Phosphorylation Sites in the Integrin beta 3 Cytoplasmic Domain in Intact Platelets*

Kenneth M. LereaDagger , Kenneth P. Cordero, Kjell S. Sakariassen§, Rita I. Kirk, and Victor A. Fried

From the Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York 10595 and the § Department of Biology, University of Oslo, 0317 Oslo, Norway

    ABSTRACT
Top
Abstract
Introduction
References

Protein seryl/threonyl phosphatase inhibitors such as calyculin A block inside-out and outside-in platelet signaling. Our studies demonstrate that the addition of calyculin A blocks platelet adhesion and spreading on fibrinogen, responses that depend on integrin alpha IIbbeta 3 signaling. We hypothesized that this reflects a change in alpha IIbbeta 3 structure caused by a specific state of phosphorylation. We show that addition of calyculin A leads to increased phosphorylation of the beta 3 subunit, and phosphoamino acid analysis reveals that only threonine residues become phosphorylated; sequence analysis by Edman degradation established that threonine 753 became stoichiometrically phosphorylated during inhibition of platelet phosphatases by calyculin A. This region of beta 3 is linked to outside-in signaling such as platelet spreading responses. The effect of calyculin A on platelet adhesion and spreading and on the phosphorylation of T-753 in beta 3 is reversed by the calcium ionophore A23187, demonstrating that these effects of calyculin A are not generally toxic ones. We propose that phosphorylation of beta 3 on threonine 753, a region of beta 3 linked to outside-in signaling, may be a mechanism by which integrin alpha IIbbeta 3 function is regulated.

    INTRODUCTION
Top
Abstract
Introduction
References

Effective hemostasis depends on three basic platelet responses (1). First, adhesion, which includes attachment and spreading reactions, initiates events that lead to formation of a hemostatic plug. Second, secretion of factors aid in the activation of additional platelets and in the repair of the damaged vessel wall. Third, aggregation of recruited platelets to each other and to adhered platelets results in growth of the hemostatic plug. Both adhesive and aggregatory responses are controlled by the coordinate actions of cell surface glycoprotein receptors. Many of these receptors are dimers of two different subunits (2). These include selective members of the integrin family of cell adhesion molecules such as alpha 2beta 1 (collagen-binding), alpha 6beta 1(laminin), alpha 5beta 1 (fibronectin), and alpha IIbbeta 3 (fibrinogen and von Willebrand factor) (for review, see Ref. 1).

The alpha IIbbeta 3 complex (glycoprotein IIb-IIIa) is unique among integrin molecules expressed on platelets; it functions as a binary switch. On resting platelets, alpha IIbbeta 3 exists in an inactive conformation in which the binding pocket for soluble forms of fibrinogen and von Willebrand factor is cryptic. Thus, before activation, the interaction of alpha IIbbeta 3 is limited to surface-bound fibrinogen, which supports adhesion under low shear forces (2-5). As a consequence of platelet activation, alpha IIbbeta 3 converts to an active conformation (via a process referred to as inside-out signaling) and becomes capable of binding other surface-bound molecules and soluble forms of fibrinogen and von Willebrand factor (6). This latter activity facilitates platelet aggregation. The molecular events that switch alpha IIbbeta 3 into a high affinity state are not known. Structural correlations have identified potential cytoplasmic motifs in beta 3 that regulate its activation. First, a naturally occurring mutation in the cytoplasmic domain of alpha IIbbeta 3 negatively modulates its activity: serine 752 to proline mutation in the cytoplasmic portion of beta 3, a variant associated with Glanzmann's thrombasthenia, inhibits alpha IIbbeta 3 signaling (7, 8). Second, site-directed mutagenesis of Asp723 in beta 3 effectively disrupts potential salt bridges between the alpha  and beta  subunits and results in a constitutively active integrin molecule (9). Thus, it appears that changes in charge in the cytoplasmic portion of beta 3 may have important consequences with respect to integrin structure and function. Phosphorylation of beta 3 on a threonine residue, which would change the charge in beta 3, has been implicated in exposing binding sites on alpha IIbbeta 3 (10, 11), although the role that this phosphorylation plays in integrin function is unknown (12).

