CAMP- and cGMP-dependent Protein Kinase Phosphorylation Sites ...

THE JOURNAL OF B I O ~ I C A L CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 20, Issue of May 20, pp. 14509-14517, 1994 Printed in U.S.A.

CAMP- and cGMP-dependent Protein Kinase Phosphorylation Sites of the Focal Adhesion Vasodilator-stimulated Phosphoprotein (VASP) in Vitro and in Intact Human Platelets*

(Received for publication, September 9, 1993, and in revised form, March 4, 1994)

Elke Butt$§, Kathrin AbelS, Monika KriegerS, Dieter Palmn, Viviane Hoppen, Jurgen Hoppen, and Ulrich Walter8 From the rnedizinische Uniuersitatsklinik, Klinische Biochemie und Pathobiochemie, Josef-Schneider Strasse 2, 0-97080 Wiirzburg and the qBiozentrum, Physiologische Chemie, A m Hubland, 0-97074 Wiirzburg, Federal Republic of Germany ~

The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for CAMP-dependent- (cAK) and cGMP-dependent protein kinase (cGK) in human platelets and other cardiovascular cells. To identify the VASP phosphorylation sites, purified VASP was phosphorylated by either protein kinase and subjected to trypsin, V8 and Lys-C proteolysis. The phosphorylated proteo- lytic fragments obtained were separated by reversed phase high performance liquid chromatography. Se- quence analysis of the phosphorylated peptides and ”P measurement of the released “P-labeled amino acids revealed three phosphorylation sites: a serine l-containing site (LRKVSKQEEA), a serine !&containing site (HI- ERRVSNAG), and a threonine-containing site (MNA- VLARRRKAT‘QVGE). Additional experiments with purified VASP demonstrated that both cAK and cGK phosphorylated serine 2 rapidly and the threonine residue slowly, whereas cGK phosphorylated the serine 1 residue more rapidly than the cAK. These differences in the phosphorylation rates of VASP by the two protein kinases were also observed with synthetic peptides corresponding to the sequences of the three identified phosphorylation sites. These experiments also established the synthetic peptide serine 1 as one of the best in vitro cGK substrates and the serine %containing site as the site responsible for the phosphorylation-induced mobility shift of VASP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with 32P-labeled platelets provided evidence that VASP is phosphorylated at the same three identified sites also in intact cells and that selective activation of cAK or cGK primarily increased the phosphorylation of both serine 2 and serine 1 but not threonine. Our results demonstrated overlapping substrate specificities of cAK and cGK in vitro and in intact cells. However, important quantitative and qualitative differences between cAK- and cGK-mediated phosphorylation of the focal adhesion protein VASP in human platelets were also observed, suggesting distinct functions of the two types of cyclic nucleotide-mediated VASP phosphorylation.

The 46/50-kDa vasodilator-stimulated phosphoprotein

* This work was supported in part by Deutsche Forschungsgemein- schaft Grants SFB 176/A10, All, A15; and KO 210/11-3. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 Recipient of Deutsche Forschungsgemeinschaft Postdoctoral Fel- lowship Bu 740/1-l. To whom correspondence should be addressed. Tel.: 49-931-201-3144; F a : 49-931-201-3153.

(VASP)’ is a major substrate of cyclic nucleotide-dependent protein kinases which is phosphorylated by both cGMP-dependent protein kinase (cGK) and CAMP-dependent protein kinase (cAK) in vitro and in intact human platelets (14). VASP phosphorylation in response to cGMP-elevating agents (e.g. nitrovasodilators including the endothelium-derived relaxing fac- tor), CAMP-elevating agents (e.g. prostaglandins E, and IJ, and selective membrane-permeant activators of cAK and cGK, respectively, correlates very well with the inhibition of platelet activatiodaggregation caused by these agents (2-8).

Recently, VASP and regulation of its phosphorylation were also discovered in other cell types including lymphocytes, leu- kocytes, endothelial cells, vascular smooth muscle cells, and fibroblasts (9,101. Furthermore, VASP was identified and char- acterized as a novel microfilament-associated protein that is particularly concentrated at sites of focal contacts (transmem- brane junction between microfilaments and the extracellular matrix) and certain cell-cell contacts (10). I n vitro phosphorylation experiments using low or physiological (high) concentrations of purified VASP and protein kinases indicated that both cAK and cGK are able to incorporate up to 1.3-2.1 mol of phosphate/mol of VASP (3, 4, 11). Phosphorylation of VASP shifts its apparent molecular mass in SDS-PAGE from 46 to 50 kDa, and transient phosphorylation of the 46-kDa VASP spe- cies is observed only with cGK i n vitro but not with cAK (3, 4, 11). These data indicated the presence of at least two distinct VASP phosphorylation sites. The aim of the present study was the identification of the cGK and cAK phosphorylation sites of VASP in vitro and in intact cells. Preliminary results of this study have been presented in abstract form (12).

EXPERIMENTAL PROCEDURES Materzul~-[y-~~PlATP (3,000 Ci/mmol) was from Amersham, Braun-

schweig, F. R. G. [32PlH,P0, (HC1-free) was from DuPont NEN. Phos- phoamino acid standards, prostaglandin E, (PGE,), sodium nitroprusside (SNP), and cyanogen bromide (CNBr) were purchased from Sigma. Trypsin, endoproteinase Glu-C (VS), and endoproteinase Lys-C (all sequencing grade) were purchased from Boehringer Mannheim, F. R. G. Ninhydrin and Silica Gel 60 thin layer chromatography plate (20 x 20 cm) were from Merck. Sequelon diisothiocyanate and Sequelon arylam-

The abbreviations used are: VASP, vasodilator-stimulated phosphoprotein; cGK, cGMP-dependent protein kinase; cAK, CAMP-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; PGE,, prostaglandin E,; SNP, sodium nitroprusside; CNBr, cyanogen bromide; HPLC, high performance liquid chromatography; Tricine, N42-hy- droxy-1,l-bis(hydroxymethy1)ethyllglycine; CAPS, 3-[cyclohexylamino]-

Dide, a peptide derived from the sequence of the phosphorylation site in 1-propanesulfonic acid; Kemptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly; BP-

the cGMP-binding cGMP-specific phosphodiesterase; 8-pCPT-cGMP, 8-(4-chlorophenylthio)-cGMP; Sp-5,6-DCI-cBiMPS, Sp-5,6-dichloro-l-P- ~-ribofuranosylbenzimidazole-3’,5’-monophosphorothioate.

