Identification of a novel 14-3-3zeta binding site within the cytoplasmic tail of platelet glycoprotein Ibalpha.

The glycoprotein Ib-V-IX (GPIb-V-IX) complex interacts with subendothelial von Willebrand factor (VWF) to ensure recruitment of platelets at sites of vascular injury, a process that culminates in integrin alpha(IIb)beta(3)-dependent stable adhesion and spreading. Interaction of the 14-3-3zeta adaptor protein with the C-terminal 606-610 phosphoserine motif of the GPIbalpha subunit has been implicated in the control of alpha(IIb)beta(3) activation and cell spreading. In this study, we have examined potentially novel 14-3-3zeta binding sites by expressing mutant forms of GPIbalpha in Chinese-hamster-ovary (CHO) cells. Analysis of a series of neighboring 11-12 residue deletions identified a critical role for the 580-LVAGRRPSALS-590 sequence in promoting GPIbalpha-14-3-3zeta interaction. Development of a phosphospecific antibody demonstrated high levels of phosphorylation of the Ser587 and Ser590 residues in resting platelets (which became dephosphorylated during platelet spreading on VWF), and peptides containing these phosphorylated residues effectively displaced 14-3-3zeta from GPIbalpha. Analysis of single and double alanine substitutions of Ser587 and Ser590 demonstrated a major role for these residues in promoting GPIbalpha-14-3-3zeta binding. Moreover, these cell lines exhibited a defect in cell spreading on immobilized VWF. These studies demonstrate the existence of a second major 14-3-3zeta binding site within the cytoplasmic tail of GPIbalpha that has an important functional role in regulating integrin-dependent cell spreading.


Introduction
The 14-3-3 protein family consists of ubiquitous homodimeric or heterodimeric intracellular adaptor proteins that take part in the signaling pathways of numerous biologic responses. 1 There have been 5 isoforms identified in human platelets, the ⑀ and isoforms being weakly expressed relative to the more abundant ␥, , and ␤ isoforms. 2 The crystal structure of 14-3-3 revealed the association of 2 monomers, each composed of a bundle of 9 antiparallel helices, to form a large negatively charged groove that could be implicated in interactions with other proteins. 3 The 14-3-3 proteins bind to specific phosphoserine-containing motifs, 4 and their dimeric nature allows them to act as intramolecular and intermolecular phosphorylation-dependent bridges. 5 The functions of the different isoforms in platelets are, however, still poorly understood.
In a seminal study, it was reported that 14-3-3 interacted with the platelet von Willebrand factor (VWF) receptor, the glycoprotein Ib-V-IX (GPIb-V-IX) complex. 6 A binding site for 14-3-3 was found at the C-terminus of GPIb␣ within a serine-rich 606-SGHSL-610 sequence bearing similarities to phosphoserine containing 14-3-3 binding motifs. 1,7 Further work showed that the serine at position 609 was predominantly phosphorylated in resting platelets and that phosphorylation was required for 14-3-3 binding. 8 Glutathione S-transferase (GST)-14-3-3 pull-down and immuno-precipitation studies of GPIb-IX-transfected cells pointed to the existence of a separate binding or regulatory site in the GPIb␣ 570-590 domain. 9 Experiments using synthetic GPIb␤ and GPV intracellular peptides suggested that these subunits could also interact with 14-3-3. 10 Phosphorylation of the GPIb␤ serine at position 166 by a protein kinase A (PKA)-dependent mechanism has been proposed to play a role in regulating 14-3-3 interactions. [9][10][11] Recent studies have demonstrated that the GPIb-14-3-3 interaction is dynamically regulated, such that shear-induced activation of platelets leads to dissociation of 14-3-3 from GPIb. 11 While the precise mechanisms regulating this have not been defined, changes in the phosphorylation status of Ser609 may play an important role. A functional correlation between 14-3-3 release from GPIb and integrin activation has been suggested from studies in Chinesehamster-ovary (CHO) cells transfected with the GPIb-IX complex following deletion of the Glu591-Leu610 GPIb␣ C-terminal domain. 8,9 This deletion prevented 14-3-3-GPIb interactions, leading to defective integrin dependent spreading. 12 However, 2 further studies, examining integrin activation using CHO cells transfected with the same constructs, have demonstrated either normal or increased cell spreading. 13,14 To establish a precise functional map of the 14-3-3 binding regions in the GPIb␣ cytoplasmic tail, CHO cells were transfected with constructs of the GPIb-IX complex containing short 11-12 residue deletions and single mutations of the GPIb␣ intracellular domain. The effects of these mutations on GPIb-14-3-3 association and cell spreading on VWF were examined. In addition to the known 605-610 region, a new binding site was identified between residues 580 and 590 that resembled other 14-3-3 ligands and required Ser residues at positions 587 and 590. Phosphorylation of the diSer587/590 motif was demonstrated in resting platelets using a specific antibody, while its dephosphorylation and that of Ser609 were observed during platelet adhesion and activation on a VWF matrix. Following phosphatase treatment, dephosphorylation led to 14-3-3 release from GPIb-IX. Furthermore, lack of 14-3-3 association with GPIb␣ in cells expressing the GPIb␣ 580-590 deletion mutant, Ser587/590 to Ala substitution, or deletion of the 605-610 sequence resulted in a reduced ability of the VWF-GPIb␣ interaction to activate endogenous integrins and induce cell spreading.