Suggesting that a phosphorylation event controls alpha IIbbeta 3 activation is in accordance with protein phosphorylation events regulating numerous platelet responses, including adhesion and aggregation (13). Phosphorylation of proteins on serine, threonine, and tyrosine residues is controlled by the competing activities of protein kinases and phosphatases. Thus, increased phosphorylations can represent increased kinase activity or decreased phosphatase activity. Platelets contain numerous protein kinases (13) and protein seryl/threonyl phosphatase types 1, 2A, and 2B (14, 15). Type 1 and 2A protein phosphatases are active in resting and stimulated platelets (14) whereas type 2B, a Ca2+-dependent, calmodulin-stimulated enzyme, is only active after platelet activation (15). To understand the physiological significance of these enzymes in platelets, membrane-permeable inhibitors have been used. Type 1 and 2A phosphatases have been implicated in controlling platelet responses including aggregations (16-23). Very little is known, however, about how these enzymes modulate molecular events linked to the aggregatory response.

Previous studies have shown that calyculin A, a serine/threonine phosphatase inhibitor, inhibits platelet aggregation, which is dependent on alpha IIbbeta 3 function. The present study tests whether calyculin A affects other alpha IIbbeta 3 functions and tests the hypothesis that altered alpha IIbbeta 3 function is linked to altered structure of this integrin. The data show that, in addition to an effect on aggregation, calyculin A blocks platelet adhesion and spreading on fibrinogen, reactions that depend on alpha IIbbeta 3 outside-in signaling. Biochemical analysis links calyculin A effects to beta 3 phosphorylation. Sequence analysis demonstrates that threonine 753, which is in the intracellular segment of beta 3, becomes stoichiometrically phosphorylated. These data suggest that structural modification of beta 3, through phosphorylation, may regulate outside-in, and perhaps inside-out, signaling through the alpha IIbbeta 3 integrin.

    EXPERIMENTAL PROCEDURES

Materials-- Prostaglandin E1, apyrases, aprotinin, leupeptin, benzamidine, protein A-Sepharose, human fibrinogen, low molecular weight heparin (Mr aprox. 3,000), phenylmethylsulfonyl fluoride, sodium vanadate, and dithiothreitol were obtained from Sigma. [32P]Orthophosphate was purchased from DuPont NEN. LC Services supplied calyculin A. Rhodamine-labeled phalloidin was purchased from Molecular Probes (Eugene, OR). Boehringer Mannheim supplied sequencing grade AspN protease and trypsin. Hirudin (BKHV recombinant) was obtained from Calbiochem. The C18 reverse phase TSK-GEL ODS-120T column was purchased from Toso Haas (Montgomeryville, PA). Anti-beta 3 polyclonal antiserum (E8) was a gift from Drs. David Phillips and Debbie Law (COR Therapeutics, San Francisco, CA).

Platelet Preparations-- Human blood (10 parts) was drawn into acid-citrate-dextrose (1 part). Platelet-rich plasma was obtained after centrifugation at 210 × g for 10 min at 21 °C. After the addition of prostaglandin E1 (0.4 µM) and apyrases (12.5 milliunits/ml), platelets were removed by centrifugation at 1100 × g for 15 min at 21 °C. Platelets were resuspended in a modified Tyrode's buffer, pH 6.4, containing 5 mM HEPES, 140 mM NaCl, 1 mM MgCl2, 2 mM KCl, 5.5 mM glucose, 12 mM NaHCO3, 0.2% (w/v) bovine serum albumin, 0.2 µM prostaglandin E1, and apyrases (6 milliunits/ml), recentrifuged at 1100 × g, and suspended in a buffer containing 10 mM HEPES, pH 7.4, 140 mM NaCl, 1 mM MgCl2, 2 mM KCl, 5.5 mM glucose, and 12 mM NaHCO3 at a concentration of 2 × 109/ml.

32P Labeling of beta 3-- Platelet suspensions (4 ml) were incubated with [32P]orthophosphate (0.2 mCi/ml) for 3 h at 37 °C before treatment with calyculin A (100 nM) for 5 min. Platelets were directly solubilized using Nonidet P-40 lysis buffer containing 1% Nonidet P-40, 137 mM NaCl, 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, 0.15 units/ml aprotinin, and 10 mM rho -nitrophenyl phosphate. Detergent-insoluble material was removed by centrifugation at 10,000 × g for 5 min, and the resulting soluble fraction was incubated with anti-beta 3 (10 µg/mg of lysate) for 16 h at 4 °C followed by a 1-h incubation with protein A-Sepharose at 4 °C. The immunoprecipitates were washed three times with Nonidet P-40 lysis buffer and solubilized with SDS sample buffer. Proteins were separated by 10% SDS-PAGE,1 stained using Coomassie Blue, destained, and either dried for autoradiography or maintained hydrated for efficient extraction of beta 3 for identification of phosphorylation sites.