14509

14510 Protein Kinase Phosphorylation Sites of VASP ine membranes were obtained from Millipore. The FluoroTrans membrane (PVDF) was obtained from Pall, Dreieich, F. R. G. All chemicals were of the highest purity grade commercially available, organic sol- vents were HPLC grade.

The type I1 cAK and the soluble type I cGK were purified from bovine heart and bovine lung, respectively, as described earlier (13, 14).

Phosphorylation of Isolated VMP-VASP, purified as described previously (31, consisted almost completely (95%) of the 46-kDa form. Pu- rified VASP (4 p) was incubated at 30 "C for 30 min with 10 lll~ Hepes (pH 7.4), 5 mM MgCl,, 1 mM EDTA, 0.2 nm dithiothreitol, 0.2 PM catalytic subunit of cAK or 0.2 p cGK plus 40 cGMP in a total volume of 100-200 pl. The reaction was started by the addition of 100 DM ATP containing 0.5 pCi of [y-32PlATP. At the times indicated, aliquots were taken, and the reaction was terminated by the addition of Laemmli SDS stop solution. Proteins were separated on a 9% SDS-PAGE gel. 32P incorporation was visualized by autoradiography.

Identification of Phosphorylation Sites-To determine the location of phosphorylation sites, VASP (50 pg) was phosphorylated by either cGK or C-subunit at 30 "C for 30 min. The reaction was stopped by the addition of trichloroacetic acid (final concentration lo%), and the precipitated protein was washed twice with ice-cold acetone. The precipi- tate was dissolved in cleavage buffer, and the digestion was started by the addition of protease yielding a protease/substrate ratio of 1:20 (w/ w). Digestion with trypsin was performed in 100 mM Tris/HCI (pH 8.5) for 18 h at 37 "C. Proteolysis by Lys-C was conducted for 18 h at 37 "C in 25 mM Tris/HCI, 1 mM EDTA (pH 8.5). Digestion with Staphylococcus aureus V8 protease was performed in 50 mM ammonium carbonate (pH 7.8) for 18 h at 25 "C.

The 32P-labeled peptides were separated by reversed phase HPLC using a Vydac C,, column (TP218, 5 pm, 4.6 x 250 mm). A linear gradient from 100% solvent A (0.1% trifluoroacetic acid) to 50% solvent B (84% acetonitrile, 0.08% trifluoroacetic acid) over 90 min was employed at a flow rate of 0.7 mumin. The absorbance was recorded at 214 nm. Fractions of 350 pl were taken, and their radioactivity was measured by the Cerenkov effect using a Packard 1900 CA scintillation analyzer. 32P-labeled peptides were covalently coupled to Sequelon diisothiocyanate or arylamine discs (Millipore) according to the manufacturer's instructions. The sequence analysis of peptides from [32PlVASP was performed with a gas phase Sequencer (Applied Biosystems A470) using a slightly modified method for solid phase sequencing described by Meyer et al. (15). In this method, anilinothiazolinone amino acids are extracted by trifluoroacetic acid, which has been described as a suitable solvent for phosphorylated amino acids and inorganic phosphate (16).

Electrophoresis-For the separation of peptides derived from digested "P-labeled VASP, SDS-PAGE was performed in 16% gels using the urea-free Tricine gel system described by Schagger and von Jagow (17).

CNBr Cleauage-The 32P-labeled VASP (or the 32P-labeled VASP gel piece) was dissolved in 200 pl of 70% formic acid containing 10 mg of CNBr/mg of protein. After 18 h at room temperature in the dark, the reaction mixture was diluted with 5 volumes of water and lyophilized. The digest was dissolved in water, its pH adjusted to 8.0 by the addition of Tris base, and the [32Plphosphopeptides were separated by SDS- PAGE. Radioactive bands were visualized by autoradiography.

Phosphoamino Acid Analysis-For phosphoamino acid analysis of the peptides resolved from an SDS-polyacrylamide gel, the gel was electroblotted to a FluoroTrans membrane using 50 mM CAPS buffer (pH 10.0), containing 10% methanol for 1 h at 260 mA. The radiolabeled peptide spots were excised and treated with 6 N HC1 at 110 "C for 4 h in a sealed tube. The samples were dried and dissolved in 20 p1 of water and analyzed for phosphoamino acids by thin layer electrophoresis on silica gel plates in 1.8% formic acid, 7.3% acetic acid (pH 1.9) at 500 V and 4 "C for 2 h. Phosphoserine and phosphothreonine were used as internal standards and visualized by staining with ninhydrin; y3'P- labeled amino acids were detected by autoradiography.

Peptide Synthesis and Purification-Peptides were synthesized from Fmoc (N-(9-fluorenyl)methoxycarbonyl)-L-amino acid derivatives using a Zinsser SMPT model 350A peptide synthesizer. The solubilized peptides were preparatively purified by HPLC on a reversed phase C,, material with a linear gradient from 25 to 75% acetonitrile in 0.06% trifluoroacetic acid. Purity and quantity of the peptides were determined by amino acid analysis and electrospray ionization mass spectrometry (Dr. Fehlhaber, Hoechst AG, Frankfurt).

Determination of Kinetic Constants-The protein kinase assay was performed as described by Roskoski (18). Briefly, phosphorylation of the synthetic peptides was assayed at 30 "C in a total volume of 100 pl containing 20 mM Tris/HCl buffer (pH 7.41, 10 mM MgCl,, 5 mM p-mer- captoethanol, 0.01% (wh) bovine serum albumin, and 10 or 50 ng of

protein kinase. The reaction was started by the addition of 50 PM [Y-~'P]ATP (100 cpndpmol). The activity of cGK in the presence of 5 WM cGMP and the catalytic subunit of cAK were measured in triplicate using peptide concentrations ranging from 1 p~ to 2 mM. Peptides were diluted in 20 mM Tris/HCI (pH 7.4). Each kinase was assayed at increas- ing synthetic peptide concentrations up to saturating levels. Initial linear rates of peptide phosphorylation were used for Lineweaver-Burk plots. K, and V,, values were derived from linear regression analysis of the reciprocal plots.

