Title: Phosphorylation of the Vasodilator-stimulated Phosphoprotein Regulates Its Interaction with Actin
Abstract: The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases in platelets and other cardiovascular cells. It promotes actin nucleation and binds to actin filaments in vitro and associates with stress fibers in cells. The VASP-actin interaction is salt-sensitive, arguing for electrostatic interactions. Hence, phosphorylation may significantly alter the actin binding properties of VASP. This hypothesis was investigated by analyzing complex formation of recombinant murine VASP with actin after phosphorylation with cAMP-dependent kinase in different assays. cAMP-dependent kinase phosphorylation had a negative effect on both actin nucleation and VASP interaction with actin filaments, with the actin nucleating capacity being more affected than actin filament binding and bundling. Replacing VASP residues known to be phosphorylated in vivo by acidic residues to mimic phosphorylation had similar although less dramatic effects on VASP-actin interactions. In contrast, phosphorylation had no significant effect on VASP oligomerization or its interaction with its known ligands profilin, vinculin, and zyxin. When overexpressing VASP mutants in eukaryotic cells, they all showed targeting to focal contacts and stress fibers. Our results imply that VASP phosphorylation may act as an immediate negative regulator of actin dynamics. The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases in platelets and other cardiovascular cells. It promotes actin nucleation and binds to actin filaments in vitro and associates with stress fibers in cells. The VASP-actin interaction is salt-sensitive, arguing for electrostatic interactions. Hence, phosphorylation may significantly alter the actin binding properties of VASP. This hypothesis was investigated by analyzing complex formation of recombinant murine VASP with actin after phosphorylation with cAMP-dependent kinase in different assays. cAMP-dependent kinase phosphorylation had a negative effect on both actin nucleation and VASP interaction with actin filaments, with the actin nucleating capacity being more affected than actin filament binding and bundling. Replacing VASP residues known to be phosphorylated in vivo by acidic residues to mimic phosphorylation had similar although less dramatic effects on VASP-actin interactions. In contrast, phosphorylation had no significant effect on VASP oligomerization or its interaction with its known ligands profilin, vinculin, and zyxin. When overexpressing VASP mutants in eukaryotic cells, they all showed targeting to focal contacts and stress fibers. Our results imply that VASP phosphorylation may act as an immediate negative regulator of actin dynamics. vasodilator-stimulated phosphoprotein cAMP-dependent kinase cGMP-dependent kinase Ena-VASP homology domain wild type VASP birch profilin sequence tag phosphate-buffered saline polyacrylamide gel electrophoresis enhanced green fluorescent protein enzyme-linked immunosorbent assay Cell morphology and motility critically depend on the remodeling of the cytoskeletal architecture in response to external stimuli. Directional locomotion requires locally confined membrane protrusion driven by actin polymerization, resulting in the formation of a leading edge. Adhesion to the extracellular matrix is mediated by distinct multi-protein complexes. The formation of these focal adhesions is initiated by the activation of integrin heterodimers, which then recruit a variety of cytoskeletal and signaling molecules (1Critchley D.R. Curr. Opin. Cell Biol. 2000; 12: 133-139Crossref PubMed Scopus (494) Google Scholar). Most of the cytoskeletal components involved, e.g. talin, α-actinin, and vinculin, are multi-ligand proteins. They may function as structural scaffolds for other cytoskeletal and signaling proteins or interact directly with the actin cytoskeleton. Given the complexity of focal adhesions, actin dynamics at these sites is not completely understood, and the final integration of integrin-mediated signaling with de novo actin polymerization remains to be elucidated. Several lines of evidence have implicated the vasodilator-stimulated phosphoprotein (VASP)1 to be involved in the regulation of filament assembly and organization. VASP was originally purified from human platelets (2Halbrugge M. Walter U. Eur. J. Biochem. 