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J Biol Chem, Vol. 274, Issue 47, 33474-33479, November 19, 1999


The Cytoplasmic Domain of the Platelet Glycoprotein Ibalpha Is Phosphorylated at Serine 609*

Richard J. Bodnar, Minyi Gu, Zhenyu Li, Graham D. Englund, and Xiaoping DuDagger

From the Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The alpha  chain of the platelet von Willebrand factor receptor, glycoprotein (GP) Ib, is not known to be phosphorylated. Here, we report that the cytoplasmic domain of GPIbalpha is phosphorylated at Ser609; this was detected by immunoblotting with an anti-phosphopeptide antibody, anti-pS609, that specifically recognizes the GPIbalpha C-terminal sequence S606GHSL610 only when Ser609 is phosphorylated. Immunoabsorption with anti-pS609 removed almost all of the GPIbalpha from platelet lysates, indicating a high proportion of GPIbalpha phosphorylation. Anti-pS609 inhibited GPIb-IX binding to the intracellular signaling molecule, 14-3-3zeta . Dephosphorylation of GPIb-IX with potato acid phosphatase inhibited anti-pS609 binding and also 14-3-3zeta binding. A synthetic phosphopeptide corresponding to the GPIbalpha C-terminal sequence (SIRYSGHpSL), but not a nonphosphorylated identical peptide, abolished GPIb-IX binding to 14-3-3zeta . Thus, phosphorylation at Ser609 of GPIbalpha is important for 14-3-3zeta binding to GPIb-IX. In certain regions of spreading platelets, particularly at the periphery, there was a reduction in GPIbalpha staining by anti-pS609 as observed under a confocal microscope, indicating that a subpopulation of GPIbalpha molecules in these regions is dephosphorylated. These data suggest that phosphorylation and dephosphorylation at Ser609 of GPIbalpha regulates GPIb-IX interaction with 14-3-3 and may play important roles in the process of platelet adhesion and spreading.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Platelet adhesion plays a critical role in thrombosis and hemostasis. Platelets in normal circulation are in a resting, nonadherent state. At sites of vascular injury, platelets adhere to the exposed subendothelial matrix. Under the high shear force of blood flow, platelet adhesion involves multiple steps. Initially, platelets adhere in a reversible manner (1). This process is mediated by the interaction between a platelet receptor for von Willebrand factor (vWF),1 the glycoprotein Ib-IX complex (GPIb-IX), and matrix-bound vWF (1-3). GPIb-IX interaction with vWF mediates signaling leading to activation of integrins that are responsible for platelet spreading and aggregation (1, 4).

GPIb-IX consists of three subunits: GPIbalpha , GPIbbeta , and GPIX. GPIb-IX is loosely associated with glycoprotein V. The N-terminal domain of GPIbalpha contains binding sites for vWF and thrombin (for reviews see Refs. 5 and 6). The cytoplasmic domain of GPIbalpha contains a binding site (residues 536-568 (7)) for filamin (also called actin-binding protein or ABP-280), which links GPIb-IX to cross-linked actin filamental structures underlying the plasma membrane (the membrane skeleton) (8, 9). We found that an intracellular signaling molecule, 14-3-3zeta , is associated with GPIb-IX (10). A binding site for 14-3-3zeta is located in a 15-amino acid residue serine-rich region (residues 595-610) at the C terminus of GPIbalpha (29). 14-3-3 binding also involves an additional 14-3-3 binding site in GPIbbeta (11, 12).