Phosphorylation and Immunoprecipitation of VASP from Intact Hu- man Platelets-Platelets were isolated from healthy volunteers and incubated with [32PlH3P0, as described previously (2). Briefly, platelets were collected by centrifugation, washed, and resuspended to a concentration of approximately 1 x lo9 celldml in 10 mM Hepes buffer (pH 7.41, 137 mM NaCl, 2.7 mM KCI, 5.0 mM glucose, and 1 mM EDTA. For VASP phosphorylation, intact platelets were incubated at 37 "C with 100 PM SNP or 10 PM PGE,. 50-pl aliquots of this suspension were removed at the times indicated, and the incubation was stopped by the addition of 50 pl of modified 2 x RIPA buffer (40 mM Tris/HCl (pH 7.4), 300 mM NaC1, 2% sodium deoxycholate, 2% Triton X-100, 1 mM sodium or- thovanadate, 20 mM NaH,PO,, 200 units/ml Trasylol, 20 mM EDTA, and 10 mM p-nitrophenyl phosphate). The samples were centrifuged at 15,000 x g and 4 "C for 30 min, and the supernatant was used for immunoprecipitation. Rabbit VASP antiserum (2 pl) described earlier (4) was added to each tube, and the reaction mixture was incubated at 4 "C for 2 h with constant agitation. Immune complexes were then precipitated after a 2-h incubation with 50 pl of 12.5 mg/ml preswollen protein A-Sepharose beads (Pharmacia LKl3 Biotechnology Inc.). Using these conditions, VASP was nearly quantitatively removed from the supernatant, and about 50% of the precipitated VASP was recovered in eluates of beads washed three times.' Here, the pelleted beads were washed sequentially two times with modified RIPA-buffer (1 x) and then once with 25 nm TridHCI, 1 mM EDTA (pH 8.5) (Lys-C digestion buffer). The washed beads were resuspended in 200 pl of Lys-C digestion buffer containing 0.035 pg of Lys-C, yielding a proteasehbstrate ratio (w/w) of 15, and the reaction mixture was incubated overnight at 37 "C. For the CNBr cleavage, the washed beads were precipitated with 10% trichloroacetic acid, washed twice with ice-cold acetone, and dissolved in 200 pl of 70% formic acid containing 10 mg of CNBr/ml, The mixture was incubated in the dark for 18 h at room temperature. Pep- tides were prepared by dilution of the hydrolysate with 5 volumes of H,O and subsequent evaporation under vacuum.

RESULTS

One of the major cGMP-dependent and CAMP-dependent protein kinase substrates in platelet membranes and in intact human platelets is the 46/50-kDa focal adhesion protein desig- nated VASP. Phosphorylation of VASP alters the apparent mobility of the protein in SDS-PAGE, resulting in the appearance of a 50-kDa form and the concomitant disappearance of the 46-kDa form (3, 4). Transient phosphorylation of the 46-kDa form of purified VASP was observed primarily with cGK but only to a limited extent with cAK (Fig. 1). To identify the sites of VASP phosphorylated by cGK and by cAK, purified VASP was phosphorylated and subjected to four different digestions (see "Experimental Procedures"). The absorbance and 32P profile of the tryptic peptides separated by reversed phase HPLC are presented in Fig. 2. Two peaks of radioactivity were detected and collected in fractions 51 and 118. Half of peptide fraction 51 was immobilized via carboxy groups to arylamine filter discs. The other half was spotted onto FluoroTrans membrane discs. The covalently immobilized peptide was sequenced using the modified protocol that enables the identification of 32P radioactivity during Edman degradation (16). Peptides spotted onto fluorotrans membranes were sequenced using standard protocol. The sequence analysis of the spotted peptide from fraction 51 yielded the sequence KVASKQEEA. The analysis of the covalently coupled peptide gave the sequence KVASXQXXA. As indicated in the inset of Fig. 2, radioactivity was predominantly recovered in step 3, corresponding to a serine residue. The 2 glutamic acid residues were not detectable

M. Reinhard and U. Walter, unpublished experiments.

Protein Kinase Phosphorylation Sites of VASP

cGK FIG. 1. Time course of VASP phos-

phorylation by cGK and the catalytic subunit of cAK. Purified VASP was

14511

CAK

phosphorylated as described under "Ex- a k D a ~

perimental Procedures" for the times in- a k D a dicated. Proteins were seDarated bv SDS- I- PAGE and detected by autoradiography of the phosphorylated proteins.

0.1

0.05 I N U

0.5 1 2 3 5 10 20 0.5 1 2 3 5 10 20 min

-- - +akDa c 46 kDa

I 1 . ,A. .

I 20

I 50

I 80

FIG. 2. Reversed phase HPLC separation of VASP peptides generated by tryptic digestion. Purified VASP (100 pg) was phosphorylated for 1 h in the presence of the C-subunit and subjected to tryptic digestion as described under "Experimental Procedures." Re- versed phase HPLC separation of the derived peptides was performed using a linear gradient from 0.1% trifluoroacetic acid to 0.09% trifluoroacetic acid, 42% CH,CN over 90 min a t a flow rate of 700 pl/min. Peptides were detected by their absorbance at 214 nm. The eluted peptides were collected in 0.5-min fractions, and their 32P radioactivity was quantitated by Cerenkov counting. The shading indicates radioactive fractions. Inset, amino acid sequence and corresponding radioactive profile of the peptide from fraction 51. A similar pattern of peptides and phosphopeptides was obtained when cGK phosphorylated VASP was digested by trypsin (not shown).

because of their immobilization onto the arylamine membrane. Unequivocal identification of the phosphopeptide in fraction 118 was not possible since this fraction contained several peptides.