Antibody production
The anti-phospho-GPIb␣ (pSer609) antibody was raised by immunizing New Zealand White rabbits with the phosphorylated CYSGHpSL peptide conjugated to keyhole limpet hemocyanin. The antipeptide antibody was affinity purified with the immunizing peptide conjugated to BSA and coupled on a 1:1 column of Affi-Gel 10/15. The antibody was then absorbed with the nonphosphorylated CYSGHSL peptide conjugated to BSA, also coupled on a 1:1 column of Affi-Gel 10/15. The specificity of the anti-pS609 antibody was verified by dot immunoblots against the CYSGH(pS)L and CYSGHSL peptides conjugated to albumin (1:10 wt/wt), versus antibody that bound to both columns, anti-Ib␣ϩ/ϪP, the reactivity of which was phosphorylation nonspecific.

Cell lines
CHO cell lines stably expressing the wild-type GPIb-IX complex or GPIb␣ deleted forms of GPIb-IX have been described previously. [17][18][19] Stable cell lines with Ser to Ala mutations at positions 587, 590, and a double 587/590 mutation were obtained following transfection of the GPIb␣ cDNA into GPIb␤IX expressing cells as previously described. 18 Oligonucleotidedirected mutagenesis was performed by polymerase chain reaction amplification of PDXGPIb␣ vector with Pfu DNA polymerase (Stratagene, La Jolla, CA), digestion with DpnI (Roche Molecular Biochemicals), and transformation in TOP10 bacteria (Invitrogen, San Diego, CA).

Platelet preparation
Washed human platelets were prepared from acid citrate dextrose (ACD) anticoagulated blood obtained from aspirin-free healthy volunteers by sequential centrifugation as previously described. 16 Biotinylation Cell biotinylation has been described in detail elsewhere. 19 Briefly, platelets were washed twice in phosphate-buffered saline (PBS), centrifuged at 1900g for 8 minutes, and incubated for 30 minutes at room temperature (RT) in PBS containing 100 g/mL NHS-Biotin at a final concentration of 10 6 platelets/L.

Immunoprecipitation and immunodepletion
Cell lysates were cleared twice by incubation for 60 minutes at 4°C with 50 L protein G-Sepharose CL4B (50% wt/vol suspension in lysis buffer). Proteins were immunoprecipitated from 50 L cleared lysate by addition of 5 g mAb and 80 L protein G-Sepharose CL4B (50% wt/vol suspension) and rotation for 2 hours at 4°C. The beads were washed 3 times in PBS containing 1% Triton X-100 before addition of 45 L of 1X Laemmli buffer (62.5 mM Tris, pH 6.8, 0.2% sodium dodecyl sulfate (SDS), 25% glycerol, 0.01% bromophenol blue) containing 10 mM DTT. Samples were boiled for 5 minutes and proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on 4% to 15% gels and transferred to PVDF membranes. Biotinylated proteins were revealed with streptavidin-HRP as previously described, 19 while nonbiotinylated proteins were identified by Western blotting as previously described. 20 In immunodepletion experiments, a first immunoprecipitation was performed using a 2-L aliquot of cleared platelet lysate to ensure an excess of anti-phospho-GPIb IgG (pSGPIb1 or pSer609) and 80 L protein G-Sepharose CL4B (50% wt/vol suspension). The remaining supernatant was reprecipitated with a mAb against GPIb␤ (RAM.1).