Phosphoamino Acid Analysis-- Phosphoproteins extracted from SDS gels were hydrolyzed in 6 N HCl for 1 h at 110 °C, and phosphoamino acids were separated by two-dimensional electrophoresis on cellulose plates using 2.5% formic acid and 7.8% acetic acid, pH 1.9, in the first dimension and 0.5% pyridine and 5% acetic acid, pH 3.5, in the second dimension (24).

Identification of Phosphorylation Sites-- Immunoprecipitated 32P-labeled beta 3 was reduced and alkylated by the method of Talmadge et al. (29) before SDS-PAGE. The Coomassie Blue-stained beta 3 was cut from the gel using a scalpel and digested with AspN in situ and extracted (25). The extracts were concentrated under vacuum, resuspended in 0.1% trifluoroacetic acid in 6 M guanidine-HCl, and fractionated on a 120T C18 reverse phase column (reverse phase HPLC), collecting fractions by peak detection (30). Isolated peptides were sequenced by automated Edman degradation on a model 477/120A pulse liquid sequencer (Applied Biosystems) using standard chemistry.

Static Platelet Adhesion Studies-- Washed platelets (5 × 108/ml) were treated with either buffer or 100 nM calyculin A for 5 min at 21 °C. After the addition of buffer or A23187 (0.25 µM), the platelets were added to fibrinogen-coated slides and allowed to settle for 20-30 min. In some experiments, apyrase (0.5 units, Sigma) or ADP (10 µM, Sigma) were included in the incubations before adding the cells to the slides. Adhered platelets were processed for confocal microscopy as described below.

Perfusion Adhesion Studies-- Perfusions were conducted using one of three parallel plate perfusion chambers as described previously (26). Blood was drawn into acid-citrate-dextrose at a ratio of 1:10 (acid-citrate-dextrose:blood), hirudin (200 units/ml), or low molecular weight heparin (6 units/ml). Under the flow conditions described below, platelets adhered poorly in the presence of low molecular weight heparin, an anticoagulant that does not chelate calcium (n = 3),2 precluding its use in subsequent studies. This is analogous to its effect on platelet adhering to fibronectin (32). The blood (4 ml) was prewarmed at 37 °C for 10 min before the addition of dimethyl sulfoxide (Sigma, 0.001% final) or varying concentrations of calyculin A. After an additional 5-min incubation at 37 °C, the blood was perfused through the chamber containing a fibrinogen-coated coverslip positioned 70 mm from the inlet valve. Whole blood was used within 3 h of withdrawal. Flow was conducted using two different approaches. In one approach, blood was allowed to recirculate for 10 min at 10 ml/min through an eight-roller precalibrated peristaltic pump (Cole Parmer). Under these conditions, wall shear rates of 650 and 100/s were achieved. Alternatively, whole blood passed through the chambers one time at a constant flow rate for 1-2 min. Flow rates of 1 and 2 ml/min produced 65 and 520/s wall shear rates, respectively, which corresponds to flow in veins and small arteries. When indicated, platelet-rich plasma was prepared by centrifugation at 1100 × g for 3 min at 21 °C. Platelet counts in the platelet-rich plasma were standardized to 350,000/µl using a modified Tyrode's buffer. Calyculin A treatment and perfusion of platelet-rich plasma through the chamber were conducted as described for whole blood. For preparation of surfaces for flow studies, suspensions of 0.1 mg/ml fibrinogen, prepared in phosphate-buffered saline, were sprayed onto plastic coverslips using a retouching airbrush (model 100; Badger, Franklin Park, IL) at a nitrogen pressure of 1 atm as described previously (26). Coverslips (18 × 22 mm) were spray-coated with ~300 ul to a final surface density of ~5 µg/cm2. The coverslips were kept at 21 °C for up to 12 h. Before perfusion with whole blood or platelet-rich plasma, coverslips were washed for 5 min with a modified Tyrode's buffer, pH 7.4. For static adhesions, drops of the fibrinogen solution were pipetted onto glass slides and dried at room temperature. The matrix was washed with the modified Tyrode's buffer before the adhesion assay. After perfusion with blood, the coverslips were immediately washed under flow conditions with the modified Tyrode's buffer for 1 min. The coverslips were removed, fixed in a 2.5% glutaraldehyde solution for 15 min, and washed twice with phosphate-buffered saline. After permeabilization with 0.1% Triton X-100 for 3 min, platelets were washed twice with TBS, blocked for 3 min with 0.1% bovine serum albumin, and stained with rhodamine-conjugated phalloidin. Adhered platelets were imaged using a Bio-Rad MRC-1000 confocal microscope. Platelets were viewed after excitation with an argon laser beam using either an inverted- or upright-staged microscope equipped with a 60× oil immersion or 40× dry objective and an epifluorescent illumination attachment. The percentage of area covered by platelets was determined using a digitized palette and Sigmascan software.