In studies with S. aureus V8, digested VASP yielded two radioactive peaks: fractions 51 and 90 (Fig. 3). Amino acid sequence analysis of the first peptide identified a threonine in cycle 12 (inset A in Fig. 3) as the phosphorylated amino acid. Automatic sequencing of the second peptide after immobilization on arylamine membranes detected the sequence HLXRRXASNAGG. The phosphorylated serine was identified at position 7 (Table I and inset B in Fig. 3).

To substantiate the above results, phosphorylated VASP was digested by the endoprotease Lys-C, and resulting fragments were analyzed by gel electrophoresis. Consistently a radioactive band with a molecular mass of 12 kDa (Fig. 4) was observed, whereas the composition and relative amount of the bands with molecular masses from 3-8 kDa varied consider- ably among the different experiments (see Figs. 4, 7, and 9). Phosphoamino acid analysis of all bands indicated a serine residue to be phosphorylated. For further analysis the peptides were separated by reversed phase HPLC (chromatogram not shown), immobilized onto diisothiocyanate membranes, and sequenced using the modified protocol. Amino acid sequence analysis of the fraction containing the 12-kDa peptide identified the sequence RQQPGPSEHIERRVASXAGGPPA. Sequence and 32P content analysis of the fraction corresponding to the 6-kDa peptide revealed the peptide sequence LRKVASKQEEA and

I I I 1 50 70 90 I 1 0

Fractlon Number

FIG. 3. Reversed phase HPLC separation of VASP peptides generated by VS digestion. Purified VASP (50 pg) was phosphorylated for 30 min in the presence of the C-subunit and subjected to V8 digestion as described under "Experimental Procedures." Separation was performed using a linear gradient from 0.1% trifluoroacetic acid to 0.09% trifluoroacetic acid, 42% CH,CN over 90 min a t a flow rate of 700 pl/min. Peptides were detected by their absorbance at 214 nm. The eluted peptides were collected in 0.5-min fractions, and their 32P radioactivity was quantitated by Cerenkov counting. The shading indicates radioactive fractions. Inset A, amino acid sequence and corresponding radioactive profile of the peptide from fraction 51. Inset B, amino acid sequence and corresponding radioactive profile of the peptide from fraction 90.

TABLE I Identified serine- and threonine-containing phosphopeptides of VASP Serine- and threonine-containing phosphopeptides obtained by V8,

Lys-C, or tryptic digestion and CNBr cleavage of phosphorylated VASP were purified and sequenced. The phosphorylated serine or threonine is shown in bold. The peptide sequences agreed with the corresponding VASP sequences deduced from a VASP cDNA clone" as indicated. The one-amino acid difference (VN) in the threonine-containing sequence may be because of polymorphism since the VASP protein was isolated from human platelet, whereas the VASP clone was obtained using a human HL60 cell cDNA library.

Serine 1 Lys-c LRKVSKQEEA Trypsin KVSKQEEA Clone KLRKVSKQEEA

V8 HIXRRxSNAGG Lys-c RQQPGPSEHIERRVSAGGPPA Clone KRQQPGPSEHIERRVSNAGGPPA

V8 MNAVLARRRKATQVG CNEk LARRRKATQVGEKXP Clone EMNAMLARRRKATQVGE

Serine 2

Threonine

The molecular cloning of VASP and the complete VASP sequence deduced from a cDNA clone will be reported elsewhere (see Footnote 3).

concomitant 32P release in cycle 5 (serine). Automatic sequencing of the 3-kDa peptide detected the sequence LRKVAS,'indicating an additional cleavage of the 6-kDa peptide after lysine at position 6.

In total, three VASP phosphorylation sites were identified which were phosphorylated by either cGK or the C-subunit of

14512 Protein Kinase Phosph

injected sample

orylation Sites of VASP Molecular mass (kDa)

102 78 33 Fraction

FIG. 4. Autoradiography of the phosphorylated VASP peptides generated by Lys-C digestion. Purified VASP (50 pg) was phosphorylated for 30 min in the presence of cGK, subjected to Lys-C digestion, and separated by reversed phase HPLC as described under “Experi- mental Procedures.” The eluted peptides were collected, and their ”P radioactivity was quantitated by Cerenkov counting. The autoradiography of the injected sample and the separated radioactive fractions 33, 78, and 102, separated on a 16% Tricine gel, are shown.

CAK: a threonine-containing site (MNAVLARRRKATQVGE) and two serine-containing sites (LRKVSKQEEA and QQPG- PSEHIERRVSNAGGPPA). These peptide sequences were con- firmed by information obtained from the recent VASP cDNA cloning in our laboratory (see Table I). Analysis of the cDNA- derived VASP sequence (not shown) indicated that none of the peptides generated by trypsin, Lys-C, or V8 digestion contained other likely cGK or cAK phosphorylation residues in addition to the amino acids identified by the sequence analysis of phosphorylated VASP. The observed lengths of the Lys-C fragments were in good agreement with the fragments predicted by the cDNA-derived sequence. For the smaller Lys-C fragments vary- ing between 3 and 8 kDa, the appearance of an 6- or 8-kDa peptide was predicted assuming incomplete digestion (not shown).

Further experimental conditions were developed to distinguish the phosphorylation of the 2 serine residues and the threonine residue by SDS-PAGE. CNBr cleavage of phosphorylated VASP incubated with the C-subunit for 30 min under standard conditions resulted in two radioactive fragments on SDS-PAGE: a highly radioactive 22-kDa peptide and a 9-kDa peptide with less radioactivity (Fig. 5) . FluoroTrans membrane blots of the 9- and 22-kDa radioactive peptides were incubated with acid and analyzed for their phosphoamino acid content (Fig. 6). Radioactivity from the 22-kDa fragment comigrated with the phosphoserine standard, whereas the phosphoamino acid derived from the 9-kDa fragment was identified as a phosphothreonine, suggesting that the 22-kDa peptide contained both serine residues. No radioactivity corresponding to phos- photyrosine was observed. Peptide sequence analysis of the 9-kDa peptide aRer transfer onto Fluoro’hans filter membranes gave the sequence (LARRRKATQVGEKXP). The two observed CNBr fragments were in perfect agreement with CNBr fragments predicted by cDNA-derived VASP sequence (not shown). Thus, cleavage by CNBr is a valuable tool for distinguishing the serine or threonine phosphopeptides of VASP.