Enzyme immunoassays
Synthetic peptides corresponding to the GPIb␣ 580-590 domain were coated from 5 g/mL solutions in 50 mM carbonate buffer (pH 9.5) onto the wells of 96-well microtiter plates (Maxi-Sorb; Nunc, Rochester, NY) for 2 hours at RT. After 3 washes in PBS-T, the wells were blocked by incubation for 30 minutes at 37°C with 200 L of 1% BSA in PBS. All subsequent incubations were carried out for one hour at RT, and each was followed by washing 3 times with PBS-Tween 0.05% (PBS-T). The wells were treated first with 100 L diluted pSGPIb1 antiserum and then with GAR-HRP (1:1000 dilution). The secondary antibody was detected by addition of a chromogenic substrate (OPD: o-phenylenediamine dihydrochloride; Pierce) and measurement of optical densities at 490 nm in a THERMO-max microplate reader (Molecular Devices, Sunnyvale, CA).

Cell adhesion to VWF
Glass coverslips were coated with HVWF (10 g/mL) in a humid chamber for 90 minutes at RT and then blocked for 60 minutes with PBS containing 1% BSA. CHO cells in PBS were incubated with the coverslips in the wells of microtiter plates (10 5 cells/well) at 37°C for 20 minutes. The coverslips were then washed 3 times in PBS, fixed for 20 minutes with 4% PFA in PBS, washed 3 times in PBS, and labeled for 30 minutes in the dark with 2 g/mL TRITC-phalloidin. After 3 more washes in PBS and a last wash in water, the adherent cells were covered with 8 L Mowiol 4-88 solution (France Biochem, Meudon, France), and the coverslips were mounted on microscope slides. Fluorescence was visualized by ultraviolet (UV) illumination at 570 nm under a Leica DMDL microscope (Leica Microsystems, Wetzlar, Germany). The cell count and morphology of the adherent cells were scored in 8 random fields (2.3 ϫ 10 3 m 2 /field).

Analysis of platelet adhesion by confocal microscopy
Glass coverslips were coated with HVWF as before, or coated with 0.01% polylysine for 5 minutes at RT and dried for one hour at 60°C. Washed platelets in Tyrode buffer were incubated with the coverslips in the wells of microtiter plates (3 ϫ 10 6 platelets/well) for different times at 37°C. When indicated, 5 mM EDTA and 10 g/mL ReoPro were added to block integrin activation. The coverslips were then washed 3 times in PBS, fixed for 15 minutes with 4% PFA in PBS, washed 3 times in PBS, and blocked with PBS containing 0.2% BSA and 1% normal goat serum (NGS) for one hour at RT. All subsequent incubations were followed by 3 washes in PBS-T. The samples were labeled for 45 minutes in the dark with 1 g/mL ALMA.12cyanin 3 (Cy3); permeabilized for one hour in PBS containing 0.2% BSA, 0.05% saponin, and 1% NGS; labeled for 45 minutes in the dark with pSGPIb1 or pSer609 antiserum diluted 1:10 000 in the blocking buffer; and finally counterstained with GAR-Cy5 (1:2000 dilution). The coverslips were then washed in PBS, rinsed with water, and mounted in Mowiol 4-88. The labeled platelets were examined under a Zeiss laser scanning microscope (LSM 510 invert; Carl Zeiss, Le Pecq, France) equipped with a Planapo oil-immersion lens (ϫ 63, numerical aperture 1.4), and the Zeiss CLSM instrument software 2.8. Cy3 emission was excited with the 543-nm He/Ne laser line and signals were filtered with a long-pass 595-nm filter.

