP (St) is shown in the first lanes in A and B. Only wild-type VASP and, to a minor extent, the mutant retaining one intact phosphorylation site (AAT), but not the triple mutant (AAA), were phosphorylated by cGK and cAK (B). Also, only wild-type VASP (A, second and third lanes), not the AAT or AAA mutant, demonstrated an upward shift in migration (from 46 to 50 kDa), a previously described indicator of VASP Ser157 phosphorylation. Data like those shown were obtained in three experiments.

Cotransfection of plasmids of cGK I and VSV-tagged wild-type VASP into HUVECs, followed by 30 min of 8-pCPT-cGMP stimulation of kinase, led to detachment of VASP from focal adhesions (Fig. 11C). In contrast to the previously described experiments using adenoviral vectors, in which virtually all cGK I-expressing cells demonstrated cGMP-dependent VASP loss from focal adhesions under the same conditions used here, experiments using plasmid vectors demonstrated that only ~43% of cGK I-expressing cells lost VSV-tagged wild-type VASP from focal adhesions. However, a similar pattern of VASP relocalization was observed as in the adenoviral vector experiments, i.e. a diffuse VASP staining in the cytosol sometimes appearing as micro-aggregates, with residual stress fiber staining devoid of periodic character. Mutant VASP could localize to focal adhesions (Fig. 11E), indicating that this basic property was not disturbed by the mutations. However, after stimulation of cotransfected cGK I, the triple phosphorylation mutant of VASP was more resistant to detachment from focal adhesions (lost from only 11 ± 7% of cGK I-expressing cells) than was wild-type VASP (lost from 43 ± 9% of cells) (Fig. 11, G and C, respectively; percentages in text represent means ± S.E. of four independent experiments in which 50-100 cells were analyzed per experiment). The detachment of the double mutant of VASP from focal adhesions was not observably different from that of wild-type VASP in response to 8-pCPT-cGMP (data not shown).

View larger version (74K): [in this window] [in a new window]   Fig. 11.   Impaired detachment of the VSV-VASP triple phosphorylation (AAA) mutant (Ser/Thr to Ala) from focal adhesions in response to treatment of HUVECs with 8-pCPT-cGMP. HUVECs were cotransfected with plasmids (1 µg) containing cDNA for cGK I and either VSV-tagged wild-type (wt) VASP (A-D) or VSV-VASP-AAA containing mutations of all three phosphorylation sites (E-H). One day after transfection, cells were incubated without (A, B, E, and F) or with (C, D, G, and H) 100 µM 8-pCPT-cGMP for 30 min and fixed for immunofluorescence. In double-labeling experiments, anti-cGK I (B, D, F, and H) and anti-VSV (A, C, E, and G; to label VASP) antibodies were used. Both wild-type (A) and mutant (E) VASP-AAA localized to focal adhesions in untreated cells coexpressing cGK I (B and F). VSV-tagged wild-type VASP disappeared from focal adhesions (C) in 43 ± 9% of cells coexpressing cGK I and stimulated with 8-pCPT-cGMP (D). In contrast, the triple phosphorylation mutant VSV-VASP-AAA (G) was more stably attached to focal adhesions in ~90% of cGK I-expressing, 8-pCPT-cGMP-stimulated cells (H); only 11 ± 7% of cells lost VSV-VASP-AAA in response to stimulation of cGK I (not shown). Data represent means ± S.E. of four independent experiments, in each of which 50-100 cells were analyzed. Bar = 20 µm.

Inhibition of HUVEC Migration by cGK I-- The functional consequence of cGK I activation on HUVEC migration was examined. In a haptotactic migration assay, HUVEC migration on fibronectin (Fig. 12A) as well as on collagen and fibrinogen matrices (Fig. 12B) was inhibited by expression and activation of cGK I, but not the catalytically inactive cGK I-K405A mutant. This was the case when the expressed kinase was activated either directly with 100 µM 8-pCPT-cGMP or indirectly with 500 nM ANP. The inhibitory effect of ANP in the 5-h migration assay was slightly weaker than that of 8-pCPT-cGMP, perhaps reflecting the observed decline in ANP activation of cGK after 3 h (Fig. 4). Our studies demonstrate for the first time in endothelial cells that cGK, and not cAK, mediates cGMP-dependent inhibition of migration since the catalytically inactive cGK I-K405A mutant did not inhibit migration. Collagen and fibrinogen are known to activate the migration of HUVECs via integrins ₂₁ and ₅₁, respectively (40, 41). Fibronectin binds to integrins ₅₁, _v3, and ₄₁ on endothelial cells (42). Thus, cGK I inhibition of haptotactic cell migration was independent of the integrins involved.