To distinguish the two phosphoserine-containing peptides, phosphorylated VASP as well as the separated 46- and BO-kDa forms of phosphorylated VASP were digested with Lys-C, resulting in a 12-kDa peptide and a 6-kDa peptide as shown previously in Fig. 4. Cleavage of the 46-kDa phosphoform resulted in a 6-kDa peptide, whereas digestion of the BO-kDa VASP phosphoform yielded both a 6-kDa peptide and a 12-kDa

- 10.6

g - 0 - 8.16

- 6.21

- 3.48 FIG. 5. CNBr cleavage of phosphorylated VASP. Purified VASP

(20 pg) was phosphorylated in the presence of the C-subunit (see ”Ex- perimental Procedures”). The reaction was stopped by trichloroacetic

taining 10 mg/ml CNBr and incubated for 18 h in the dark. Samples acid precipitation. The pellet was resuspended in 70% formic acid con-

were analyzed by electrophoresis on a 16% Tricine gel. The two major radioactive fragments had an apparent molecular mass of 22 and 9 kDa as indicated by the autoradiogram. Similar data were obtained with cGK-phosphorylated VASP (not shown).

@ - Ser

Origin 0 9 kDa 22 kDa

FIG. 6. Phosphoamino acid analysis of phosphorylated VASP fragments obtained by CNBr cleavage. Radioactive fragments obtained by CNBr cleavage of purified VASP that had been phosphorylated by the C-subunit were hydrolyzed in 6 N HCI, separated on thin layer chromatography, and analyzed by autoradiography as described under “Experimental Procedures.” The migration positions of phosphoserine and phosphothreonine standards determined by ninhydrin staining are indicated. The resulting autoradiography is shown. Similar data were obtained with cGK-phosphorylated VASP (not shown).

fragment (Fig. 7). Therefore Lys-C digestion is a valuable tool to distinguish the phosphorylation of the 2 serine residues.

To analyze the time course ofVASP peptide phosphorylation, purified VASP was phosphorylated with either the C-subunit or cGK for different lengths of time, followed by CNBr cleavage of the separated 46-kDa form and the BO-kDa form of VASP. As shown in Fig. 8, most of the radioactivity was located in the 22-kDa band, which contains the 2 phosphoserines. The 9-kDa phosphopeptide (threonine) appeared much later than the 22- kDa phosphorylation and contained a minor amount of radio-

Protein Kinase Phosphorylation Sites of VASP 14513

12 kDa

- 6kDa

46 50 46/50 kDaVASP FIG. 7. Lys-C digestion of phosphorylated VASP. Purified VASP

(2 pg) was phosphorylated for 30 min in the presence of cGK as described under "Experimental Procedures." The 46- and 50-kDa forms of VASP were separated by SDS-PAGE, corresponding gel pieces were cut out and incubated with 0.5 pg of Lys-C/pg of VASP in 25 mM TrisIHC1 (pH 8.51, 1 mM EDTA at 37 "C for 18 h. The samples were separated on a 16% Tricine gel and visualized by autoradiography.

activity, indicating that the 2 serines (22-kDa fragment) are the predominant phosphorylation sites for both protein kinases in vitro.

For further analysis, purified VASP (phosphorylated with either cGK or the C-subunit of cAK), was digested with Lys-C and separated on a 16% Tricine gel. The results of this experiment are shown in Fig. 9. cGK initially phosphorylated serine 1 (6-kDa fragment) followed by a delayed incorporation of phosphate into serine 2 (12-kDa fragment). In contrast, cAK first phosphorylated serine 2 and then serine 1. The initial rate of VASP phosphorylation by cGK appeared to be faster than that by cAK. Despite differences in phosphorylation rates, cGMP- and CAMP-dependent protein kinases phosphorylated the same 2 serines. In control experiments, Lys-C-digested autophospho- rylated cGK yielded different phosphopeptides compared with Lys-C-digested phosphorylated VASP. The cAK C-subunit was not significantly phosphorylated under our experimental conditions. Therefore, cGK or cAK autophosphorylation did not affect the analysis of VASP phosphopeptides.

For further kinetic analysis, synthetic peptides corresponding to three phosphorylation sites of VASP were synthesized and tested as protein kinase substrates over a wide range of peptide concentrations. Fig. 10 shows the initial rate of peptide 1 (LRKVSKQE) phosphorylation by cGK as a function of peptide concentration. The corresponding Lineweaver-Burk plot is also shown. A summary of the kinetic constants (each obtained in a total of two to four experiments) in comparison with kinetic constants of other cGK substrates such as Kemptide, G-substrate, and BPDide is presented in Table 11. The serine 1 peptide (peptide 1) was a much better substrate for the cGK than for the catalytic subunit of CAK. The apparent K,,, of peptide 1 for phosphorylation by cGK was 94 p ~ , at least 15-fold lower than that for cAK (1,395 p~); the V,,, value of cGK was 3.7 pmol/min/mg. The K,,, and V,,, values for cAK could only be estimated by calculation since saturating substrate concentrations could not be achieved experimentally (Table 11). To study this cGK-specific phosphorylation site in more detail we also synthesized the peptide RRKVSKQE. The apparent K,,, of this peptide was 27 and 264 w in the phosphorylation reaction catalyzed by cGK or cAK, respectively. The V,, for cGK phosphorylation (6.8 pmol/min/mg) was more than 2-fold higher than that for cAK (3.22 pmol/min/mg), resulting in a specificity index of 21 (data not shown in Table 11). Thus, the substrate RRKVSKQE retained the cGK specificity observed for peptide 1. The kinetic data for peptide 2 (serine 2) suggested that it is

a phosphorylation site for both protein kinases. The K,,, and V,,, values for this substrate were 26 PM and 2.7 pmol/midmg for cAK and 30 w and 2.2 pmol/min/mg for cGK, respectively. Peptide 3 (containing threonine) was also a substrate for both protein kinases with reasonable K,,, values (35 p~ for cGK and 89 w for cAK) but low V,,, values (0.23 pmol/min/mg for cGK and 0.44 pmol/min/mg for cAK). The kinetic phosphorylation data are summarized in Table 11.