Shape change of CHO cells adhering to VWF
CHO cells (1 ϫ 10 6 /mL) suspended in PBS containing 2 mM Ca 2ϩ , 2 mM Mg 2ϩ , and 5 g/mL botrocetin were allowed to adhere to an HVWF matrix (deposited from a 10 g/mL solution) for 30 minutes at 37°C. After fixation, the adherent cells were stained with 2 g/mL TRITC-phalloidin for 30 minutes, and the coverslips were mounted in Mowiol 4-88 solution on microscope slides. The cell morphology was examined by fluorescence microscopy (UV illumination at 570 nm, Leica DMDL microscope, PL Fluotar 40ϫ/1.00-0.5 oil immersion objective) and visualized using a Micromax 1300Y digital camera (Princeton Instruments, Tucson, AZ) and Metamorph 4.5 software (Princeton Instruments), and numbers of adherent cells and spreading cells were scored in 5 random fields.

Statistical analyses
The statistical significance of differences between means was evaluated using Student t test for paired samples or Fisher exact test, and P values of less than .05 were considered significant.

Results
The GPIb␣ 580-590 intracytoplasmic sequence is required for the interaction between the GPIb-IX complex and 14-3-3 Interaction of 14-3-3 with the GPIb-IX complex was studied in human platelets and transfected CHO cells, following biotinyla-tion and immunoprecipitation with an antibody against GPIb␣ or GPIb␤. As shown in Figure 1A, coimmunoprecipitation of the 2 proteins was confirmed in human platelet lysates. To identify potentially novel 14-3-3 binding sites on GPIb␣, this subunit was subjected to a series of truncations covering the 535-610 intracellular region and stably transfected into GPIb␤-IX CHO cells. The GPIb-IX-14-3-3 binding capacity was then evaluated by coimmunoprecipitation with an anti-GPIb␤ mAb. Deletion of the known 605-610 binding site (⌬605-610) and larger truncations eliminating this domain (⌬518-610, ⌬569-610, ⌬576-610, and ⌬595-610) prevented GPIb-IX-14-3-3 coprecipitation ( Figure 1B). Conversely, deletion of the more central 535-569 region led to wild-type behavior and allowed efficient association of GPIb-IX with 14-3-3 ( Figure 1B). To examine additional sites upstream of the 605-610 region, cells with shorter deletions spanning the 535-590 domain were evaluated ( Figure 1C). All the deletions allowed for efficient GPIb-IX-14-3-3 binding with the exception of ⌬580-590, which prevented association with 14-3-3. These results suggested the existence of a second 14-3-3 binding site in addition to the previously identified 605-610 sequence. 8

Deletion of the GPIb␣ 580-590 sequence does not prevent phosphorylation of Ser609
Phosphorylation of Ser609 in the 605-610 sequence has been reported to be critical for the interaction of GPIb␣ with 14-3-3. 8 Therefore, the possibility that the 580-590 deletion could indirectly block 14-3-3 interactions by interfering with Ser609 phosphorylation was explored. An antibody (pSer609) was raised and immunopurified against the YSGHpSL peptide. This antibody was specific for the phosphorylated form of the peptide in dot blot experiments ( Figure 2A) and recognized GPIb␣ from lysates of resting platelets ( Figure 2B) and CHO cells transfected with normal GPIb-IX ( Figure 2C). pSer609 also recognized the 580-590 deleted form of GPIb␣ in CHO cell lysates, indicating normal phosphorylation at Ser609, whereas the 518-610 deleted form of GPIb␣ was not revealed by the antibody (Figure 2C).

The 580-590 domain of GPIb␣ is phosphorylated in resting platelets
To further analyze the role of the phosphoserines of the GPIb␣ 580-590 sequence in 14-3-3 binding, antibodies were raised against the P 1 P 2 peptide. The pSGPIb1 antiserum specifically bound to P 1 P 2 as detected by enzyme-linked immunosorbent assay (ELISA) but did not recognize the nonphosphorylated peptide ØP ( Figure 4A). pSGPIb1 also recognized the monophosphorylated P 1 (LVAGRRPpSALS) and P 2 (LVAGRRPSALpS) peptides. pSG-PIb1 revealed a band corresponding to GPIb␣ in lysates from resting platelets, suggesting phosphorylation of this domain in the native protein ( Figure 4B). The pSer609 antibody also recognized GPIb␣, confirming previous findings that Ser609 is phosphorylated in resting platelets. 8 The specificity of pSGPIb1 for the phosphorylated motif in the native sequence was demonstrated by a drop in reactivity in platelet lysates treated with alkaline phosphatase ( Figure 4C). Similarly pSer609 labeling decreased following phosphatase treatment. Hence in the resting state, GPIb␣ appeared to be doubly phosphorylated at the 580-590 and 609 sites. The degree of phosphorylation at these 2 positions was then evaluated in resting platelets by immunodepletion of lysates with the pSGPIb1 antiserum or pSer609 antibody. After pSGPIb1 or pSer609 depletion, reprecipitation with a GPIb␤ antibody yielded significantly less GPIb␣ than following nonimmune ( Figure 4D), suggesting that GPIb␣ is similarly phosphorylated at high levels in the 580-590 and 609 domains. To confirm the importance of GPIb␣ phosphorylation for 14-3-3 binding, GST-14-3-3 pull-down experiments were performed using phosphatase-treated platelet lysates. The results demonstrate that dephosphorylation prevented GPIb␣ association with 14-3-3 ( Figure 4E), an effect that correlated with the decrease in pSGPIb1 and pSer609 labeling.

