A Novel Ligand-binding Site in the z-Form 14-3-3 ProteinRecognizing the Platelet Glycoprotein Iba and Distinctfrom the c-Raf-binding Site*

(Received for publication, August 21, 1998)

Minyi Gu and Xiaoping Du‡

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

We reported previously that the z-form 14-3-3 protein(14-3-3z) binds to a platelet adhesion receptor, glycopro-tein (GP) Ib-IX, and this binding is dependent on theSGHSL sequence at the C terminus of GPIba. In thisstudy, we have identified a binding site in the helix Iregion of 14-3-3z (residues 202–231) required for bindingto GPIb-IX complex and to the cytoplasmic domain ofGPIba. We also show that phosphorylation-dependentbinding of c-Raf to 14-3-3z requires helix G (residues163–187) but not helix I. Thus, the GPIba-binding site isdistinct from the binding sites for RSXpSXP motif-de-pendent ligands. Furthermore, we show that wild type14-3-3z has a higher affinity for GPIb-IX complex thanrecombinant GPIba cytoplasmic domain. Deletion ofhelices A and B (residues 1–32) disrupts 14-3-3z dimer-ization and decreases its affinity for GPIb-IX. Disrup-tion of 14-3-3z dimerization, however, does not reduce14-3-3z binding to recombinant GPIba cytoplasmic do-main. This suggests a dual site recognition mechanismin which a 14-3-3z dimer interacts with both GPIba andGPIbb (known to contain a phosphorylation-dependentbinding site), resulting in high affinity binding.

A platelet receptor for von Willebrand factor, the glycopro-tein (GP)1 Ib-IX-V complex (GPIb-IX-V), mediates initial plate-let adhesion to the subendothelial matrix and triggers plateletactivation under high shear rate conditions (for reviews, seeRefs. 1 and 2). GPIb-IX-V also binds thrombin and is importantin thrombin-induced platelet activation (3–5). GPIb-IX-V con-sists of four different transmembrane subunits as follows: dis-ulfide-linked GPIba and GPIbb forms a 1:1 complex with GPIX(6); the GPIb-IX complex (GPIb-IX) forms a 2:1 complex withGPV which may dissociate in certain detergents such as TritonX-100 (7). Accumulating evidence indicates that ligand bindingto GPIb-IX-V triggers transmembrane signaling events includ-ing activation of protein kinase C (8, 9) and tyrosine kinases(10), elevation of intracellular calcium (8, 11, 12), synthesis ofthromboxane A2 (8), and activation of phosphoinositol 3-kinase(10), leading to activation of ligand binding function of theresponsive adhesion receptor, integrin aIIbb3. We found that

GPIb-IX is physically associated with an intracellular signalingprotein, the z-form 14-3-3 protein (14-3-3z) (13), suggesting apotential role for 14-3-3z in GPIb-IX-V mediated signaling. Wefurther identified a C-terminal 5-residue sequence of GPIba(SGHSL) that is critical for the binding of 14-3-3z (14), a resultconfirmed by Andrew et al. (15). Interestingly, in addition tothe C-terminal sequence of GPIba, GPIb-IX binding to 14-3-3zalso involves a protein kinase A (PKA)-phosphorylated bindingsite in the cytoplasmic domain of GPIbb (15, 16).

14-3-3z belongs to the 14-3-3 family of highly conservedintracellular proteins (17). The 14-3-3 proteins are dimeric;each monomer is composed of nine anti-parallel a-helices form-ing a large ligand binding groove as revealed by crystal struc-ture analysis (18, 19). The 14-3-3 proteins bind and regulate avariety of intracellular signaling molecules, including variousprotein kinase C isotypes (17, 20), c-Raf (21–24), Bcr (25),middle T antigen (26), c-Cbl (27), cdc25 (28), and the cell deathfactor BAD (29). The 14-3-3 proteins also have been implicatedin the assembly of protein kinase complexes (30). Thus, it ispossible that binding of GPIb-IX to 14-3-3z links GPIb-IX tointracellular signaling pathways. In addition, the 14-3-3-bind-ing site in GPIba has been shown (31) to be important inregulating the lateral movement of GPIb-IX-V in plasma mem-brane, suggesting its possible involvement in regulating GPIbainteraction with the cytoskeleton. Thus, structural definition ofthe 14-3-3z-GPIb-IX interaction may serve as a basis for un-derstanding the role of 14-3-3 in signaling mediated byGPIb-IX.