For the analysis of VASP phosphorylation sites in intact cells, human platelets were metabolically labeled with 32Pi and then incubated with either 100 p~ SNP or 10 PM PGE, followed by cell lysis, immunoprecipitation of VASP from the cell lysate (Fig. 111, and VASP digestion with Lys-C or cleavage by CNBr. The results shown in Fig. 12 demonstrate that the same set of phosphopeptides is generated from phosphorylated VASP (radiolabeled in intact cells) as from the purified protein by cleavage with Lys-C and CNBr. However, the phosphorylation in intact cells appears to proceed with different kinetics. In the case of VASP phosphorylation in response to PGE, (which activates the cAK) the autoradiography of Lys-C digestion revealed a time course similar to that already observed with purified proteins: fast incorporation of phosphate into serine 2 followed by delayed phosphorylation of serine 1 (Fig. 12, lower panel). Analysis of the VASP peptides obtained by Lys-C cleavage after SNP incubation (which activates the cGK) demonstrated that serine 1 and 2 are phosphorylated in intact platelets by the cGK with a similar time course, which contrasts with the preferential phosphorylation of serine 1 by cGK in vitro (Fig. 12, upper panel). The observed basal phosphorylation of the threonine peptide was not significantly stimulated by either SNP/cGK or PGE,/cAK in intact cells (Fig. 12).

DISCUSSION The specificity and recognition sites for cAK and cGK have

been studied extensively (24-26). The most typical motif for the consensus phosphorylation site with respect to the cAK is RW KXS*/T*. Previous cGK phosphorylation site comparisons suggest a more stringent requirement of this kinase for basic residues, and the consensus phosphorylation motif for the cGK is therefore FV&.JS*/'I'* (27). However, it should be emphasized that the cGK consensus phosphorylation site is based entirely on sequences established by in vitro studies using purified kinase and purified proteindpeptides, which have not been demonstrated to be true cGK substrates in intact cells. In contrast, stoichiometric VASP phosphorylation by both cAK and cGK has been demonstrated not only with purified proteins but also in intact cells (4 , l l ) . Moreover, use of membrane-permeant selective cGK activators and most recently the comparison of cGK- deficient and normal platelets established that the VASP phosphorylation observed in intact human platelets in response to nitric oxide-generating nitrovasodilators is essentially cGMP- mediated and caused by the activation of the platelet cGK (7,8, 28). Furthermore, VASP phosphorylation in intact cells in response to CAMP-elevating agents (e.g. PGE,) has been shown to be mediated by the cAK. In agreement with previous results that both cAK and cGK are capable of incorporating up to 1.3-2.1 mol of phosphate/mol of VASP in vitro (3, 4, 111, this study now reports the identification of three in vitro VASP phosphorylation sites (Figs. 2 and 3 and Table I), which are used by both cAK and cGK. The three identified phosphorylation sites are consistent with the cAK and cGK consensus sequences mentioned above. However, the three VASP phosphorylation sites are phosphorylated by cAK and cGK with distinct kinetics. Serine 2 of VASP (HIERRVSNAG) is phosphorylated by both cyclic nucleotide-dependent protein kinases with similar kinetics, and when compared with the two other phosphorylation sites, it is the VASP phosphorylation site preferred by

14514 Protein Kinase Phosphorylation Sites of VASP

9 -

COK c - r.b..ic

VASP was phosphorylated by cGK or the C-subunit for the time points indicated, and the 46- and 50-kDa forms were separated by SDS-PAGE, FIG. 8. CNBr cleavage analysis of the 46- and SO-kDa VASP phosphorylation time course with either cGK or the C-subunit of CAR.

excised, and cleaved by CNBr in 70% formic acid. The samples were separated on a 16% Tricine gel and visualized by autoradiography. The locations of serine-containing (22-kDa fragment) and threonine-containing (9-kDa fragment) peptides are indicated.

cGK cAK

4 6 5 0 4 5 5 0 4 6 5 0 4 6 5 0 4 6 5 0 kDaVASP 4 6 5 0 4 6 5 0 4 6 5 0 4 6 5 0 4 6 5 0

”“- Phosphorylation - - - - - 0.5 1 7 15 30 time (min) 1 3 7 15 30

FIG. 9. Lys-C digestion analysis of the 46- and SO-kDa VASP phosphorylation time course with either cGK or the C-subunit of CAR. The 46- and BO-kDa forms of purified VASP phosphorylated by cGK or the C-subunit for the time points indicated were separated by SDS-PAGE and digested with 0.5 pg of Lys-C/pgofVASP in 25 mM TrisJHCl (pH 8.5), 1 mM EDTAat 37 “C for 18 h. The samples were separated on a 16% Tricine gel and visualized by autoradiography.

terminal to the threonine but appears to be a phosphorylation site of minor importance since its phosphorylation is only ob-

2.5

II)

c 0.5

. 0 100 200 300 400 500

lPeptide 11 b”

FIG. 10. Plot of kinase activity as function of substrate concentration and Lineweaver-Burk plot for the phosphorylation of peptide 1 by cGK. Peptide 1 was phosphorylated by cGK. Initial velocities (pmoVmidmg) were determined as described under “Experi- mental Procedures” and are plotted against the substrate concentration and in double-reciprocal form (Lineweaver-Burk plot). The results shown are the mean of closely agreeing triplicate determinations of one experiment.

the cAK (Fig. 9). In contrast, serine 1 of VASP (LRKVSKQEEA) with its higher basicity is the phosphorylation site preferred by the cGK (Fig. 9). The threonine site (MNAVLARRRKATQVGE) is very basic because of its multiple arginine residues amino-

served when the phosphorylation of serines 1 and 2 has been essentially completed (Fig. 8).