Phosphorylation status of the GPIb␣ 580-590 and 609 domains in resting and adherent platelets
To evaluate phosphorylation of GPIb␣ in intact platelets, dual-label confocal analysis was performed using either pSGPIb1 or pSer609  For personal use only. on August 21, 2017. by guest www.bloodjournal.org From (detected with fluorescein isothiocyanate [FITC]) and the pan-anti-GPIb␣ mAb ALMA.12 (coupled to Cy3). Platelets fixed in a resting discoid state and allowed to adhere to a polylysine surface were predominantly double labeled (yellow), indicating the colocalization of pSGPIb1 or pSer609 with ALMA.12 ( Figure 5A,D). This confirmed that most of the GPIb␣ molecules were phosphorylated at the 580-590 and Ser609 sites in the resting state, in agreement with the biochemical data.
In previous studies it has been demonstrated that when platelets adhere to VWF in the presence of ␣ IIb ␤ 3 inhibitors, they become activated, change shape, and extend filopodia. The cell body of platelets adhering to a VWF matrix under these conditions remained double labeled (yellow). However, all the filopodia appeared in red ( Figure 5B,E), indicating a lack of pSGPIb1 or pSer609 labeling and hence a significant dephosphorylation of Ser587/590 and Ser609. In the absence of integrin blockade, platelets can spread on a VWF surface and such conditions resulted in a mixture of red and yellow labeling over the entire cell surface ( Figure 5C,F). These results show that a significant proportion of GPIb␣ molecules become dephosphorylated during the processes leading to integrin activation.

Critical role of Ser590 in the 589-590 domain for 14-3-3 binding and integrin activation
To more precisely define the role of the serine residues in the 580-590 region in promoting GPIb␣ association with 14-3-3, Ser587 and Ser590 were substituted to Ala either individually or together and GPIb␣ was introduced into CHO cells containing GPIb␤-IX. Coimmunoprecipitation studies demonstrated a decreased 14-3-3 association for all the mutants that was almost completely abolished for the 2 cell lines containing the Ser590Ala substitution ( Figure 6A). This defect in 14-3-3 binding was similar to that observed with the GPIb␣⌬580-590 deletion, suggesting a major role for Ser590 in 14-3-3 binding with a secondary role contributed by Ser587. In order to then assess the functional importance of the 14-3-3 pool bound to the two 14-3-3 binding Triton X-100 lysates of resting platelets were separated by 4% to 15% SDS-PAGE and immunoblotted with pSGPIb1, pSer609, or an antibody against the 3 GPIb-IX subunits. GPIb␣ was heavily labeled by pSGPIb1, suggesting Ser phosphorylation of the 580-590 domain. In accordance with previous reports, labeling with pSer609 showed that GPIb␣ was also phosphorylated at Ser609. (C) Triton X-100 lysates of resting platelets were treated or not with 0.3 U/mL PAP for 30 minutes at 37°C. Proteins were separated by 4% to 15% SDS-PAGE, transferred to PVDF membranes, and probed with pSer609 and pSGPIb1. Reactivity to the antibodies was lost in samples treated with PAP, confirming phosphorylation at both sites. (D) The proportion of phosphorylated GPIb␣ was estimated by immunodepleting the cell lysates with an excess of the phosphospecific antibody pSGPIb1 (middle panel) or pSer609 (right panel) and performing a second immunoprecipitation with an anti-GPIb␤ antibody. Products of the first and second immunoprecipitations were revealed with a polyclonal anti-GPIb-IX antibody. In both cases, a similar small proportion of GPIb-IX was revealed following the second immunoprecipitation, indicating that most GPIb␣ subunits were phosphorylated at both sites. The left panel corresponds to a control where the first depletion was performed in the presence of a nonimmune serum, and results are from 1 experiment representative of 4 independent assays. (E) In GST-14-3-3 pull-down experiments, proteins were precipitated from the same lysates as in panel C and probed with an antibody against the GPIb-IX complex. GST-14-3-3 precipitated GPIb␣ in the absence of PAP treatment but not after its dephosphorylation by PAP. A negative control using GST alone is shown in the left lane. Results in panels B-E are from 1 experiment representative of 3.