    RESULTS AND DISCUSSION

beta 3 Phosphorylation Correlates with Decreased Outside-in Signaling-- Previous studies have shown that calyculin A, an inhibitor of protein seryl/threonyl phosphatase types 1 and 2A, markedly reduces platelet aggregation induced by collagen (18) and low doses of thrombin (17). These data support the idea that calyculin A blocks inside-out signaling linked to alpha IIbbeta 3. In the presence of high thrombin doses, calyculin A does not inhibit aggregation but does block cytoskeletal assembly that is usually coupled to an aggregatory response (17). This indicates that calyculin A blocks outside-in signaling by activated alpha IIbbeta 3. To further test this hypothesis, we examined the effect of calyculin A on platelet adhesion to immobilized fibrinogen, a process that depends on outside-in alpha IIbbeta 3 signaling. Platelet adhesion was measured in both flow and static assays.

To determine the effect of calyculin A on platelet adhesion in a physiological setting, we used flow conditions. Citrated blood was treated with either vehicle or 100 nM calyculin A and recirculated through a parallel plate perfusion chamber at a wall shear rate of 650/s. The percent area covered by vehicle-treated platelets was ~40% (n = 5) on fibrinogen-coated surfaces (Fig. 1A). Adhesion was blocked by 80 ± 5% (mean ± S.E.; n = 4) by preincubating the blood with 100 nM calyculin A (Fig. 1B). Similarly, calyculin A added directly to platelet-rich plasma inhibited platelet deposition to fibrinogen, establishing that the drug was directly affecting platelets.


View larger version (77K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of calyculin A on platelet adhesion to fibrinogen under flow. Citrated blood was treated with dimethyl sulfoxide (0.001%) or 100 nM calyculin A for 5 min before perfusion over fibrinogen-coated coverslips. Perfusion at 650/s was conducted in a recirculating setting for 10 min, and adhered cells were viewed with a 60× immersion objective. The data are representative of five experiments.

As seen in Fig. 1, substantial aggregation occurred when a recirculating system was used. This likely reflects the activation of platelets as a consequence of being exposed to the fibrinogen matrix and to activating agents release from adhered cells. To minimize this aggregatory response, whole blood was passed through the chambers one time at wall shear rates of 65 and 520/s. Under these conditions platelets adhered as singlets (Fig. 2, A and C). Regardless of the flow rate, a decrease in deposition by >90% (n = 4) was observed by pretreating whole blood with calyculin A (Fig. 2, B and D). The decrease in platelet adhesion in citrated blood occurred in the presence of 100 nM calyculin A. As discussed later, the response to calyculin A occurs at a very sharp concentration threshold. These findings were confirmed using varying preparations of calyculin A. 


View larger version (55K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of calyculin A on platelet adhesion: single pass conditions and various anticoagulants. Blood drawn into acid-citrate-dextrose (ACD; A-D) or hirudin (E and F) were treated without (A, C, and E) or with (B, D, and F) 100 nM calyculin (Cal A). Perfusions were conducted for 2 min at 65/s (A and B) or for 1 min at 520/s (C-F), and adhered cells were viewed with a 40× objective. The data are representative of three to five experiments.

The dramatic effect of phosphatase inhibitors on the adhesive properties of platelets observed in citrated blood might possibly be exaggerated by the effects of citrate itself in the experimental system. It is known that divalent cations modulate the binding of alpha IIbbeta 3 to fibrinogen, and it is possible that citrate, as a divalent cation chelator, is modifying platelet interactions that are exaggerating the effect of calyculin A (31). To determine whether the effect of phosphatase inhibition was dependent on citrate, we repeated the adhesion studies using the anticoagulant hirudin, which is not an ion chelator. Platelets from hirudin-treated blood adhered as singlets in the absence of calyculin A (Fig. 2E; n = 2), and, as with the citrated blood, adhesion was reduced by 90% in the presence of calyculin A (Fig. 2F; n = 2). Therefore, the effect of phosphatase inhibition by calyculin A on platelet adhesion is not dependent on the anticoagulant, strongly suggesting that phosphorylation plays a direct role in regulating platelet adhesion under physiological conditions.