The 14-3-3 family of highly conserved intracellular proteins interacts with several intracellular serine/threonine kinases and other signaling molecules (13-22) and regulates their functions (15, 23-26). A recognition motif, RSXpSXP, has been identified in c-Raf and several other 14-3-3 ligands, requiring a phosphorylated serine residue (27, 28). Thus, interaction of many intracellular signaling proteins with 14-3-3 is regulated by phosphorylation. We hypothesized previously that serine residues in the 14-3-3 binding site of GPIbalpha might be important for 14-3-3 binding (29). However, it is not clear whether 14-3-3 binding is regulated by phosphorylation of these serine residues, as 14-3-3 can interact with synthetic nonphosphorylated peptides corresponding to GPIbalpha cytoplasmic domain (11, 29). Further, GPIbalpha has been thought to be a nonphosphorylated protein because previous studies failed to show phosphorylation of GPIbalpha (30). In this study, we re-examined phosphorylation states of GPIbalpha using a phosphoserine-specific anti-GPIbalpha antibody. We report here that the cytoplasmic domain of GPIbalpha is phosphorylated at Ser609 and that phosphorylation at this site is important for 14-3-3 binding to intact platelet GPIb-IX. Furthermore, we show that GPIbalpha dephosphorylation occurs at the edge of spreading platelets, suggesting that phosphorylation and dephosphorylation of Ser609 in the cytoplasmic domain of GPIbalpha is involved in regulating GPIb-IX functions during platelet adhesion and spreading.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- Anti-peptide antibody anti-Ibalpha C, recognizing the C-terminal domain of GPIbalpha , was described previously (29). Monoclonal antibody WM23 against GPIbalpha and purified vWF were kindly provided by Dr. Michael Berndt (Baker Medical Research Institute, Melbourne, Australia) (31). Monoclonal antibody SZ2 against GPIbalpha was kindly provided by Dr. Changgeng Ruan (Suzhou Medical College, Suzhou, China) (32). Recombinant 14-3-3zeta protein and 14-3-3-conjugated Sepharose beads were prepared as described previously (29, 33). Peptides and phosphopeptides were synthesized by the Protein Research Laboratory, University of Illinois at Chicago and purified by high performance liquid chromatography, and their molecular mass was verified by electron-spray mass spectrometry.

Antibody against the Phosphopeptide Corresponding to the C Terminus of GPIbalpha -- A phosphopeptide (CSGHpSL) corresponding to a C-terminal 5-residue sequence of GPIbalpha (plus an N-terminal cysteine for conjugation) was conjugated to keyhole limpet hemocyanin (Sigma) as described previously (34). An anti-peptide antibody was raised by immunizing New Zealand White rabbits with this peptide conjugate. To verify the specificity of the antibody (anti-pS609), phosphorylated GPIbalpha C-terminal peptides (CSGHpSL and SIRYSGHpSL) and nonphosphorylated versions of the same peptides were dissolved in 0.1 M NaHCO3, pH 9.2, and coated onto the microtiter plates by incubation at 4 °C overnight. Comparable amounts of the phosphorylated and nonphosphorylated peptides were coated as indicated by the binding of an anti-peptide antibody, anti-Ibalpha C, against the nonphosphorylated C-terminal sequence of GPIbalpha . The wells were blocked with 5% bovine serum albumin in phosphate-buffered saline and then incubated with anti-pS609 antiserum or control preimmune serum from the same rabbits at 22 °C for 2 h. After three washes, microtiter wells were further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG and then washed six times. Bound antibody was quantitated by incubation with peroxidase substrate (0.04% O-phenylenediamine, 0.012% H2O2 in 0.1 M citrate-phosphate buffer, pH 5.0) at 22 °C for 30 min. The reaction was stopped by the addition of 50 µl/well 2 M H2SO4 and then visualized by determining optical density at 490 nm wavelength.

14-3-3 Binding to GPIb-IX and Immunoabsorption-- Platelets were separated from whole blood by centrifugation and then washed three times with CGS buffer (0.12 M sodium chloride, 0.0129 M tri-sodium citrate, and 0.03 M glucose, pH 6.5) (35). Washed platelets were resuspended in Hepes buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 5.6 mM D-glucose, 3.3 mM Na2HPO4, 3.8 mM Hepes, pH 7.35) and solubilized by adding an equal volume of solubilization buffer (2% Triton X-100, 0.1 M Tris, 0.01 M EGTA, and 0.15 M NaCl, pH 7.4) containing 0.2 mM E64 (calpain inhibitor, Roche Molecular Biochemicals) and 1 mM phenylmethylsulfonyl fluoride. For potato acid phosphatase (PAP) treatment, the platelets were solubilized by adding an equal volume of 2% Triton X-100, 0.01 M sodium citrate, pH 5.6, containing 0.2 mM E64, 1 mM phenylmethylsulfonyl fluoride, and 1 unit/ml aprotinin. After centrifugation to remove Triton X-100-insoluble materials, the lysates (200 µl) were preincubated with 6 units/ml potato acid phosphatase (Calbiochem) at 22 °C for 15 min. Binding of GPIb-IX to 14-3-3-conjugated beads was described previously (29, 33). Platelet lysates were incubated with 25 µl (50% (v/v)) of maltose-binding protein (MBP)-conjugated control beads or 14-3-3-conjugated Sepharose 4B beads at 4 °C for 1 h. In some experiments, beads were preincubated with various concentrations of synthetic phosphorylated or nonphosphorylated peptides (SIRYSGHpSL, SIRYpSGHSL, or SIRYSGHSL) before incubation with platelet lysates. The beads were then washed three times. Bound proteins were extracted by adding SDS-sample buffer (0.125 M Tris, pH 6.8, 20% (v/v) glycerol, 0.004% (w/v) bromphenol blue, 4% (w/v) SDS, 5% beta -mercaptoethanol) and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) followed by Western blot with a monoclonal antibody against GPIbalpha , WM23. The specificity of GPIb-IX binding to 14-3-3-conjugated beads has been demonstrated previously (10, 29). For immunoabsorption, platelet lysates (150 µl) were incubated with preimmune or anti-pS609 serum (25 µl) at 4 °C for 1 h and further incubated for 1 h after adding protein A-conjugated Sepharose beads (Sigma). Beads were separated from the lysates by centrifugation. This procedure was repeated once, and then the immunoabsorbed platelet lysates were analyzed by SDS-PAGE and immunoblotting with an anti-GPIbalpha monoclonal antibody, WM23 or SZ2, and in some experiments anti-Ibalpha C. The reactions of the antibodies were visualized with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).