Synthetic peptides corresponding to the three identified VASP phosphorylation sites were also phosphorylated by both cAK and cGK. Both protein kinases phosphorylated peptide 2 (containing serine 2) with similar kinetic constants, whereas peptide 1 (containing serine 1) was an excellent cGK substrate and a relatively poor cAK substrate (Table 11). Peptide 3 (containing threonine) was phosphorylated by cAK and cGK with similar kinetic constants but appeared to be a relative poor substrate (low V,,) for both protein kinases.

The data with the three synthetic peptides not only support the interpretation of the phosphorylation kinetics obtained with the complete VASP protein (see above) but also establish peptide 1 (the serine 1-containing VASP phosphorylation site) as one of the best and most selective cGK substrates (Table 11). Most cAK substrates are poor cGK substrates, whereas some proteins are phosphorylated relatively well by both protein kinases. For example, DARPP-32, a dopamine and AMP-regu- lated phosphoprotein, has been shown to be phosphorylated on a threonine (ThP4) residue with high efficiency by both cAK and cGK both in vitro and with intact preparations of certain brain regions (29-31). There are only a few examples of pro-

Protein Kinase Phosphorylation Sites of VASP 14515 TABLE I1

Kinetic constants for the phosphorylation of synthetic VASP peptides and other peptide substrates by cGK and cAK Synthetic VASP peptides 1-3 and Kemptide were used as kinase substrates as described under "Experimental Procedures." Kinetic constants

were obtained from initial velocity data by double-reciprocal plots. The specificity index (21) of cGK is expressed as V,JK,,, for cGK divided by V,,,/K, for cAK. A value >1 indicates that the peptide is a better substrate for the cGK. The K,,, and V,, values are averages from two or three experiments. The amino acid sequence at the phosphorylation site of G-substrate is shown. The kinetic constants were obtained using the whole protein (23). The phosphorylated serine or threonine is shown in bold.

Peptide Sequence K m vm, Specificity

cGK CAK cGK CAK index Ref.

BM ymollminlmg 1 LRKVSKQE 94 1,395" 3.7 2.6" 21 2

This study IERRVSNAG 30 26 2.2 2.7 0.71

3 This study

RRRKATQVGE 35 89 0.23 0.44 1.33 This study Kemptide LRRASLG 125 9.1 2.49 3.53 0.05 This study Kemptide LRRASLG 120 4.5 4.5 16 19, 20 BPDide RKISASEFDRPLR 68 320 11 3.2 16 21 G-substrate -RRKDTPALH- 0.21 5.8 2.2 2.3 26 22

For peptide 1 phosphorylation by c A K , saturating substrate concentrations were experimentally not possible because of the high K,,,. The highest peptide concentration used was 2 mM.

S N P P G - E l

- "" - ""

5 0 kDa

4 6 kDa> "- - -- c 5 0 kDa

0 ' 1' 3' 5' 10' min 0' 1' 3' 5' 10' min

and PGE,. 32Pi-Labeied intact human platelets were incubated with 100 PM SNP or 10 p~ PGE, for the times indicated, and VASP was FIG. 11. Immunoprecipitation analysis of the time dependent VASP phosphorylation in intact human platelets in response to SNP

subsequently immunoprecipitated from the cell lysates. Immunoprecipitates were separated on a 9% SDS gel, and radioactive bands were visualized by autoradiography.

teins and corresponding peptides which are specific cGK substrates. Certain sites in histone H1 and H2B are selectively phosphorylated by the cGK (24,32,33). A 23-kDa protein from cerebellum termed G-substrate (22,23) and more recently the bovine lung cGMP-binding cGMP-specific phosphodiesterase (34) were identified by in vitro experiments as selective substrates for cGK in comparison with cAK. Unfortunately, experiments with intact cells are lacking in these examples. There- fore, it was of considerable importance to analyze whether the three identified VASP phosphorylation sites are also used by cAK and cGK in intact cells.

The data presented (Fig. 9) indicate that phosphorylation at serine 2 is responsible for the reduced mobility of the 50-kDa phospho-VASP compared with the 46-kDa form in SDS-PAGE (3, 4; see also Fig. 1). The observed mobility shift probably reflects a phosphorylation-induced conformational change and altered SDS binding capacity of VASP, resulting in decreased VASP mobility in SDS-PAGE. Interestingly, the phosphorylation site containing serine 2 (in contrast to the two other phosphorylation sites, which do not affect VASP mobility in SDS- PAGE) is in the close vicinity of a proline-rich VASP domain? This phosphorylation-induced shift in the apparent molecular mass of VASP from 46 to 50 kDa has been very useful for examining the regulation of VASP phosphorylation in a variety of intact cell preparations (4-11). These studies and the data obtained here indicate that both cAK and cGK are capable of phosphorylating VASP at serine 2 in intact platelets and other cells. However, analysis of the other VASP phosphorylation sites in experiments with intact cells was previously not possible but could only now be performed with the methods developed in this study.

VASP immunoprecipitation experiments using extracts of

C. Haffner, M. Reinhard, J. Hoppe, S. M. Lohman, T. Jarchau, and U. Walter, manuscript in preparation.

32P-labeled platelets established that some basal VASP phosphorylation is already present in resting platelets (both in the 46-kDa form and in the 50-kDa form) which is increased in response to the CAMP-elevating PGE, and the cGMP-elevating agent SNP (Fig. 11). CNBr cleavage analysis of VASP immunoprecipitated from extracts of 32P-labeled platelets demonstrated that neither PGE, nor SNP increased the phosphorylation of the threonine residue (9-kDa VASP peptide) in intact platelets but strongly stimulated the phosphorylation of serines 1 and 2 (22-kDa VASP peptide) (Fig. 12). Additional Lys-C digestion analysis demonstrated that PGE, and SNP increased the phosphorylation of serine 2 (12-kDa peptide) and serine 1 (6-kDa peptide) in intact platelets. Interestingly, PGE, rapidly increased serine 2 phosphorylation (12-kDa peptide) followed by a somewhat delayed serine 1 phosphorylation (6- kDa peptide). In contrast, the time course of SNP-induced serine 1 (6-kDa peptide) and serine 2 phosphorylation (12 kDa) was very similar. Since nitric oxide-generating agents may el- evate platelet CAMP and activate cAK due to the cGMP-inhib- ited phosphodiesterase type I11 (35) we also performed experiments with cell membrane-permeant selective activators of platelet cGK (8-PCP"-cGMP) (7) and cAK (Sp-5,6-DCl-cBiMPS) (6). The pattern and relative time course of VASP peptide phosphorylation in 32P-labeled human platelets stimulated by Sp- 5,6-DCl-cBiMPS or 8-pCPT-cGMP (data not shown) resembled those evoked by PGE, or SNP, respectively (Fig. 12). These data indicate that the selectivity of cAK-mediated VASP phosphorylation in vitro (with purified protein) and in intact human platelets is very similar, whereas the cGK selectivity for serine 1 in intact human platelets (Fig. 11) is not as strong as observed in vitro (Fig. 9).