Figure 5. The GPIb␣ intracellular domain is dephosphorylated after platelet adhesion and shape change on a VWF matrix.
Resting platelets were either fixed and allowed to adhere to a polylysine matrix (A,D), or allowed to adhere to a VWF matrix in the presence (B,E) or absence (C,F) of EDTA by incubation with the matrix for 20 minutes in the presence of botrocetin followed by washing and fixation. GPIb␣ was labeled with ALMA.12-Cy3 (red), and phosphorylated GPIb␣ was labeled with pSGPIb1 (A-C) or pSer609 (D-F), which was revealed with a secondary FITC-coupled antibody (green). Samples were analyzed by dual-label confocal microscopy. (A,D) Discoid platelets appeared predominantly yellow after staining with either pSGPIb1 or pSer609, indicating that GPIb␣ was mainly phosphorylated in resting platelets. (B,E) When platelets adhered to a VWF matrix and integrin activation was blocked with EDTA and ReoPro, they extended numerous filopodia. The staining of the cell body remained yellow, whereas the filopodia appeared in red, suggesting a lack of recognition by pSer609 or pSGPIb1 and dephosphorylation of pSer609 and pSer587/590 within the membrane extensions. (C,F) In the absence of ReoPro and EDTA, platelets spread on the VWF matrix and the labeling appeared as a mixture of yellow and red over the entire cell surface, pointing to the existence of separate pools of phosphorylated and dephosphorylated GPIb␣. Shown is 1 representative experiment of 3 performed. Scale bars indicate 10 m. sites of GPIb␣ for integrin activation, GPIb-IX CHO cell lines containing either the deletion mutants (GPIb␣⌬605-610 and GPIb␣⌬580-590) or the specific alanine mutants (␣A587, ␣A590, and ␣A587A590) were studied in VWF adhesion assays. Cells expressing wild-type GPIb/IX underwent extensive cell spreading, indicating that intact GPIb/IX is able to mediate efficient integrin activation, observations consistent with previous studies. 12,13 A comparison with the deletion mutant cells, which had completely lost the ability to support GPIb␣-14-3-3 association (Figure 1C), indicated that although these cells were still able to adhere as efficiently as wild-type control cells ( Figure 6B) they were significantly less susceptible to spread on the VWF matrix ( Figure  6B-C). This suggested inefficient integrin activation possibly due to an absence of mobilizable 14-3-3. Similar assays were performed with the alanine mutants that demonstrated a profound and specific decrease in cell spreading for the 2 clones mutated at Ser590, reproducing the defect observed in GPIb␣⌬ 580-590 cells ( Figure 6D). In contrast, the single Ser587Ala substitution did not prevent cell spreading. These observations suggested a central role for Ser590 to induce efficient integrin activation potentially as a result of a mobilizable pool of 14-3-3.