Confocal Microscopy-- Washed platelets in modified Tyrode's buffer (35) were allowed to adhere and spread on vWF-coated glass chamber slides (Nunc) for various lengths of time. Nonadherent cells were removed by three washes. Adherent platelets were fixed by adding 4% paraformaldehyde in phosphate-buffered saline and then permeabilized by incubation for 30 min at 22 °C in 0.1 M Tris, 0.01 M EGTA, 0.15 M NaCl, and 5 mM MgCl2, pH 7.4, containing 0.1% Triton X-100, 0.5 mM leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mM E64. Platelets were then incubated with 10 µg/ml of various antibodies at 22 °C for 1 h. After three washes, platelets were further incubated with fluorescein- or rhodamine-labeled secondary antibodies at 22 °C for 30 min. After additional washes, cells were scanned under a Zeiss LSM510 confocal microscope (×2520).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Serine 609 in the Cytoplasmic Domain of GPIbalpha Is Phosphorylated-- We previously showed that the C-terminal 15-residue serine-rich region of GPIbalpha contains a 14-3-3zeta binding site in which 5 amino acid residues (S606GHSL610) at the C terminus are important (35). To investigate the possibility that 14-3-3 binding is regulated by phosphorylation, a phosphorylated peptide, CS606GHpSL610, was synthesized. This peptide incorporates a phosphoserine (pS) at the residue corresponding to Ser609 of GPIbalpha . An antibody against this peptide was developed. This antibody (anti-pS609) reacted specifically with the phosphorylated peptides CSGHpSL and S602IRYSGHpSL610 corresponding to C-terminal 5- and 9-residue sequences of GPIbalpha but failed to react with the identical nonphosphorylated peptides CSGHSL or SIRYSGHSL (Fig. 1, A and B). Anti-pS609 also failed to react with the SIRYpSGHSL peptide with phosphorylation at Ser606 (Fig. 1B). As a positive control, anti-Ibalpha C antibody against the nonphophorylated GPIbalpha C-terminal sequences was shown to interact with both the nonphosphorylated and phosphorylated peptides coated on the microtiter plates (Fig. 1C). These data indicate that anti-pS609 specifically binds to GPIbalpha C-terminal sequences only when Ser609 is phosphorylated (Fig. 1).