Adhesion represents the attachment and spreading of platelets on matrices. This latter response confers tight adhesion, allowing platelets to withstand high shear forces associated with arterial and venous flow. To test whether the effect of calyculin A on platelet adhesion seen under flow conditions correlates with decreased platelet spreading, we monitored the effect of calyculin A on platelet spreading to fibrinogen. On adherence, >90% nontreated platelets spread (Fig. 3A). In contrast, calyculin A-treated platelets adhered, but only as singlets (Fig. 3B). Thus, under conditions in which type 1 and 2A phosphatases are inhibited, platelets attach but do not spread. Their ability to attach was further demonstrated by Western analysis of adhered "control" and "calyculin A-treated" platelets using beta 3 antibodies; similar levels of protein were found, indicating that equal numbers of platelets adhered (data not shown). Increased spreading of calyculin A-treated platelets can be restored by applying calcium ionophore (A23187) to the platelet suspension before they settle onto fibrinogen. The addition of 0.25 µM A23187 to a suspension of calyculin A-treated platelets resulted in increased platelet spreading (Fig. 3C). The reversal of calyculin A inhibition by A23187 demonstrates that inhibition of phosphatases has a specific effect on platelet functions. The apparent effect of A23187 did not result from the release of ADP; the addition of apyrases did not reverse the ionophore effect, and the addition of 10 µM ADP did not overcome the effect of calyculin A. The effect of the calcium ionophore appears to be calmodulin-dependent; trifluoperazine prevents the effect of A23187 (Fig. 3D).


View larger version (121K):
[in this window]
[in a new window]
 
Fig. 3.   Light micrographs of platelet adhesion to fibrinogen under static conditions: effect of A23187 on reversing the effect of calyculin A. Washed platelets were treated with dimethyl sulfoxide (A) or 100 nM calyculin A (Cal A; B-D) for 5 min before treatments with A23187 alone (C), or A23187 plus 10 µM trifluoperazine (D). Platelets were allowed to settle onto fibrinogen-coated slides for 20 min. Nonadhered cells were removed by aspiration, and the adhered cells were fixed, permeabilized, stained with phalloidin-labeled rhodamine, and visualized using confocal microscopy with a 60× immersion lens. These images are representative micrographs of four to six separate experiments. The phosphorylation states of beta 3 from cells treated with buffer (A), calyculin A alone (B), or calyculin A and A23187 (C) are shown adjacent to each panel title. Phosphorylation was determined by immunoprecipitation as described under "Experimental Procedures," and the bands corresponding to beta 3 are shown.

Identifying Phosphorylation Sites in alpha IIbbeta 3-- Because the platelet responses affected by calyculin A are mediated by alpha IIbbeta 3, we hypothesized that hyperphosphorylation of alpha IIbbeta 3 could be the structural change that modifies alpha IIbbeta 3 function. Thus, studies were undertaken to determine the phosphorylation state of alpha IIbbeta 3 in calyculin A-treated platelets. The cytoplasmic portion of beta 3 (residues 716-762) contains 8 potential serine and threonine phosphorylation sites: 7 threonines and 1 serine (27). To determine its phosphorylation state, beta 3 was immunoprecipitated from platelets that had been labeled with [32P]orthophosphate and treated with calyculin A. Immunoprecipitated beta 3 was analyzed by SDS-PAGE followed by autoradiography. As seen in Fig. 3, little beta 3 was phosphorylated in the absence of calyculin A (Fig. 3A), whereas a loss of phosphatase activity by calyculin A treatment led to increased phosphorylation of beta 3 (Fig. 3B). This correlated with a decrease in spreading. The addition of a calcium ionophore, which partially restores spreading, caused a corresponding dephosphorylation in beta 3 (Fig. 3C). The major protein band that becomes phosphorylated by calyculin A treatment is beta 3, as identified by Western analysis (Fig. 4A). Maximal phosphorylation occurred at 100 nM calyculin A (Fig. 5; n = 3), a concentration that inhibits adherence to fibrinogen (as shown in Figs. 1, 2, and 5) and collagen-induced aggregations (18). The same concentration of ionophore that reversed the effect of calyculin A on platelet spreading decreased the phosphorylation of beta 3 (shown in Fig. 3), which is likely attributable to the activation of the calmodulin-dependent phosphatase, protein phosphatase 2B. Together, the data indicate that an inverse relationship exists between beta 3 phosphorylation and platelet adhesion reactions.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 4.   Phosphorylation of beta 3 in calyculin A-treated platelets. A, [32P]-labeled platelets were incubated with either vehicle (lane 1) or 100 nM calyculin A (lane 2) for 5 min. Immunoprecipitated beta 3 was subjected to SDS-PAGE and visualized by autoradiography. beta 3 was identified by Western analysis as the phosphorylated band migrating at ~110 kDa. The entire gel is shown in A. This autoradiogram is representative of six different experiments. B, phosphorylated beta 3 was subjected to phosphoamino acid analysis as described under "Experimental Procedures." PT, phosphothreonine; PS, phosphoserine; PY, phosphotyrosine.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 5.   Dose responsiveness of beta 3 phosphorylation and platelet adhesion to calyculin A. Washed 32P-labeled platelets and whole blood were treated with varying concentrations of calyculin A before phosphorylation studies or adhesion assays, respectively. In both cases, calyculin A treatment lasted 5 min. Bands corresponding to immunoprecipitated beta 3 are shown and are representative of three different experiments. For platelet adhesions, perfusions were conducted for 2-8 min using both recirculating and single pass conditions and chambers that mimic arterial flow. Platelet adhesion values are means ± S.E. of three independent experiments.