Discussion
The 96-amino acid GPIb␣ intracellular domain (residues 515-610) was originally described as a bridging sequence between the GPIb-V-IX complex and the platelet actin cytoskeleton via filamin. 23 Deficient association with the cytoskeleton likely contributes to the giant platelet phenotype observed in Bernard-Soulier syndrome. 24 It is now becoming apparent that this domain is multifunctional and can assemble additional partners potentially involved in signal transduction. 25 GPIb/VWF signaling leads to the mobilization of intracellular Ca 2ϩ , platelet shape change, and activation of integrin ␣ IIb ␤ 3 . 12,26 The precise mechanism whereby binding of VWF to GPIb-IX triggers ␣ IIb ␤ 3 -dependent functions such as platelet spreading and aggregation nevertheless remains unclear. Recruitment of the 14-3-3 adaptor protein to the GPIb␣ 605-610 C-terminal region has been proposed to play a role in integrin activation. 7,12 The present study provides evidence for the existence of a second 14-3-3 binding site in the upstream 580-590 region and suggests the implication of both the 605-610 and 580-590 domains for functional GPIb␣-14-3-3 association and in the control of integrin dependent spreading.
An interaction of 14-3-3 with the GPIb-V-IX complex was initially revealed by their coisolation during affinity chromatography purification of GPIb from platelet lysates. 6 Subsequent synthetic peptide binding experiments and deletion mutagenesis in GPIb-IX-transfected CHO cells located a binding site for 14-3-3 in the last 6 amino acids of the GPIb␣ subunit (residues 605-610). 7,10 A second binding site for 14-3-3 has been assigned to the 557-575 region of the GPIb␣ subunit, mainly from studies of synthetic peptide binding. 10 More recently, the existence of yet another binding site upstream of the 605-610 domain was suggested from the lack of GPIb␣-14-3-3 coprecipitation in cells deleted of the 570-590 region. 9 In this work, a closer deletion scanning of the 535-610 region revealed defective GPIb-14-3-3 association only for the 580-590 deletion mutant, in addition to the C-terminal truncation. Together, this study and previous work 19 indicate that in fact the 570-590 sequence contains separate functional sites involved in filamin (570-580) and 14-3-3 binding (580-590), respectively.
It was of interest that the 580-590 site contained serines at positions that agreed with a consensus 14-3-3 binding motif. 22 The capacity of a phosphorylated 580-590 synthetic peptide to inhibit GPIb␣-14-3-3 coimmunoprecipitation provided supporting evidence that the GPIb␣ 580-590 domain is involved in 14-3-3 binding, and also agreed with the mode of interaction of most 14-3-3 ligands, which require phosphorylation at key Ser or Thr residues for efficient binding. 4 In a previous study, 2 large peptides (571-589 and 585-603) that partially overlapped the 580-590 sequence failed to bind purified 14-3-3. 10 This apparent discrepancy could be related to the use of nonphosphorylated peptides, as the nonphosphorylated 580-590 peptide competed less efficiently for 14-3-3 association (Figure 3). It can also be inferred from the present results that the 14-3-3 molecule can accommodate 2 peptide sequences from the GPIb␣ intracellular domain. This mode of interaction might be necessary to allow for efficient binding of the adaptor protein and for a fine control of its release. There are previous evidences that 14-3-3 can bind more than one peptide in other systems. For example, there was a report on the crystal structure of dimeric 14-3-3 in complex with 2 peptides from the kinase Raf-1, 27 and, more recently, 3 separate binding sites were identified for the phosphatase cdc25B peptides. 5 The C-terminal ␣-helix of 14-3-3 (helix I) 12,28 appears to contain the GPIb␣ binding motifs, but the precise mode of interaction and in particular its capacity to interact with the 580-590 and 605-610 sequences remain to be determined.
Functional relevance of the 580-590 domain and its phosphorylation was indicated in a series of experiments in platelets with the use of an antibody against the diphosphorylated peptide. These studies revealed that the GPIb␣ 580-590 domain is phosphorylated in resting platelets. Phosphorylation was observed both in platelet lysates by immunoblotting and in intact cells by confocal microscopy analysis. Use of an antibody against the pSer609 sequence confirmed that this domain is likewise phosphorylated in resting platelets. Phosphorylation at both sites appears to be required to support efficient 14-3-3-GPIb-V-IX association in resting platelets. Firstly, phosphatase treatment of platelet lysates prevented 14-3-3-GPIb-V-IX association and was accompanied by dephosphorylation of pSer587/590 and pSer609. Studies in GPIb-IX-transfected CHO cells were also consistent with this model and showed that deletion of either binding site prevented association of GPIb␣ with 14-3-3. Ala mutagenesis revealed that residues 587 and 590 were both required for correct association.