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  		Fig. 1.   Specific binding of anti-pS609 antibody to Ser609-phosphorylated peptides corresponding to GPIbalpha C-terminal sequences. A, synthetic phosphopeptide CSGHpSL or nonphosphorylated identical peptide CSGHSL was coated onto microtiter plates. Anti-pS609 antiserum or preimmune serum (negative result not shown) was incubated in the microtiter wells for 2 h at 22 °C. After further incubation with horseradiash peroxidase-conjugated goat anti-rabbit IgG, bound antibody was detemined by measuring A490 nm as described under "Experimental Procedures." B, synthetic phosphopeptide SIRYSGHpSL with phosphorylated Ser609, an identical nonphosphorylated peptide (SIRYSGHSL), or an identical peptide with phosphorylation at Ser606 but not Ser609 (SIRYpSGHSL) were coated onto the microtiter well, and binding of the anti-pS609 serum to each of these peptides was measured as described in A. Note that anti-pS609 reacts only with the Ser609-phosphorylated peptide. C, microtiter plates were coated with synthetic phosphopeptides SIRYSGHpSL, an identical nonphosphorylated peptide SIRYSGHSL, a phosphopeptide SIRYpSGHSL (phosphorylation at Ser606), or a negative control peptide corresponding to GPIbbeta C-terminal 14-amino acid sequence (Ibbeta C). The microtiter wells were incubated with an antiserum, anti-Ibalpha C, directed against the nonphosphorylated GPIbalpha C-terminal sequence. The comparable amounts of binding of this antibody to various GPIbalpha peptides indicates that comparable amounts of these peptides were coated on the microtiter wells. Shown in the figure are the results from three samples (mean ± S.D.).

To examine whether Ser609 of the platelet GPIbalpha is phosphorylated, washed resting platelets were solubilized directly into SDS-containing sample buffer and immunoblotted with anti-pS609. Fig. 2A shows that anti-pS609 specifically reacted with a band with its molecular weight identical to that of GPIbalpha . To verify that anti-pS609 indeed reacted with GPIbalpha , platelets were solubilzed and GPIbalpha was immunoprecipitated with a monoclonal antibody, SZ2, directed against GPIbalpha . The immunoprecipitates were then immunoblotted with anti-pS609. Fig. 2B shows that anti-pS609 indeed reacted with immunoprecipitated GPIbalpha . As the binding of anti-pS609 requires phosphorylation at Ser609, these results indicate that the Ser609 in the cytoplasmic domain of GPIbalpha is phosphorylated. To further verify that the reactivity of anti-pS609 with GPIbalpha requires phosphorylation, platelets were solubilized and treated with PAP to dephosphorylate proteins. As shown in Fig. 2C, treatment of platelet lysates with PAP dramatically inhibited the binding of anti-pS609 to GPIbalpha . Inhibition in anti-pS609 binding did not result from the loss of GPIbalpha because such treatment did not affect the recognition of GPIbalpha by the antibody (anti-Ibalpha C) that reacts with both the phosphorylated and nonphosphorylated GPIbalpha C-terminal sequence. Thus, binding of anti-pS609 indeed requires phosphorylation of GPIbalpha . Taken together, the above results indicate that Ser609 in the cytoplasmic domain of platelet GPIbalpha is phosphorylated in resting platelets.


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  		Fig. 2.   Phosphorylation-dependent binding of anti-pS609 to the platelet GPIbalpha . A, washed platelets were directly solubilized into SDS-sample buffer, analyzed by SDS-PAGE, and then immunoblotted with a control preimmune serum (Control) or anti-pS609 serum. B, platelets were solubilized in Triton X-100-containing buffer as described under "Experimental Procedures." GPIb-IX were immunoprecipitated with control IgG or an anti-GPIbalpha monoclonal antibody, SZ2 (SZ-2). Control IgG and SZ-2 immunoprecipitates as well as platelet lysates were immunoblotted with anti-pS609. C, the lysates were treated with (PAP) or without (No PAP) 6 units/ml potato acid phosphatase as described under "Experimental Procedures" and then analyzed by SDS-PAGE and immunoblotting with anti-pS609 or anti-Ibalpha C (reactive with both phosphorylated and nonphosphorylated GPIbalpha C-terminal sequence). Note that potato acid phosphatase treatment inhibited anti-pS609 binding to GPIbalpha but did not affect the binding of anti-Ibalpha C.

Stoichiometry of Ser609 Phosphorylation-- To examine the stoichiometry of Ser609 phosphorylation, washed platelets were solubilized and lysates were immunoprecipitated with anti-pS609 to deplete the GPIbalpha population containing phosphorylated Ser609. The GPIbalpha that remained in platelet lysates was then detected by immunoblotting with the antibody anti-Ibalpha C. Fig. 3A shows that preabsorption by anti-pS609, but not by preimmune serum, removed most of the GPIbalpha molecules (>95%) from platelet lysates. In contrast, anti-pS609 failed to remove GPIbalpha from the PAP-dephosphorylated platelet lysates (Fig. 3B). Thus, the majority of the GPIbalpha molecules in Triton X-100-soluble platelet lysates are phosphorylated at Ser609. As a population of GPIb-IX is associated with the Triton X-100-insoluble cytoskeleton of platelets, we also examined whether phosphorylated GPIbalpha is present in the Triton X-100-insoluble fractions corresponding to the cytoskeleton (precipitated by centrifugation at 15,000 × g) and the membrane skeleton (precipitated at 100,000 × g) using the method reported by Fox (9). Fig. 3C shows that anti-pS609 reacted with GPIbalpha in the cytoskeleton and the membrane skeleton, and the distribution pattern of anti-pS609-binding GPIbalpha in these different fractions is similar to that reactive with anti-Ibalpha C. This finding suggests that a majority of the GPIb-IX population in both the Triton X-100 soluble and insoluble fractions of platelet lysates is phosphorylated at Ser609.