Phosphoamino acid analysis of beta 3 showed the presence of only phosphothreonine (Fig. 4B). The sites of phosphorylation of beta 3 were determined directly by a two-dimensional peptide isolation and sequencing approach. beta 3 was immunoprecipitated from 32P-labeled platelets exposed to 100 nM calyculin A, reduced, alkylated, and separated from coprecipitating polypeptides by SDS-PAGE. The Coomassie Blue-stained beta 3 band was excised and digested in situ with AspN. Proteolytic fragments were separated by reverse phase HPLC. Peptide peaks (and gaps between peaks) were collected, and their content of 32P was determined by Cerenkov counting. Two prominent 32P-labeled peptides were recovered (Fig. 6A, N-1 and N-2). Approximately 40% of the 32P was recovered in N-1, and 60% was recovered in N-2. These radiolabeled peptide fractions were digested with trypsin and refractionated by reverse phase HPLC. All the radioactivity from each digest was recovered in single peaks, indicating the presence of only one phosphopeptide from each digest. Both of these phosphopeptides were sequenced, and the resulting analysis revealed that both peptides were residues 749-760 of beta 3 (Fig. 6A). The sequences of these peptides differed in that the peptide generated from N-1 was missing a detectable signal at threonine 753, whereas the peptide generated from N-2 was missing the threonine residues at positions 751 and 753. Because a phosphorylated residue is not released for detection in the Applied Biosystems protein sequencers, the absence of a signal at these positions is consistent with these threonine residues being the sites of phosphorylation. Using quantitative regression to extrapolate the initial yield of each peptide (Fig. 6B), the singly (Thr753) phosphorylated peptide, N-1, was ~ 3 pmol, and the doubly (Thr751 and Thr753) phosphorylated peptide, N-2, was ~2 pmol. The amount of peptide 32P sequenced was initially 160 cpm for N-1 peptide and 230 cpm for N-2 peptide, and, assuming equal efficiencies for initial coupling during sequencing of these samples, the specific activity for N-1 is 53 cpm/pmol, and that for N-2 is 115 cpm/pmol. These results are consistent with one phosphorylation site in peptide N-1 and two in peptide N-2; this independently confirms the phosphothreonine assignment derived from direct Edman sequencing.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 6.   Sequence analysis of beta 3 phosphorylation sites. A, 32P-labeled beta 3 was excised from gels, subjected to digestion with AspN protease, and chromatographed by reverse phase HPLC. Peptides were eluted with an acetonitrile gradient, and phosphopeptides were detected by Cerenkov counting. Peptides N-1 and N-2 were further digested with trypsin and subjected to sequence analysis. The amino acid sequences of the tryptic fragments generated from N-1 and N-2 are presented in the box. Similar results were obtained in two separate experiments. B, quantitative regression analysis of amino acids released from the tryptic peptides of N-1 and N-2 during each cycle of the Edman degradation. The recovery of threonines in cycle 5 from N-1 and cycles 3 and 5 for N-2 were below delectability (<0.1 pmol).