1989; 185: 41-50Crossref PubMed Scopus (114) Google Scholar). It belongs to a protein family including the Drosophila protein Enabled (Ena), its mammalian homologue Mena, and the Ena-VASP-like protein (Evl) (3Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). They all share a common domain structure comprising a central proline-rich core flanked by two highly conserved Ena-VASP homology domains (EVH1 and EVH2; Fig. 1and Ref. 3Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). Ena/VASP proteins target to the leading edge and focal adhesions in fibroblasts (3Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar, 4Reinhard M. Halbrugge M. Scheer U. Wiegand C. Jockusch B.M. Walter U. EMBO J. 1992; 11: 2063-2070Crossref PubMed Scopus (289) Google Scholar), which is mediated by the EVH1 domain recognizing the consensus motif (D/E)FPPPPXD (5Niebuhr K. Ebel F. Frank R. Reinhard M. Domann E. Carl U.D. Walter U. Gertler F.B. Wehland J. Chakraborty T. EMBO J. 1997; 16: 5433-5444Crossref PubMed Scopus (332) Google Scholar, 6Carl U.D. Pollmann M. Orr E. Gertler F.B. Chakraborty T. Wehland J. Curr. Biol. 1999; 9: 715-718Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). This motif is present in several VASP ligands, including zyxin and vinculin. The central region of Ena/VASP proteins harbors proline-rich stretches that are recognized by the G-actin-binding protein profilin (3Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar, 7Reinhard M. Giehl K. Abel K. Haffner C. Jarchau T. Hoppe V. Jockusch B.M. Walter U. EMBO J. 1995; 14: 1583-1589Crossref PubMed Scopus (416) Google Scholar). VASP oligomerization and F-actin binding are confined to the C-terminal EVH2 domain (3Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar, 8Huttelmaier S. Harbeck B. Steffens O. Messerschmidt T. Illenberger S. Jockusch B.M. FEBS Lett. 1999; 451: 68-74Crossref PubMed Scopus (104) Google Scholar, 9Bachmann C. Fischer L. Walter U. Reinhard M. J. Biol. Chem. 1999; 274: 23549-23557Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). VASP may thus control the actin cytoskeleton by three different mechanisms: (i) it may recruit G-actin via its binding to profilin, (ii) it may stabilize and possibly organize newly formed filaments by direct binding to F-actin, and (iii) oligomerization may potentiate both effects.Figure 1Murine VASP structure, ligand binding, and phosphorylation sites. VASP consists of a central proline-rich domain (PR) that is flanked by two Ena-VASP homology domains (EVH1 and EVH2). The EVH1 domain binds to zyxin, vinculin, and the bacterial surface protein ActA, whereas binding to profilin involves three GP5 motifs (shaded boxes) located in the proline-rich region. VASP oligomerization and F-actin binding is confined to the EVH2 domain. Phosphorylation sites for the cyclic nucleotide-dependent kinases PKA and PKG are located in the proline-rich region (S153) and the EVH2 domain (S235 and T274). The preferences described for each kinase in vitro (11Butt E. Abel K. Krieger M. Palm D. Hoppe V. Hoppe J. Walter U. J. Biol. Chem. 1994; 269: 14509-14517Abstract Full Text PDF PubMed Google Scholar) are indicated by the thickness of the arrows; Ser153 is preferentially phosphorylated by PKA, whereas Ser235 is the preferred phosphorylation site for PKG.View Large Image Figure ViewerDownload Hi-res image Download (PPT) VASP is highly enriched in platelets (10Eigenthaler M. Nolte C. Halbrugge M. Walter U. Eur. J. Biochem. 1992; 205: 471-481Crossref PubMed Scopus (146) Google Scholar), and it is phosphorylated in response to vasodilators and platelet inhibitors, substances that raise intracellular cAMP and cGMP levels. VASP has been shown to be an immediate target for PKA and PKG in vitro and in vivo (11Butt E. Abel K. Krieger M. Palm D. Hoppe V. Hoppe J. Walter U. J. Biol. Chem. 1994; 269: 14509-14517Abstract Full Text PDF PubMed Google Scholar), and its phosphorylation correlates with the inhibition of platelet aggregation (12Horstrup K. Jablonka B. Honig-Liedl P. Just M. Kochsiek K. Walter U. Eur. J. Biochem. 