The occurrence of dephosphorylation of these 14-3-3 binding sites during the process leading to platelet spreading supports their role in a pathway leading to integrin activation. Platelets adhering to a VWF matrix initially extend thin filopodia in a process that has been found to be integrin independent. 26 This is followed by platelet spreading, which requires integrin ␣ IIb ␤ 3 activation. Strikingly, the GPIb␣ molecules recruited to filopodial extensions were no longer recognized by pSer587/590 or pSer609 phosphospecific antibodies. This points to an early and localized dephosphorylation mechanism that precedes integrin activation and occurs concomitant with cytoskeletal reorganization. Deletion mutant cells lacking 14-3-3 binding are still able to extend filopodia, suggesting that the release of 14-3-3 from GPIb␣ is not required for this early cytoskeletal reorganization (data not shown). On the other hand, their inefficient spreading suggests a role in the late integrindependent shape change. In fully spread platelets, confocal microscopy indicated that a majority of the GPIb␣ subunits were dephosphorylated at this later integrin-dependent stage. Since phosphatase treatment leads to a decreased GPIb␣-14-3-3 interaction, one likely consequence of the pSer587/590 and pSer609 dephosphorylation in platelets adhering to VWF will be the release of GPIb␣-associated 14-3-3. Consistent with this proposal, it has been reported that 14-3-3 was released from the GPIb-V-IX complex in shear activated platelets. 9 Although it cannot be excluded that the loss of antibody labeling during shape change could be indirect, due for example to a change in GPIb␣ conformation or to the binding of other molecules that mask the epitopes, the fact that it was observed concomitantly for distinct GPIb␣ domains makes this appear less likely.
Deletion of the C-terminal end of GPIb␣ that contains the 605-610 14-3-3 binding site has led to discordant conclusions concerning the role of this adaptor protein in integrin activation. CHO cells transfected with integrin ␣ IIb ␤ 3 and GPIb-IX containing the same GPIb␣ 591-610 deletion were found to undergo defective 12 or on the contrary normal or increased spreading 13,14 following adhesion to a VWF matrix. These discrepancies are not easily explained but could be due to the use of larger deletions (591-610), which could affect other mechanisms, to the use of cells expressing different levels of integrin or 14-3-3, or to differences in the adhesive assays (adhesive proteins, botrocetin, time-course. . .). In the present study, deletion of the minimal GPIb␣ 605-610 binding site or the second binding motif in the 580-590 region prevented normal spreading of GPIb-IX-transfected cells on VWF. Furthermore, a single amino acid change of Ser590 to Ala, which has a low probability to alter the protein structure, yielded the same result. Collectively these findings favor a model in which a population of 14-3-3 molecules bound at the resting state to the intracellular face of GPIb-IX through 2 phosphorylated motifs is required for integrin activation following its release from the GPIb-IX complex upon GPIb/VWF interaction.
The concomitant phosphorylation/dephosphorylation of the 580-590 and 605-610 regions of GPIb␣ in resting versus VWF-activated platelets suggests the possibility of a coordinated control by specific kinases and phosphatases. PKA and protein kinase G (PKG) were recently implicated in the control of platelet activation following GPIb/VWF interaction. 29,30 However, it has been reported that inhibitors of PKA and PKG have no effect on Ser609 phosphorylation. 8 The 580-590 sequence and the Ser587 site display good homology with a consensus PKA phosphorylation site (RRXSX). 30 However, incorporation of 32 P was not observed in the presence of agents increasing cyclic adenosine monophosphate (cAMP) levels such as forskolin (data not shown), and phosphorylation of GPIb␣ could be documented only with phosphospecific antibodies. This was surprising in view of the large proportion of phosphorylated GPIb␣ detected with the phosphospecific antibodies. This could be related to a slow rate of phosphate exchange during the washing procedure, which is designed to avoid platelet activation. 16 In conclusion, the present study describes a new 14-3-3 binding site located in the 580-590 region of GPIb␣, which requires Ser phosphorylation in resting platelets. Adhesion of platelets to a VWF matrix led to dephosphorylation of GPIb␣ pSer587/590 and pSer609. This would seem to be a common regulatory mechanism mediating release of 14-3-3 from the GPIb-IX complex and thereby integrin activation and subsequent cell spreading.