View larger version (28K): [in this window] [in a new window]   Fig. 12.   Inhibition of HUVEC migration by cGK I expression from adenoviral vectors. HUVECs were uninfected (control) or infected with equal amounts (5 × 109 particles/ml) of Ad5-cGK I or the catalytically inactive Ad5-cGK I-K405A mutant. One day later, HUVECs were analyzed in migration assays using modified Boyden chambers. Shown is the migration in response to the following extracellular matrix proteins: A, fibronectin; B, collagen (left) and fibrinogen (right). In the presence of either 100 µM 8-pCPT-cGMP (black bars) or 500 nM ANP (hatched bars) to activate cGK I, HUVEC migration was ~1.5-2-fold less than that observed for untreated cells (white bars). In contrast, the migration of HUVECs expressing mutant cGK I-K405A was not significantly inhibited in response to 8-pCPT-cGMP. Migration of control uninfected cells (A) in the absence of 8-pCPT-cGMP or ANP (open bar) was designated as 100% for normalizing data for cell migration in response to fibronectin (similar control data for collagen and fibrinogen are not shown). Each bar represents the mean ± S.E. of three individual experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression and activation of cGK I (but not a catalytically inactive cGK I-K405A mutant) in primary cultures of HUVECs resulted in depletion of several cytoskeletal proteins from focal adhesions, beginning with VASP and zyxin within 30 min and later vinculin, followed by microfilament disruption, thus causing a destabilization of motility structures that may contribute to the observed cGK I-dependent inhibition of HUVEC migration. Of the cytoskeletal proteins affected, only VASP is a known cGK I substrate, suggesting that perhaps VASP phosphorylation leads to the subsequent chain of events. In the past, few effects of VASP phosphorylation have been reported. The localization of the Ser¹⁵⁷ phosphorylation site in the central domain of VASP, in close proximity to the binding site for profilin, suggested an influence of VASP phosphorylation on VASP/profilin interaction; however, in vitro binding of VASP to matrix-bound profilin was not grossly altered by VASP phosphorylation (22). Recently, only VASP phosphorylation on Ser¹⁵⁷ was analyzed and suggested to enhance VASP affinity for F-actin in vitro (20); however, the contribution of the other VASP phosphorylation sites was not investigated.

In this study, all three VASP phosphorylation sites were investigated in intact cells using a combination of site-specific antibodies and site-specific mutation. In particular, VSV-tagged VASP mutants that could be expressed and distinguished from endogenous VASP were used to demonstrate that direct VASP phosphorylation was required for VASP detachment from focal adhesions since the VSV-tagged VASP-AAA triple phosphorylation mutant was highly resistant to 8-pCPT-cGMP-dependent detachment from focal adhesions in comparison with wild-type VASP. However, in the absence of 8-pCPT-cGMP, all phosphorylation site mutants of VASP were targeted to focal adhesions and showed a periodic distribution along stress fibers, suggesting that the mutations did not induce any major changes in the tertiary structure of VASP. VASP detachment from focal adhesions was inhibited only if all three phosphorylation sites were mutated, suggesting that all three sites are important for regulation of VASP interaction with cytoskeletal components. Ser¹⁵⁷ is located just distal to the VASP EVH1 domain, and Ser²³⁹ and Thr²⁷⁸ are located in the VASP EVH2 domain (12, 23). Specific functions ascribed to these domains are consistent with our results that point mutations of phosphorylation sites close to and within these domains affect VASP localization at focal adhesions. VASP truncation and other experiments have shown that both the EVH1 and EVH2 domains are involved in VASP localization at focal adhesions and that the EVH2 domain affects the function and binding properties of the EVH1 domain (discussed in Ref. 23). Additional data clearly demonstrated that both EVH1 and EVH2 domains of Ena/VASP family members are essential but alone not sufficient for the overall function of these proteins (43). Furthermore, during the review process for our manuscript, another publication appeared demonstrating that expression of the EVH2 domain in keratinocytes inhibited VASP function in adhesion zipper and epithelial sheet formation; and in keratinocytes of transgenic mice overexpressing the EVH2 domain, abnormalities in intercellular adhesion were observed (44). Thus, it is highly conceivable that Ser²⁷⁸ phosphorylation, in combination with Ser¹⁵⁷ and Ser²³⁹ phosphorylation, could affect important VASP functions, ultimately resulting in a major effect like VASP displacement from focal adhesions.