1994; 225: 21-27Crossref PubMed Scopus (220) Google Scholar). These data are further supported by genetic analyses from VASP knockout mice that display enhanced agonist-induced platelet aggregation (13Aszodi A. Pfeifer A. Ahmad M. Glauner M. Zhou X.H. Ny L. Andersson K.E. Kehrel B. Offermanns S. Fassler R. EMBO J. 1999; 18: 37-48Crossref PubMed Scopus (281) Google Scholar, 14Hauser W. Knobeloch K.P. Eigenthaler M. Gambaryan S. Krenn V. Geiger J. Glazova M. Rohde E. Horak I. Walter U. Zimmer M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8120-8125Crossref PubMed Scopus (191) Google Scholar). How platelet inhibition is mediated by VASP is currently unknown, but phosphorylation seems to be a key factor. VASP is phosphorylated in vitro and in intact human platelets at three residues by both PKA and PKG (11Butt E. Abel K. Krieger M. Palm D. Hoppe V. Hoppe J. Walter U. J. Biol. Chem. 1994; 269: 14509-14517Abstract Full Text PDF PubMed Google Scholar), corresponding to residues Ser153, Ser235, and Thr274 in murine VASP (8Huttelmaier S. Harbeck B. Steffens O. Messerschmidt T. Illenberger S. Jockusch B.M. FEBS Lett. 1999; 451: 68-74Crossref PubMed Scopus (104) Google Scholar). All three phosphorylation sites are positioned close to ligand-binding modules (Fig. 1): Ser153 is located N-terminal to the (GP5)3 motif in the proline-rich region that has been shown to bind to profilin (15Lambrechts A. Verschelde J.L. Jonckheere V. Goethals M. Vandekerckhove J. Ampe C. EMBO J. 1997; 16: 484-494Crossref PubMed Scopus (111) Google Scholar). In the EVH2 domain, Ser235 and Thr274 neighbor basic stretches that seem to mediate VASP-actin interactions (8Huttelmaier S. Harbeck B. Steffens O. Messerschmidt T. Illenberger S. Jockusch B.M. FEBS Lett. 1999; 451: 68-74Crossref PubMed Scopus (104) Google Scholar, 9Bachmann C. Fischer L. Walter U. Reinhard M. J. Biol. Chem. 1999; 274: 23549-23557Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Hence the ligand binding properties of VASP may significantly be altered by phosphorylation. So far, VASP-ligand interactions and VASP phosphorylation have mainly been investigated separately, yielding little information about how these two are related. The aim of the present study was to directly analyze the influence of phosphorylation on VASP-ligand complex formation. Recombinant VASP was phosphorylated by PKA in vitro and tested in different assays for actin binding, oligomerization, and the interaction with its known ligands profilin, vinculin and zyxin. These experiments reveal that VASP phosphorylation by PKA diminishes binding to F-actin and even suppresses actin nucleation, whereas oligomerization and ligand binding remain unaffected. Our data lead to a model in which phosphorylation serves as a direct regulatory switch for VASP-mediated actin polymerization at adhesion sites. Cloning of murine VASP and its EVH1 and EVH2 domains has already been reported (8Huttelmaier S. Harbeck B. Steffens O. Messerschmidt T. Illenberger S. Jockusch B.M. FEBS Lett. 1999; 451: 68-74Crossref PubMed Scopus (104) Google Scholar). The constructs comprising either EVH domain and the proline-rich domain (EVH1-P; P-EVH2) were generated accordingly by polymerase chain reaction using full-length VASP as a template. Amplification primers introducedEcoRI and XhoI restriction sites for further cloning into the following vectors: pQE30 (Qiagen, Hilden, Germany) for the generation of recombinant His-tagged proteins in bacteria, pEGFP-C2 (CLONTECH, Palo Alto, CA) for expression of EGFP fusion proteins in eukaryotic cells, and a derivative of pcDNA3 (CLONTECH) bearing a sequence tag derived from birch profilin (BiPro-tag) (16Rudiger M. Jockusch B.M. Rothkegel M. BioTechniques. 