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  		Fig. 3.   Stoichiometry and distribution of GPIbalpha Ser609 phosphorylation. A, washed platelets were solubilized, immunoabsorbed with anti-pS609 to remove Ser609-phosphorylated GPIbalpha , and then analyzed by SDS-PAGE and immunoblotting with the monoclonal anti-GPIbalpha antibody, WM23. B, platelets were first treated with (+PAP) or without (No PAP) PAP and then immunoabsorbed with anti-pS609 or control preimmune serum. Lysates were then analyzed by SDS-PAGE and immunoblotting with anti-GPIbalpha monoclonal antibody SZ2. C, washed platelets were solubilized as described previously (9). The platelet lysates were centrifuged at 14,000 × g for 5 min (low speed). and the supernatant was again centrifuged at 100,000 × g for 3 h (high speed). The pellets from low speed and high speed centrifugations as well as the final supernants were solubilized in identical final volumes of SDS-sample buffer and Western blotted with anti-pS609 (phosphorylation-specific) or anti-Ibalpha C (reactive with both phosphorylated or nonphosphorylated GPIbalpha ). Note the similar distribution patterns of GPIbalpha as detected with anti-pS609 or anti-Ibalpha C.

Anti-pS609 Inhibits 14-3-3 Binding to Platelet GPIb-IX-- To examine whether the phosphoserine-dependent epitope of anti-pS609 is involved in 14-3-3 binding, platelet lysates were preincubated with anti-pS609 and then with 14-3-3-coated Sepharose beads. As a control, platelet lysates were preincubated with preimmune serum from the same rabbit. Preincubation with anti-pS609 but not the control serum inhibited the binding of GPIb-IX to 14-3-3-coated beads, suggesting that the SGHpSL sequence recognized by anti-pS609 is proximate to the 14-3-3 binding site (Fig. 4).


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  		Fig. 4.   Inhibition of 14-3-3 binding to GPIb-IX by anti-pS609. Platelet lysates were first incubated with anti-pS609 serum or preimmune negative control serum and then further incubated with control MBP-conjugated beads or 14-3-3 conjugated beads using the methods described previously (29). After three washes, the 14-3-3-bound GPIbalpha were detected by Western blotting with monoclonal antibody WM23.

Phosphorylation at Ser609 of GPIbalpha Is Important for the Binding of Platelet GPIb-IX to 14-3-3 Protein-- To investigate whether 14-3-3zeta binding to GPIb-IX is regulated by phosphorylation, the platelet lysates were pretreated with PAP to dephosphorylate proteins. This treatment inhibited the binding of anti-pS609 to GPIbalpha (Fig. 2B), suggesting that Ser609 is dephosphorylated. PAP-treated platelet lysates were then allowed to interact with recombinant 14-3-3zeta -conjugated beads. As shown in Fig. 5, GPIb-IX from platelet lysates bound to 14-3-3zeta -coated beads, and this binding was dramatically reduced by PAP treatment. Thus, phosphorylation of GPIb-IX is required for high affinity binding between GPIb-IX and 14-3-3zeta .


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  		Fig. 5.   Phosphorylation of GPIb-IX regulates 14-3-3 binding. Platelet lysates were first incubated with (+PAP) or without (No PAP) potato acid phosphatase as described under "Experimental Procedures" and then further incubated with 14-3-3- or MBP-conjugated beads. Bead-bound GPIb-IX was detected by Western blot with anti-GPIbalpha monoclonal antibody, WM23. GPIbalpha in platelet lysates (Lysate) treated with (+PAP) or without PAP (No PAP) were also immunoblotted with WM23 to show that amounts of GPIb-IX were not significantly changed following PAP incubation.