The exact role of each individual VASP phosphorylation site in either VASP loss from or recovery to focal adhesions is not clear. It was noted, however, that after 3 h of ANP treatment, VASP localization at focal adhesions began to recover concomitant with a relative loss of Ser¹⁵⁷ phosphorylation. This was not observed after 3 h of 8-pCPT-cGMP treatment, most likely due to the greater metabolic stability of this cGMP analog in comparison with unmodified cGMP formed in response to ANP. It is possible that incomplete VASP phosphorylation is insufficient for initiating VASP removal from focal adhesions and perhaps sufficient for its recovery. All available data support the interpretation that all three VASP phosphorylation sites are critical for the interaction of VASP with focal adhesion components. This complexity is perhaps not surprising since VASP interacts via its EVH1 and EVH2 domains with at least two distinct components present in focal adhesions, FP₄ motif-containing proteins (e.g. zyxin and vinculin) and F-actin, as discussed below.

In comparison with VASP, VASP-related proteins show conservation of only Ser¹⁵⁷ and Ser²³⁹ (Mena), only Ser¹⁵⁷ (Evl), or no sites (Ena), suggesting that VASP homologs may display different regulation in response to phosphorylation. Phosphorylation of these sites has not been extensively studied, but a cAK-induced shift in Mena migration in SDS-polyacrylamide gel electrophoresis has been shown (14), and our 16C2 antibody detects phosphorylation of Mena, probably at Ser³⁷⁶ homologous to VASP Ser²³⁹.³ Interestingly, tyrosine phosphorylation of Ena on other sites by Abl tyrosine kinase attenuates Ena association with profilin or SH3 domain-containing proteins, and mutation of these phosphorylation sites partially impairs the in vivo function of Ena, indicating that phosphorylation is required for optimal Ena function (45).

Phosphorylation state has also been shown to regulate focal adhesion localization of other proteins. Paxillin dephosphorylation on tyrosine resulted in its loss from focal adhesions in response to cAMP (46), whereas serine and threonine phosphorylation has been shown to accelerate fibronectin-induced localization of paxillin to focal adhesions (47). In contrast, serine phosphorylation of talin in response to interleukin-1 decreased talin's focal adhesion localization (48), and phorbol ester stimulation of protein kinase C increased talin serine phosphorylation and talin removal from focal adhesions (49). Thus, cGK I regulation of focal adhesion constituents exemplifies "inside-out" regulation of adhesion sites by yet a new signal transduction pathway.

Our results in intact cells provide a physiological correlate to the previous demonstrations of in vitro interactions between VASP and either zyxin or vinculin (26, 32). These interactions were demonstrated using one solid phase (nitrocellulose, column, or microtiter plate)-bound partner to bind another from solution. More recently, VASP and vinculin have also been shown to co-immunoprecipitate after in situ cross-linking experiments (50). The direct binding partner that attaches VASP to focal adhesions is unclear, although vinculin and zyxin are likely candidates since they have been shown to directly interact, via their FP₄ motifs, with the N-terminal EVH1 domain of proteins of the Ena/VASP protein family (15, 26, 32), and injection of peptides containing the FP₄ motif into human fibroblasts and HeLa cells caused a depletion of VASP from focal adhesions, resulting in a diffuse cytosolic distribution of VASP (15, 17). Although the role of the Ena/VASP family EVH2 domain is not entirely clear, it does appear to regulate tetramerization, which is important for EVH1 domain interactions with other proteins, including ones present at focal adhesions (23). Detachment of VASP and zyxin from focal adhesions may also have additional cellular consequences since zyxin has been shown to shuttle between focal contacts and the nucleus in fibroblasts (51).

Prolonged effects of 8-pCPT-cGMP treatment were also observed after VASP phosphorylation and disappearance from focal adhesions. A progressive reduction of the actin microfilament system and vinculin at focal adhesions was observed, which was complete after 3 h of 8-pCPT-cGMP treatment. VASP influence on the microfilament system may be related to the role shown for VASP in enhancing actin assembly required for Listeria motility (15-21). FP₄ domain-containing proteins were also implicated in bacterial motility since motility was blocked by peptides containing the FP₄ motif (52). In mammalian aortic smooth muscle and endothelial cells, disassembly of focal adhesions induced by thrombospondin and tenascin has recently been suggested to be mediated by cGK I, although GMP-dependent protein kinase I alone had no effect (53). In our studies, cGK I stimulation resulted in loss of proteins from focal adhesions, and this was not mimicked by a catalytically inactive cGK I-K405A mutant. This mutant is not only itself inactive, but could also compete with wild-type cGK I for the cGMP analog. Although it was not clear whether mutant and wild-type cGKs I could form heterodimers, this did not seem to be the case since a large ratio of mutant to wild-type cGK I was required for inhibition of wild-type cGK I, and inhibition could be overcome by increasing the cGMP analog concentration. Furthermore, a heterodimer holoenyzme might still display activity since even cGK I monomers demonstrate catalytic activity (54).