1997; 23: 96-97Crossref PubMed Scopus (30) Google Scholar) to yield sequence-tagged proteins for immunoprecipitation experiments from HeLa cells. To mimic phosphorylation, VASP constructs were generated, in which Ser153, Ser235, and Thr274 were replaced by acidic residues. Site-directed mutagenesis was performed according to manufacturer's instructions using the Quick-change kit (Stratagene, Heidelberg, Germany). First, single phospho-mutants (S153D, S235D, and T274E) were generated using the following primer pairs: 5′-GGAGCGCCGGGTCGACAATGCAGGAGGCCCACC-3′ (S153Dfwd); 5′-GGTGGGCCTCCTGCATTGTCGACCCGGCGCTCC-3′ (S153rev); 5′-CAAACTCAGGAAAGTGGACAAGCAGGAGGAGGCC-3′ (S235Dfwd); 5′-GGCCTCCTCCTGCTTGTCCACTTTCCTGAGTTTG-3′ (S235Drev); 5′-GGAGAAGAAAAGCCGAACAGGTTGGGGAGAAG-3′ (T274fwd); and 5′-CTTCTCCCCAACCTGTTCGGCTTTTCTTCTCCC-3′ (T274rev). After sequencing, these constructs were further altered via additional site-directed mutagenesis to yield double mutants and the triple mutant. Murine VASP and its derivatives were expressed in the Escherichia colistrain M15(pREP4). Bacteria were transformed with VASP expression vectors (pQE30) and were grown in 2× YT medium at 30 °C. Protein expression was induced in late log phase with 1 mmisopropyl-1-thio-β-d-galactopyranoside. Bacteria were harvested after 3 h post-induction. Recombinant proteins were purified essentially as described in the manufacturer's protocol (Qiagen). Protein elution was achieved by a stepwise histidine gradient (20, 30, 40, 50, and 150 mm) in VASP elution buffer (50 mm sodium phosphate, pH 7.0, 100 mm KCl, 0.5 mm EDTA, 0.1% Triton X-100, 20 mmβ-mercaptoethanol, 5 mm benzamidine, 20 μmleupeptin, 50 μm Pefabloc SC, 1 μmpepstatin A, and 20 units/ml aprotinin). All fractions were analyzed by SDS-PAGE. VASP containing fractions with 70% purity as judged by densitometric analysis (E.A.S.Y. RH apparatus, E.A.S.Y. Image Plus Software; Herolab, Wiesloch, Germany) were transferred into 50 mm sodium phosphate buffer, pH 7.0, containing 100 mm KCl, 2.5 mm EGTA, 0.75 mmdithioerythritol, 0.1% Triton X-100, and protease inhibitors (see above). 2–3 mg of VASP were purified from 1 liter of bacterial culture by this method. VASP proteins were stored in sodium phosphate buffer with 20% glycerol added at −80 °C for up to 2 months. Recombinant mouse profilin I and II (17Witke W. Podtelejnikov A.V. Di Nardo A. Sutherland J.D. Gurniak C.B. Dotti C. Mann M. EMBO J. 1998; 17: 967-976Crossref PubMed Scopus (285) Google Scholar) were purified by poly-l-proline affinity chromatography as described previously (18Schluter K. Schleicher M. Jockusch B.M. J. Cell Sci. 1998; 111: 3261-3273PubMed Google Scholar) with slight modifications: profilin I and profilin II were eluted in 6 and 8 m urea, respectively. Proteins were dialyzed against 10 mm Tris-HCl, pH 7.2, 0.2 mmCaCl2, and 1.25 mm dithiothreitol. Rabbit skeletal muscle actin was prepared from acetone powder (19Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar) with an additional gel filtration step as described (20Giehl K. Valenta R. Rothkegel M. Ronsiek M. Mannherz H.G. Jockusch B.M. Eur. J. Biochem. 1994; 226: 681-689Crossref PubMed Scopus (64) Google Scholar). Purified VASP (3.75 μm) was incubated at 30 °C in buffer A (50 mm KCl, 5 mmMgCl2, 0.2 mm ATP, 1 mmdithiothreitol, 0.2 mm EGTA, 10 mm HEPES, pH 7.4, 20 units/ml aprotinin, and 1 μm pepstatin A). For radioactive assays, buffer A was supplemented with [γ-32P]ATP yielding a specific activity of 100 Ci/mol ATP. Phosphorylation was initiated by the addition of 0.5 μm catalytic subunit of PKA (Promega, Madison, WI) and stopped at times indicated with 25 μm PKA inhibitor (Promega). Radiolabeled VASP was separated on a 10% polyacrylamide gel, and phosphate incorporation was visualized by autoradiography using BioMax film (Eastman Kodak Co.). For quantitative analysis, gel pieces containing VASP were excised from the gel and measured by Cerenkov counting in a scintillation analyzer (Wallac 1409 liquid scintillation counter, EG&G Berthold, Isernhagen, Germany). One-dimensional phosphoamino acid analysis on thin layer cellulose plates (Macherey and Nagel, Düren, Germany) was performed in pH 1.9 buffer (2.2% formic acid and 7.8% glacial acetic acid) essentially as described in Ref. 21Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1275) Google Scholar. Phospho-serine and phospho-threonine (Sigma) were used as internal standards and stained with ninhydrin. Radiolabeled phosphoamino acids were detected by autoradiography. The influence of VASP or profilin I or II on actin polymerization was determined by fluorimetry with 10% pyrene-labeled actin (22Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (719) Google Scholar) added to unlabeled actin. Actin polymerization assays were performed essentially as described (8Huttelmaier S. Harbeck B. Steffens O. Messerschmidt T. Illenberger S. Jockusch B.M. FEBS Lett. 1999; 451: 68-74Crossref PubMed Scopus (104) Google Scholar). 