The data of this study are also in agreement with previous publications from our group (2,3) in which tryptic fingerprints of VASP phosphorylated in vitro and in intact cells were re-

14516 Protein Kinase Phosphorylation Sites of VASP

C - Digallom

ma

s e r l - --- 6 9

0 0.5 1 3 5 1 o m h

SNP

Lyn C - Diguliom

ma

P

s e r l - a 6

0 1 3 5 1 o m h PO-E,

12

CNBr Cleavage - serl+2

o a5 1 3 5 10 ’ m h

SNP

CNBr Clunge

s e r l + Z

FIG. 12. Lys-C digestion and CNBr cleavage analysis of the time-dependent VASP phosphorylation in intact human platelets. 32Pi-Labeled intact human platelets were incubated with 100 p~ SNP or 10 p~ PGE, for the times indicated, and VASP was subsequently immunoprecipitated from the cell lysates and digested with either Lys-C or cleaved by CNBr as described under “Experimental Procedures.” Samples were separated on a 16% Tricine gel, and radioactive bands were visualized by autoradiography. The locations of serine- and threonine- containing peptides are indicated (Ser I + 2,22-kDa fragment; Ser 2, 12-kDa fragment; Ser I , 6-kDa fragment; Thr, 9-kDa fragment).

P P

P P I I

Scr2 Scr 1 Thr + P P P I l l

S n Z Scr 1 Tbr

FIG. 13. Proposed model of VASP phosphorylation by cGMP- or cAMP-dependent protein kinase. The order of phosphorylation of the three sites by the two protein kinases is indicated as well as the shift in the apparent molecular mass of VASP in SDS-PAGE caused by serine 2 phosphorylation. As discussed in the text, the cGK selectivity for serine 1 phosphorylation is stronger in vitro (purified protein) than in intact human platelets. Recent cloning data (see Footnote 3) indicate that the phosphorylated amino acids Thr, Ser I , and Ser 2 correspond to ThP*, SeP9, SerLS7 of the VASP protein, respectively.

ported. Tryptic phosphopeptide maps of VASP phosphorylated by cAK and cGK in vitro and in intact human platelets were identical and closely resembled those of 46-kDa VASP tran- siently phosphorylated by cGK in vitro (2, 3).

An important difference between in vitro experiments (with purified proteins) and experiments with intact cells is the extent of cGK-mediated VASP phosphorylation. Zn vitro, both purified cGK and cAK are capable of converting essentially all of

46-kDa VASP to the BO-kDa form (3, 4, 11; see also Fig. 11, which is also the case with &-mediated VASP phosphorylation in intact cells (5,6,9,10,36; see also Fig. 11). In contrast, activation of cGK in intact cells by nitric oxide-generating agents (SNP and other nitrovasodilators) and membrane-permeant cGK activators maximally converts up to 50% of VASP to its BO-kDa form (4,7,8,10,11; see also Fig. 11). In this respect, it is of considerable interest that CAMP-elevating agents and cGMP-elevating nitrovasodilators inhibit the agonist-evoked fibrinogen receptor (glycoprotein IIbDIIa) expression in human platelets up to 100 and 50%, respectively (37, 38). These data suggest a correlation between fibrinogen receptor down-regulation and platelet inhibitor-caused phosphorylation of VASP, which has been recently identified as a novel focal adhesion- associated protein (10). Since focal adhesions are important structures for the regulation of inside-out and outside-in sig- naling through the cell membrane (39), elucidation of VASP structure and function, its regulation by phosphorylation, and its possible association with other focal adhesion proteins are of considerable interest.

The data presented here compared for the first time the phosphorylation kinetics and protein kinase selectivity of cAU and cGK for the three VASP phosphorylation sites both in vitro and in an intact cell preparation. As schematically shown in Fig. 13, the sides include a typical cAK consensus phosphorylation site (Ser Z), a phosphorylation site preferred by the cGK in vitro (Ser 11, and a weaker phosphorylation site common for both cyclic nucleotide-dependent protein kinases (Thr). The sequential phosphorylation of the three sites as indicated in Fig. 13 represents best the results obtained with purified proteins since the selectivity of the cGK for serine 1 in intact platelets is less significant. Our data indicate that in vitro data cannot be simply extrapolated to the situation in intact cells

Protein Kinase Phosphorylation Sites of VASP 14517

since many additional factors (e.g. intracellular concentration of second messengers, kinases and substrates, compartmental- ization, substrate accessibility, interaction with other proteins) modify phosphorylation specificity and kinetics (11). Our data demonstrate that cAK and cGK have overlapping substrate specificity not only in vitro but also in intact cells. Our results also demonstrate important quantitative and qualitative differences between cAK- and cGK-mediated VASP phosphorylation in intact human platelets. However, the role of VASP phosphorylation for the structure and regulation of focal adhesion sites and their associated proteins remains to be elucidated.

Acknowledgments-We are grateful to Dr. Fehlhaber for the electrospray ionization mass spectrometry and amino acid analysis. We also thank A. Wolpert for help with the peptide synthesis, C. Dees for tech- nical assistance, several members of our clinical research unit for con- structive criticism of this work, and Dr. T. Jarchau for help in computer analysis of peptide cleavage and phosphorylation sites.

1.

2.

3. 4.

5.

6 .

7.

8.

9.

10.

11.

12.

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