Direct sequence analyses of other peptide peaks obtained from the same AspN digests gave recoveries of ~5-8 pmol (data not shown). Because these peptides represented extracellular regions of beta 3 not related to phosphorylation, the recovery of phosphorylated peptides (~5 pmol) is consistent with a nearly stoichiometric phosphorylation (63-100%) of platelet beta 3. Thus, inhibiting protein seryl/threonyl phosphatase activity in platelets results in the phosphorylation of almost all beta 3 molecules at Thr753 and the phosphorylation at Thr751 in ~50% of beta 3 molecules.

Conclusion-- The present study tests the hypothesis that decreased type 1 and 2A phosphatase activity affects platelet responses by altering the structure of alpha IIbbeta 3. Indeed, the addition of calyculin A decreases platelet adhesion and spreading on fibrinogen. A major finding of these studies is that phosphatase inhibition leads to increased phosphorylation of beta 3 on threonine residues, linking this phosphorylation event to decreased outside-in signaling. These data suggest that protein phosphatases are critical for regulating alpha IIbbeta 3 activity. The phosphorylation of beta 3 occurs on threonine residues adjacent to serine 752. Given that a point mutation of this serine to proline adversely affects alpha IIbbeta 3 function and is linked to reduced aggregation (7), outside-in signaling (8), and ability of beta 3 to recruit signaling proteins (33), it is tempting to speculate that phosphorylation of threonines 753 and 751 disrupts alpha IIbbeta 3 signaling in an analogous fashion. Finally, being that threonine 753 in beta 3 is conserved in the other beta -integrin subunits (28), we hypothesize that phosphorylation of this conserved residue may be a common mechanism to negatively modulate integrin adhesiveness to ligands. In support of this, preliminary studies indicate that calyculin A blocks platelet adhesion to collagen and laminin, which bind via integrins that contain beta 1 subunits.

    ACKNOWLEDGEMENTS

We thank Drs. David Phillips and Debbie Law for kindly providing anti-beta 3 antiserum and Dr. Phillips for critically evaluating the manuscript, Drs. Sansar Sharma and Marisa Cotrina for expertise with confocal microscopy, Anne Marie Snow for preparation of the figures, and Steve Mills, Kelly Pun, and Paul Thur for discussions throughout these studies.

    FOOTNOTES

* This work was supported by Grant HL4489301 from the NHLBI, National Institutes of Health, and Grant NS29542 from the NINDS, National Institutes of Health (to V. A. F.).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.

Dagger Recipient of a Genentech established investigatorship from the American Heart Association. To whom correspondence should be addressed: Dept. of Cell Biology and Anatomy, Basic Science Bldg., New York Medical College, Valhalla, NY 10595. Tel.: 914-594-4097; Fax: 914-594-4653; E-mail: ken_lerea{at}nymc.edu.

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.