To further investigate whether phosphorylation at Ser609 of GPIbalpha is important for the interaction between the GPIbalpha C-terminal sequence and 14-3-3zeta , a nonphosphorylated peptide with a sequence corresponding to the C-terminal region of GPIbalpha , S602IRYSGHSL610, and identical peptides phosphorylated at Ser609 or Ser606 were synthesized. Sepharose beads conjugated with recombinant 14-3-3zeta were preincubated with these peptides (1 mM) and then allowed to interact with GPIb-IX. As shown in Fig. 6, preincubation with nonphosphorylated or Ser606-phosphorylated peptides did not significantly affect the binding of GPIb-IX to 14-3-3zeta -conjugated beads. In contrast, preincubation with the Ser609-phosphorylated peptide almost completely abolished GPIb-IX binding. Inhibition by the Ser609-phosphorylated GPIbalpha cytoplasmic domain peptide was concentration-dependent, with the half-maximal inhibition at ~50 µM (Fig. 6B). These data suggest that phosphorylation at Ser609 of GPIbalpha is required for the high affinity binding of platelet GPIb-IX to 14-3-3zeta .


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  		Fig. 6.   A GPIbalpha C-terminal peptide phosphorylated at Ser609 inhibits 14-3-3 binding to GPIb-IX. A. synthetic peptides (1 mM) corresponding to the GPIbalpha C-terminal sequence SIRYSGHSL, a Ser606-phosphorylated identical peptide, SIRYpSGHSL, a Ser609-phosphorylated form of the same peptide, SIRYSGHpSL, or a negative control peptide corresponding to the GPIbbeta C-terminal 14 amino acid residues (Ibbeta C) were incubated with 14-3-3zeta -conjugated Sepharose beads (14-3-3) or MBP-conjugated beads (Control) at 4 °C for 1 h. Platelet lysates (150 µl) were then added and incubated for additional 1 h at 4 °C, and after washing, the bead-bound GPIb-IX was detected by immunoblotting with anti-GPIbalpha antibody, anti-Ibalpha C. B, control MBP-conjugated beads or 14-3-3-conjugated beads were preincubated with increasing concentrations of nonphosphorylated GPIbalpha C-terminal peptide (SIRYSGHSL) or the Ser609-phosphorylated peptide (SIRYSGHpSL) and then allowed to bind to GPIb-IX as described in A. The relative quantity of bead-bound GPIb-IX was estimated by scanning the GPIbalpha bands and then analyzing them by NIH Image for optical density. Percentages of inhibition by the peptides were calculated by the formula: Inhibition % = [1 - OD (sample)/OD (positive control)] × 100. Shown in the figure are the results (mean ± S.D.) from three experiments.

In Situ Distribution of Ser609-phosphorylated and Dephosphorylated GPIbalpha in Spreading Platelets-- To examine whether phosphorylation of GPIbalpha is regulated in intact platelets, freshly washed platelets were allowed to adhere to a vWF- or fibrinogen-coated surface and were then fixed and permeabilized. These platelets were double-stained with anti-pS609 and a monoclonal antibody, WM23, against the extracellular region of GPIbalpha and were then scanned under a confocal microscope. Platelets adherent on fibrinogen and vWF were similar in staining patterns (Fig. 7). Although anti-pS609 stain (red) and WM23 stain (green) were colocalized in most parts of the platelet as indicated by the orange and yellow colors (depending on the relative intensity of each color), WM23 stain (green) was also observed in regions where there was no or very weak staining of anti-pS609 (red), suggesting that the GPIbalpha population in these regions was mostly dephosphorylated. In particular, the lamellipodium-like edge of spreading platelets was strongly stained by WM23 only. This WM23-only staining pattern was also seen at the tips of pseudopodia. Thus, it appears that dephosphorylated GPIbalpha is distributed at the leading edge of spreading platelets. These data suggest that the phosphorylation state of GPIbalpha at Ser609 is dynamically regulated in intact platelets, and regulation of GPIbalpha phosphorylation may be involved in regulating GPIb-IX function during platelet adhesion and spreading.