1 μm actin was preincubated in the absence or presence of 1 μm profilin I or II, respectively, at 25 °C in buffer B (25 mm HEPES, pH 7.0, 0.2 mmCaCl2, 0.5 mm dithioerythritol, and 1 mm ATP) for 30 min. Polymerization was initiated by adjusting the solution to 25 mm NaCl, 2 mmMgCl2, and 15 mm KCl and adding 0.25 μm VASP protein (wild type, VASP phosphorylated by PKA, or VASP phospho-mutants). Fluorescence was monitored for 1 h at 366 nm excitation (slid width, 10 nm) and 384 nm emission (slid width, 10 nm) using a 150-μl cuvette in an LS50B fluorimeter (Perkin-Elmer, Langen, Germany). Co-sedimentation assays were performed essentially as described in Ref. 8Huttelmaier S. Harbeck B. Steffens O. Messerschmidt T. Illenberger S. Jockusch B.M. FEBS Lett. 1999; 451: 68-74Crossref PubMed Scopus (104) Google Scholar. 10 μm actin was prepolymerized in buffer C (25 mm HEPES, pH 7.0, 0.2 mmCaCl2, 0.5 mm dithioerythritol, 1 mm ATP, 25 mm NaCl, and 2 mmMgCl2, KCl (15 mm or 50 mm) for 1 h at 37 °C. 2 μm F-actin was incubated with 2 μm VASP in buffer C for 1 h at room temperature. After high speed centrifugation (100,000 × g, 60 min in an Airfuge; Beckman, München, Germany) pellets and supernatants were analyzed by SDS-PAGE. Coomassie Blue-stained gels were analyzed densitometrically as described above. The percentage of VASP remaining in the supernatant compared with the total amount of VASP used in the experiment was calculated. In sedimentation assays to test for ternary VASP-profilin-actin complexes, prepolymerization was omitted, and actin filaments were polymerized in the presence of VASP and profilin. 2 μm unlabeled actin was polymerized in buffer C in the presence of 0.5 μm wild type VASP, the triple mutant, and VASP phosphorylated by PKA, respectively, at 37 °C for 1 h. Filaments were stained with rhodamine-labeled phalloidin (Sigma) and directly analyzed by fluorescence microscopy (Axiophot; Zeiss, Jena, Germany) using a cooled CCD camera (Roper Scientific, Tucson, AZ) and the MetaMorph Software package (Visitron Systems, Puchheim, Germany). Yeast two-hybrid analysis was performed with a GAL4-based MATCHMAKER System 3 (CLONTECH) with yeast strains HF7C and Y187 according to manufacturer's instructions. VASP constructs were cloned into the "bait" vector pGBKT7 as well as the "prey" vector pGADT7 by use of EcoRI and XhoI/SalI restriction sites in the multiple cloning sites of either vector. A mouse cDNA library (embryonic day 17.5;CLONTECH) was screened using either VASP or the VASP triple mutant as bait. DNA from positive clones was prepared from yeast and transformed into competent E. coli(XL1 blue; Stratagene) according to standard protocols. DNA sequencing was performed on an ABI PRISM™ 310 genetic analyzer (Perkin-Elmer). C2C12 cells (mouse myogenic cell line) were grown in Dulbecco's minimum essential medium supplemented with 10% calf serum at 10% CO2. 16 h prior to transfection, the cells were seeded onto collagen-coated coverslips. Transfection with EGFP-VASP constructs was achieved by calcium phosphate precipitation according to standard protocols. 48 h after transfection, cells were fixed with 4% formaldehyde followed by a 30-min permeabilization with 0.2% Triton X-100 in phosphate-buffered saline (PBS). Actin filaments were stained with coumarin-labeled phalloidin, and vinculin was detected with a monoclonal anti-vinculin antibody (Sigma). Samples were analyzed with a Zeiss Axiophot microscope (Zeiss) equipped for triple immunofluorescence. Images were taken with a cooled CCD camera (Roper Scientific) using the MetaMorph Software package (Visitron Systems). Immunoprecipitations from HeLa cells using the membrane permeant cross-linker dithiobis[succinimidyl propionate] (Pierce) were performed as described (8Huttelmaier S. Harbeck B. Steffens O. Messerschmidt T. Illenberger S. Jockusch B.M. FEBS Lett. 1999; 451: 68-74Crossref PubMed Scopus (104) Google Scholar) using a monoclonal antibody against the BiPro sequence (16Rudiger M. Jockusch B.M. Rothkegel M. BioTechniques. 