2 R. I. Kirk and K. M. Lerea, unpublished observation.

    REFERENCES
Top
Abstract
Introduction
References

  1. Kroll, M. H., Hellums, J. D., McIntire, L. V., Schafer, A. I., and Moake, J. L. (1996) Blood 88, 1525-1541[Free Full Text]
  2. Rosenfeld, S. J., and Gralnick, H. R. (1997) Acta Haematol. 97, 118-125[Medline] [Order article via Infotrieve]
  3. Zaidi, T. N., McIntire, L. V., Farrell, D. H., and Thiagarajan, P. (1996) Blood 88, 2967-2972[Abstract/Free Full Text]
  4. Ruggeri, Z. M. (1997) Thromb. Haemost. 78, 611-616[Medline] [Order article via Infotrieve]
  5. Savage, B., and Ruggeri, Z. M. (1991) J. Biol. Chem. 266, 11227-11233[Abstract/Free Full Text]
  6. Goto, S., Salomon, D. R., Ikeda, Y., and Ruggeri, Z. M. (1995) J. Biol. Chem. 270, 23352-23361[Abstract/Free Full Text]
  7. Chen, Y.-P., Djaffar, I., Pidard, D., Steiner, B., Cieutat, A.-M., Caen, J. P., and Rosa, J. P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10169-10173[Abstract]
  8. Chen, Y.-P., O'Toole, T. E., Ylanne, J., Rosa, J.-P., and Ginsberg, M. H. (1994) Blood 84, 1857-1865[Abstract/Free Full Text]
  9. Hughes, P. E., Diaz-Gonzalex, F., Leong, L., Wu, C., McDonald, J. A., Shattil, S. J., and Ginsberg, M. H. (1996) J. Biol. Chem. 271, 6571-6574[Abstract/Free Full Text]
  10. Parise, L. V., Criss, A. B., Nannizzi, L., and Wardell, M. R. (1990) Blood 75, 2363-2368[Abstract]
  11. van Willigen, G., Hers, I., Gorter, G., and Akkerman, J.-W. N. (1996) Biochem. J. 314, 769-779[Medline] [Order article via Infotrieve]
  12. Hillery, C. A., Smyth, S. S., and Parise, L. V. (1991) J. Biol. Chem. 266, 14663-14669[Abstract/Free Full Text]
  13. Siess, W. (1989) Physiol. Rev. 69, 58-178[Free Full Text]
  14. Lerea, K. M. (1991) Biochemistry 30, 6819-6824[Medline] [Order article via Infotrieve]
  15. Tallant, E. A., and Wallace, R. W. (1985) J. Biol. Chem. 260, 7744-7751[Abstract/Free Full Text]
  16. Lerea, K. M. (1992) Biochemistry 31, 6553-6561[Medline] [Order article via Infotrieve]
  17. Hoyt, C. H., and Lerea, K. M. (1995) Biochemistry 34, 9565-9570[Medline] [Order article via Infotrieve]
  18. Chiang, T. M. (1993) Arch. Biochem. Biophys. 302, 56-63[CrossRef][Medline] [Order article via Infotrieve]
  19. Sakon, M., Murata, K., Fujitani, K., Yano, Y., Kambayashi, J., Uemura, Y., Kawasaki, T., Shiba, E., and Mori, T. (1993) Biochem. Biophys. Res. Commun. 195, 139-143[CrossRef][Medline] [Order article via Infotrieve]
  20. Murata, K., Sakon, M., Kambayashi, J., Yukawa, M., Yano, Y., Fujitani, K., Kawasaki, T., Shiba, E., and Mori, T. (1993) J Cell. Biochem. 51, 442-445[Medline] [Order article via Infotrieve]
  21. Koike, Y., Ozaki, Y., Qi, R., Satoh, K., Kurota, K., Yatomi, Y., and Kume, S. (1994) Cell Calcium 15, 381-390[Medline] [Order article via Infotrieve]
  22. Yano, Y., Kambayashi, J., Shiba, E., Sakon, M., Oiki, E., Fukuda, K., Kawasaki, T., and Mori, T. (1994) Biochem. J. 299, 303-308[Medline] [Order article via Infotrieve]
  23. Nishikawa, M., Toyoda, H., Saito, M., Morita, K., Tawara, I., Deguchi, K., Kuno, T., Shima, H., Nagao, M., and Shirakawa, S. (1994) Cell. Signal. 6, 59-71[Medline] [Order article via Infotrieve]
  24. Boyle, W. J., van der Geer, P., and Hunter, T. (1991) Methods Enzymol. 201, 110-149[Medline] [Order article via Infotrieve]
  25. Rosenfeld, J., Capdevielle, J., Guillemot, J. C., and Ferrara, P. (1992) Anal. Biochem. 203, 173-179[Medline] [Order article via Infotrieve]
  26. Sakariassen, K. S., Joss, R., Muggli, R., Kuhn, H., Tschopp, T. B., Sage, H., and Baumgartner, H. R. (1990) Arteriosclerosis 10, 276-284[Abstract]
  27. Fitzgerald, L. A., Steiner, B., Rall, S. C., Lo, S., and Phillips, D. R. (1987) J. Biol. Chem. 262, 3936-3939[Abstract/Free Full Text]
  28. Pasqualini, R., and Hemler, M. E. (1994) J. Cell Biol. 125, 447-460[Abstract]
  29. Talmadge, K., Kaufman, J., and Gilbert, W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3988-3992[Abstract]
  30. Deshpande, K. L., Fried, V. A., Ando, M., and Webster, R. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 36-40[Abstract]
  31. Kirchhofer, D., Gailit, J., Ruoshlahti, E., Grzesiak, J., and Pierschbacher, M. D. (1990) J Biol. Chem. 265, 18525-18530[Abstract/Free Full Text]
  32. Beumer, S., Ijsseldijk, M. J. W., de Groot, P. G., and Sixma, J. J. (1994) Blood 84, 3724-3733[Abstract/Free Full Text]
  33. Law, D. A., Nannizzi-Alaimo, L., and Phillips, D. R. (1996) J. Biol. Chem. 271, 10811-10815[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.