cGK I effects on focal adhesion integrity may also underlie the observation that cGK I and VASP have been implicated in inhibition of fibrinogen binding to its integrin receptor, _IIb3. (7-9). The time course and concentration of cGMP that caused VASP phosphorylation were very similar to those that inhibited fibrinogen binding. However, VASP phosphorylation may be only one of the effects of cGK I on the cytoskeletal system. cGK I in platelets is known to inhibit phospholipase C and myosin light chain (MLC) phosphorylation (10, 55), which are suggested to be involved in tension- and stress fiber-mediated focal adhesion assembly (56, 57). Inhibition of MLC phosphorylation is unlikely to be mediated by cGK I phosphorylation of MLC kinase since this did not affect MLC kinase activity (58). Alternatively, a role for cGK in MLC phosphatase activation has been discussed (59-61). Another potential target of cGK could be the small GTP-binding protein RhoA, which has been shown to mediate the thrombin-induced increase in MLC phosphorylation in endothelial cells (62). RhoA can be inhibited by phosphorylation through cAK, which correlates with an inhibition of lymphocyte motility by cAMP (63).

The regulation of focal adhesions is intimately involved in the process of cell migration, which is a dynamic process involving polarized formation and disassembly of focal adhesions at opposite cell ends (64, 65). Lack of focal adhesions and stress fibers in Chinese hamster ovary cells expressing a mutant integrin resulted in decreased migration in haptotactic assays like that used here, although the cells were motile in a random migration assay (65). However, others have reported that decreasing vinculin and -actinin via antisense RNA increased 3T3 cell motility in random migration assays (66, 67). In our experiments on haptotaxis, 8-pCPT-cGMP-dependent vinculin loss was observed at the time of inhibition of HUVEC migration. The cGK I-dependent reorganization of focal adhesion proteins and ultimate destabilization of the microfilament system that we observed may facilitate cGK I-dependent inhibition of cell migration. This inhibition was independent of which extracellular matrix or integrins were involved. In agreement with our observations, Ikeda et al. (68) demonstrated that migration of rat aorta endothelial cells was inhibited by 8-Br-cGMP as well as by natriuretic peptides that elevate cGMP. However, focal adhesions and the presence of endogenous cGK I were not investigated. The migration of smooth muscle cells stably transfected with cGK I was also inhibited by cGMP analogs (69). Furthermore, overexpression of endothelial nitric-oxide synthase in balloon-injured carotid arteries resulted in impaired endothelial regeneration (70), consistent with possible nitric oxide (or cGMP)-dependent inhibition of endothelial cell proliferation and migration.

Collectively, our data demonstrate that cGMP has effects on VASP phosphorylation and localization and on cell migration that are mediated by cGK and not cAK. Use of a combination of VASP phosphorylation site-specific antibodies and mutations demonstrated the importance of cGK I-dependent VASP phosphorylation in inhibiting VASP localization at focal adhesions. Continued studies are necessary to more closely define the far-reaching consequences of cGK I-dependent inhibition of focal adhesion integrity and cellular processes that depend on them.

	ACKNOWLEDGEMENTS

We thank the staff of the Theresienklinik for providing umbilical cords; Susanne Stumpf, Petra Thalheimer, and Petra Hönig-Liedl for expert technical assistance; Christiane Bachmann for single site VASP mutants; Matthias Reinhard for valuable discussions and advice; and Martin Eigenthaler for an introduction to the migration assay.

	FOOTNOTES

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 355/B4.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Cardiology and Pneumonology, University Hospital Benjamin Franklin, Free University Berlin, 12200 Berlin, Germany.

^§ To whom correspondence should be addressed. Tel.: 49-931-201-3457; Fax: 49-931-201-3153; E-mail: slohmann@klin-biochem.uni-wuerzburg.de.

Published, JBC Papers in Press, June 12, 2000, DOI 10.1074/jbc.M909632199

² U. Walter and H. Hoschuetsky, manuscript in preparation.

³ A. Smolenski, unpublished data.

	ABBREVIATIONS

The abbreviations used are: ANP, atrial natriuretic peptide; cGK, cGMP-dependent protein kinase; cAK, cAMP-dependent protein kinase; VASP, vasodilator-stimulated phosphoprotein; EVH, Ena/VASP homology; HUVEC, human umbilical vein endothelial cell; HEK, human embryonic kidney; VSV, vesicular stomatitis virus; Ad5, adenovirus type 5; 8-pCPT-cGMP, 8-(4-chlorophenylthio)guanosine 3':5'-monophosphate; PBS, phosphate-buffered saline; MLC, myosin light chain.

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