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  		Fig. 7.   In situ distribution of phosphorylated GPIbalpha . Freshly washed platelets were allowed to spread on vWF- or fibrinogen-coated glass chamber-slides at 37 °C for 60 min, fixed, and then permeabilized as described under "Experimental Procedures." The platelets were then double-stained with anti-pS609 (red), and WM23 (green). The slides were scanned under a Zeiss LSM510 confocal microscope (amplification factor = 2520). The orange and yellow colors indicates the colocalization of the red and green stains. Note the green color at the edge of the spreading platelets, which indicates the presence of nonphosphorylated GPIbalpha .


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we provide the first evidence that GPIbalpha is phosphorylated and that a phosphorylation site is at Ser609. Previously, phosphorylation of platelet membrane proteins has been studied (30, 36). The only serine/threonine-phosphorylated major membrane glycoprotein identified was GPIbbeta (30). GPIbbeta is phosphorylated at Thr166 when platelets are stimulated with agents that enhance intracellular cAMP level (36, 37). However, previous studies used a 32P labeling technique to detect protein phosphorylation, which is dependent upon incorporation of exogenous 32P into the phosphorylated proteins and thus is not sensitive to phosphoproteins that are protected from dephosphorylation or rephosphorylation during the procedure. In this study, we used an anti-phosphopeptide antibody, anti-pS609, recognizing the SGHpSL sequence at the C terminus of GPIbalpha but not the nonphosphorylated peptide. The phosphorylation-dependent antibody can be used to detect protein phosphorylation whether or not phosphorylation is metabolically active and thus is capable of detecting GPIbalpha phosphorylation that has not been detected by the 32P labeling technique. Use of the phosphorylation-dependent antibody also enabled us to detect phosphorylation at a specific residue. Two serine residues (Ser606 and Ser609) are present at the C-terminal SGHSL region of GPIbalpha , which is important for 14-3-3 binding (29). We showed that the phosphoserine 609-specific antibody, anti-pS609, specifically bound to platelet GPIbalpha and that its binding was inhibited by dephosphorylation of GPIb-IX with potato acid phosphatase. In addition, an antibody raised against the phosphorylated Ser606-containing sequence (SIRYpSGH) did not react with GPIbalpha from resting platelets (data not shown). These data indicates that the C-terminal domain of GPIbalpha is phosphorylated at Ser609.

Phosphorylation states of proteins are balanced by the actions of protein kinases and phosphatases. In platelet lysates, the percentage of phosphorylated GPIbalpha in the whole GPIb-IX population is high, as indicated by the removal of nearly all GPIb-IX by the anti-phosphopeptide antibody (Fig. 3A); this suggests that balance under these conditions is tilted toward phosphorylation of GPIbalpha . Thus, it appears that, unlike many other phosphoproteins, the default state of GPIbalpha is a phosphorylated state. One possible mechanism for this default phosphorylation state is that 14-3-3 may play a protective role, since the phosphoserine 609 is located in the 14-3-3 binding site. 14-3-3 has previously been shown to protect 14-3-3 ligands from dephosphorylation (38). The protein kinase that catalyzes phosphorylation of GPIbalpha remains to be identified. Several protein kinase inhibitors had no effect on GPIbalpha phosphorylation, including inhibitors of protein kinase A, protein kinase G, and protein kinase C (data not shown). It is thus possible that these kinases are not involved in GPIbalpha phosphorylation. However, as the default state of GPIbalpha appears to be a phosphorylated form, it is also possible that the ineffectiveness of these protein kinase inhibitors is due to the fact that GPIbalpha is already in a relatively stable phosphorylated state and thus immune to the effects of protein kinase inhibitors.