1997; 23: 96-97Crossref PubMed Scopus (30) Google Scholar). Endogenous proteins were detected after Western blotting with the following antibodies: anti-profilin (2H11) (23Mayboroda O. Schluter K. Jockusch B.M. Cell Motil. Cytoskelet. 1997; 37: 166-177Crossref PubMed Scopus (51) Google Scholar), anti-vinculin (hVIN-1; Sigma), and antibodies against human VASP and zyxin, which were a kind gift of J. Wehland, Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany. Horseradish peroxidase-coupled secondary antibodies (Dianova, Hamburg, Germany) were used for detection by enhanced chemoluminescence (Amersham Pharmacia Biotech). The interaction between VASP and both profilin isoforms was monitored by an ELISA assay. Microcolon ELISA plates (Greiner, Frickenhausen, Germany) were coated with 50 pmol of profilin I or II/well, washed three times with 0.1% Tween-20 in PBS, and blocked with 1% bovine serum albumin in PBS for 2 h at room temperature. After an additional wash with PBS, increasing amounts of recombinant Bipro-tagged VASP were added (0.1–100 pmol in 100 μl of PBS with 0.05% Tween-20 and 0.5 mm dithiothreitol) and incubated with either profilin I or profilin II for 2 h at room temperature. Unbound VASP was removed by three washing steps (PBS, 0.1% Tween-20). Bound VASP was detected with a monoclonal antibody (4A6) specific for the BiPro-tag derived from birch profilin (16Rudiger M. Jockusch B.M. Rothkegel M. BioTechniques. 1997; 23: 96-97Crossref PubMed Scopus (30) Google Scholar, 24Wiedemann P. Giehl K. Almo S.C. Fedorov A.A. Girvin M. Steinberger P. Rudiger M. Ortner M. Sippl M. Dolecek C. Kraft D. Jockusch B. Valenta R. J. Biol. Chem. 1996; 271: 29915-29921Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). After incubation with a peroxidase-conjugated polyvalent anti-mouse secondary antibody, enzymatic activity was measured using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) as a substrate at 410 nm using an ELISA reader (Dynatech Laboratories, Billingshurst, UK). To determine the stoichiometry and dissociation constants (K D) of VASP-profilin complexes, surface plasmon resonance studies were performed on a BIACORE 2000 analyzer (Biacore, Uppsla, Sweden). VASP (the ligand) was immobilized in 10 mm sodium acetate, pH 6.0, on a CM5 sensor chip byN-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chemistry, following the manufacturer's instructions. Profilin I and II (the analytes) were passed over the sensor chip with a flow rate of 10 μl/min at increasing concentrations as indicated. A different flow cell without VASP was used as a reference. Kinetics were analyzed by Biacore evalution software 3.0. The response from the reference cell was substracted from the response of the VASP cell to correct for refractive index changes and nonspecific binding. Thek on and k off values for association and dissociation and RU max, exp(the maximum increase of response units that can be obtained by complete binding of the analyte to the immobilized ligand) were calculated by the software from the association and dissociation phase of the kinetic curve. Best fitting of the data was obtained by assuming a 1:2 complex of VASP to profilin. A global fitting procedure (fitting of k on, k off, andRU max, exp to all measured curves) was used for profilin I, a local fitting was applied for profilin II (fitting of binding data to one curve only). The quality of the obtained binding data was judged by comparing the calculated, fitted curve with the measured curves. An indicator for the differences between the calculated and the measured curves is the chi2 value, which should be <10 for a global and <1 for a local fitting procedure. Furthermore RU max, exp was compared with a therotical RU max, theor value, which was calculated from the amount of VASP coupled to the sensor chip.RU max, theor can be obtained by the formulaRU max, theor=M P/M V·RU coupled·N, where M P = molecular mass profilin (15 kDa),M V = molecular mass VASP (40 kDa),RU coupled = response units of VASP coupled to the sensor chip surface (1045 and 1280 for studies with profilin I and profilin II, respectively), and n = the number of binding sites of VASP for profilin, which is 2 according to data from Ref. 15Lambrechts A. Verschelde J.L. Jonckheere V. Goethals M. Vandekerckhove J. Ampe C. EMBO J. 1997; 16: 484-494Crossref PubMed Scopus (111) Google Scholar. To analyze VASP phosphorylation by PKA under the experimental conditions chosen (see "Experimental Procedures"), 1 μg of recombinant murine VASP was phosphorylated by the catalytic subunit of PKA in the presence of [γ-32P]ATP. Samples were taken after 0.5, 1.5, 3, 10, and 60 min of incubation. The phosphorylation reaction was terminated by addition of excess amounts of the PKA inhibitory peptide. Aliquots were analyzed by SDS-PAGE. The Coomassie-stained gel (Fig.2 A, upper panel) revealed that His-tagged VASP was completely shifted from 50 kDa to an apparent molecular mass of approximately 54 kDa (phospho-VASP), already after 0.5 min. As has been described previously (11Butt E. Abel K. Krieger M. Palm D. Hoppe V. Hoppe J. Walter U. J. Biol. Chem. 1994; 269: 14509-14517Abstract Full Text PDF PubMed Google Scholar), this shift is caused by phosphorylation of Ser153 in murine or Ser157 in human VASP, respectively, located in the central proline-rich domain of VASP (Fig. 1). Prolonged phosphorylation did not cause additional changes in the electrophoretic mobility, even though an increase in phosphate incorporation was observed by autoradiography (Fig. 2 A, lower panel). Protein bands were excised from the gel and phosphate incorporation was analyzed by Cerenkov counting. After 60 min 1.8 mol phosphate/mol VASP had been incorporated (Fig. 2 B). Phosphoamino acid analysis (Fig.2 C) revealed that VASP was mainly phosphorylated at serine residues even though some phospho-threonine was detectable. These results are in good agreement with a previous study on human VASP (11Butt E. Abel K. Krieger M. Palm D. Hoppe V. Hoppe J. Walter U. J. Biol. Chem. 1994; 269: 14509-14517Abstract Full Text PDF PubMed Google Scholar) showing that VASP becomes phosphorylated at three residues, both in human platelets and in vitro: Ser157, Ser239, and Thr278, corresponding to Ser153, Ser235, and Thr274 in murine VASP (8Huttelmaier S. Harbeck B. Steffens O. Messerschmidt T. Illenberger S. Jockusch B.M. FEBS Lett. 1999; 451: 68-74Crossref PubMed Scopus (104) Google Scholar). In analogy to the data obtained by Butt and co-workers (11Butt E. Abel K. Krieger M. Palm D. Hoppe V. Hoppe J. Walter U. J. Biol. Chem. 1994; 269: 14509-14517Abstract Full Text PDF PubMed Google Scholar), we conclude that PKA first phosphorylates murine VASP at Ser153 in the proline-rich domain and subsequently at Ser235 and, to a much lesser extent, at Thr274. To analyze the influence of phosphorylation by PKA on VASP interaction with actin, we used unlabeled samples that had been prepared in parallel with the samples for the phosphate incorporation analysis. In a previous study we demonstrated that actin nucleation by VASP as well as its binding to actin filaments is salt-sensitive (8Huttelmaier S. Harbeck B. Steffens O. Messerschmidt T. Illenberger S. Jockusch B.M. FEBS Lett. 1999; 451: 68-74Crossref PubMed Scopus (104) Google Scholar), indicating that the complex formation is based on electrostatic interactions. Hence all experiments were performed under low salt conditions (15 or 50 mm KCl). Actin nucleation was monitored in a standard actin polymerization assay where 10% of the G-actin used is labeled with pyrene (Fig.3 A). Actin filament formation causes an increase in fluorescence intensity giving a direct measurement of actin polymerization. When 1 μm G-actin was transferred into a buffer promoting actin polymerization, only negligible filament formation was observed. In the presence of 0.25 μm VASP, PKA, and PK