Phosphorylation at Ser609 of GPIbalpha is important for GPIb-IX interaction with 14-3-3. This conclusion is supported by our finding that the Ser609-phosphorylated GPIbalpha C-terminal domain peptide (SIRYSGHpSL), but not the identical nonphosphorylated or Ser606-phosphorylated peptides, inhibited GPIb-IX interaction with 14-3-3 in a concentration-dependent manner (Fig. 6), suggesting that interaction between GPIb-IX and 14-3-3 involves a binding site in 14-3-3 that interacts with the Ser609-phosphorylated GPIbalpha C-terminal sequence. This result is consistent with the previous result of Andrews et al. (11) showing that a nonphosphorylated GPIbalpha C-terminal peptide failed to abolish the binding between 14-3-3 and GPIb-IX. Furthermore, dephosphorylation of GPIb-IX by PAP or preincubation with anti-pS609 inhibited 14-3-3 binding (Figs. 4 and 5). Thus, it is likely that high affinity interaction between the intact platelet GPIb-IX and 14-3-3 requires phosphorylation of Ser609 of GPIbalpha . It is interesting to note that the 14-3-3 binding site of GPIbalpha (RYSGHSL) shares similarities with the RSXpSXP-like motifs of other phosphorylated 14-3-3 ligands; they all contain an arginine and a serine at the N-terminal side of the phosphorylated serine (27, 28). Most of the identified RSXpSXP motifs are present in the middle of the protein sequence, and the proline in the motif may possibly form a turn exposing the phosphoserine. Because the 14-3-3 binding site in GPIbalpha is already exposed at the C terminus, it may not require the presence of a proline residue. However, despite the similarities, there are striking differences between GPIbalpha and the RSXpSXP-like ligands. The prototype RSXpSXP-like ligand of 14-3-3, c-Raf, requires the helix G region of 14-3-3 (33), and the crystal structure data suggest that phosphoserine in the RSXpSXP motif may interact with residues in the more N-terminal helix C and E region of 14-3-3 (28, 39). In contrast, GPIbalpha binds to the helix I region of 14-3-3 (33), which forms an amphiphilic ligand contact surface (40). Furthermore, synthetic peptides corresponding to C-terminal 15 residues of GPIbalpha bound to 14-3-3 without requiring phosphorylation (11, 29), and the recombinant GPIbalpha cytoplasmic domain, which is not phosphorylated at Ser609 (data not shown), also binds to 14-3-3 but with a much lower affinity than GPIb-IX from platelets (33). This suggests that the interaction of 14-3-3 with GPIbalpha may involve both phosphorylation-dependent and phosphorylation-independent binding mechanisms. However, in intact platelet GPIb-IX, Ser609 phosphorylation is required for the high affinity binding of 14-3-3.

Phosphorylation of the Ser609 of GPIbalpha is likely to play important roles in GPIb-IX-mediated platelet adhesion and signaling. First, phosphorylation of Ser609 of GPIbalpha regulates 14-3-3 binding (Figs. 4 and 5), and we have evidence that 14-3-3 binding to GPIb-IX plays an important role in GPIb-IX signaling.2 Furthermore, our data indicate that a population of GPIbalpha becomes dephosphorylated at the periphery of platelets during platelet spreading on vWF or fibrinogen (Fig. 7), suggesting that the phosphorylation state of GPIbalpha can be dynamically regulated and that phosphorylation or dephosphorylation of GPIbalpha may have a functional role during platelet spreading. Although further studies are required to understand how phosphorylation of GPIbalpha may play a role in GPIb-IX function, one possibility is that phosphorylation regulates GPIb-IX-associated membrane skeleton organization and thus regulates the movement of GPIb-IX. This possibility is supported by the finding of Dong et al. (41) that a GPIb-IX mutant, lacking the C-terminal 4 amino acid residues including Ser609 is more likely to move laterally on the membrane. However, we show in Fig. 3 that Ser609-phosphorylated GPIbalpha is distributed in both cytoskeleton and non-cytoskeleton fractions, suggesting that Ser609 phosphorylation does not directly regulate association between GPIb-IX and the membrane skeleton. Consistent with this result, GPIb-IX association with the membrane skeleton has been shown to be mediated by filamin, which binds to the central region of the GPIbalpha cytoplasmic domain distinct from the C terminus (7), and mutant GPIb-IX that lacks the C-terminal domain of GPIbalpha is still associated with filamin and the membrane skeleton (42). However, it is possible that GPIb-IX-associated cytoskeleton organization or movement of GPIb-IX may be indirectly regulated by phosphorylation of GPIbalpha and 14-3-3 binding via intracellular signaling pathways.

    FOOTNOTES

* This work is supported in part by Grant HL52547 from the National Institutes of Health.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 Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Pharmacology (M/C868), University of Illinois, 835 S. Wolcott Ave., Chicago, IL 60612. Tel.: 312-355-0237; Fax: 312-996-1225; E-mail: xdu@uic.edu.

2 M. Gu, R. J. Bodnar, Z. Li, G. D. Englund, and X. Du, unpublished data.

    ABBREVIATIONS

The abbreviations used are: vWF, von Willebrand factor; GP, glycoprotein; GPIb-IX, glycoprotein Ib-IX complex; PAGE, polyacrylamide gel electrophoresis; MBP, maltose-binding protein; PAP, potato acid phosphatase; pS, phosphoserine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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