Many of the 14-3-3-binding proteins contain an Arg-Ser-X-phosphoserine-X-Pro (RSXpSXP) motif, originally found in c-Raf (32). At least two of the RSXpSXP motif-containing ligandsc-Raf and tryptophan hydroxylase can be induced to bind 14-3-3 by PKA-catalyzed phosphorylation (32, 33). Other serineprotein kinases have also been shown to induce 14-3-3 bindingto various proteins (27, 34). Recently, several different 14-3-3binding sequences have been identified in 14-3-3 ligands in-cluding RX(Y/F)XpSXP, RXXSXpSXP, and RXSX(pS/pT)XP(where pT is phosphothreonine) (15, 27, 35). In the cytoplasmicdomain of GPIba, a 5-residue sequence, SGHSL, is both neces-sary and sufficient for interaction with 14-3-3z (14, 15). Inter-estingly, in contrast to RSXpSXP-containing ligands c-Raf andtryptophan hydroxylase, the cytoplasmic domain of GPIba ap-pears to be a poor PKA substrate in intact platelets (36, 37).Thus, characterization of the structural basis of the GPIbainteraction with 14-3-3 will clarify the differences betweenRSXpSXP-containing 14-3-3 ligands and GPIba and furtherour understanding of how 14-3-3 regulates functions of differ-ent types of ligand proteins.

In this study, we have identified a binding site in 14-3-3z inthe helix I region of 14-3-3z encompassing residues 202–231that interact with the GPIba cytoplasmic domain and the in-tact GPIb-IX complex. We also show in vitro that c-Raf binds to

* This work was supported in part by Grant HL52547 from theNational Institutes of Health. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

‡ Established Investigator of the American Heart Association. Towhom correspondence should be addressed: Dept. of Pharmacology(M/C868), University of Illinois, Chicago, 835 S. Wolcott Ave., Chicago,IL 60612. Tel.: 312-355-0237; Fax: 312-996-1225; E-mail: xdu@uic.edu.

1 The abbreviations used are: GP, glycoprotein; GPIb-IX, glycoproteinIb-IX complex; PKA, protein kinase A; PAGE, polyacrylamide gel elec-trophoresis; MBP, maltose-binding protein; PCR, polymerase chain re-action; pS, phosphoserine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 50, Issue of December 11, pp. 33465–33471, 1998© 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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14-3-3z in a PKA-dependent manner and that this bindingrequires helix G but not helix I. Thus, the GPIba-binding site in14-3-3z is distinct from the binding site for RSXpSXP-contain-ing ligand c-Raf. Furthermore, we show that 14-3-3z dimeriza-tion is required for high affinity binding to GPIb-IX complex,suggesting that a dual site recognition mechanism involvingGPIba and b subunits and dimerized 14-3-3z.

MATERIALS AND METHODS

Recombinant 14-3-3z, 14-3-3z Mutants, and Recombinant GPIba Cy-toplasmic Domain—Cloning of the cDNA encoding wild type 14-3-3zwas described previously (14). The 14-3-3z cDNA was subcloned into apmalC2 vector (New England Biolabs, Beverly, MA). The construct(pmal1433z) encodes a fusion protein with the N-terminal region cor-responding to the Escherichia coli maltose-binding protein (MBP) andC-terminal region corresponding to 14-3-3z. Mutagenesis of pmal1433zwas performed using PCR techniques (38). In all mutants except T3,stop codons were introduced into reverse primers at designated sites ofthe 14-3-3z. The stop codon in mutant T3 was introduced inadvertentlyby PCR error. The mutants were subcloned into a pmalC2 vector at theEcoRI and XbaI sites. Correct sequences were verified by automatedsequencing. The wild type 14-3-3z and 14-3-3 truncation mutants werepurified by affinity chromatography using a cross-linked amylose-Sepharose column (New England Biolabs, Beverly, MA). Equivalentamounts (71 mmol of protein/ml Sepharose) of the purified wild type ormutant 14-3-3z or MBP were conjugated onto cyanogen bromide-acti-vated Sepharose 4B (Amersham Pharmacia Biotech), respectively. Cou-pling efficiencies in all cases were better than 99% as assessed byoptical density at 280 nm wave length.

The cDNA encoding GPIba in a pBlueScript vector was a generousgift from Dr. Jerry Ware at the Scripps Research Institute, La Jolla, CA.The cDNA fragment encoding the cytoplasmic domain of GPIba (resi-dues 518–610) was generated by PCR with EcoRI and XbaI sitesincorporated in the forward and reverse primers, respectively. The PCRproduct was subcloned into the pmalC2 vector. The correct sequencewas verified by automated sequencing. The protein was expressed andpurified as described previously (38).

Binding of Platelet GPIb-IX and Mal-IbaC to 14-3-3z—Specific bind-ing of the platelet GPIb-IX complex to 14-3-3z-conjugated beads hasbeen described previously (14). Briefly, washed platelets were resus-pended 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 byadding an equal volume of the solubilization buffer (2% Triton X-100,0.1 M Tris, 0.01 M EGTA, 0.15 M NaCl, and 1 mM dithiothreitol, pH 7.4)containing 0.2 mM E64 (Boehringer Mannheim) and 1 mM phenylmeth-ylsulfonyl fluoride (13). In some experiments, platelets were solubilizedin the presence of 1 mM CaCl2 but in the absence of EGTA and E64 toallow calpain cleavage of GPIba and thus generation of the C-terminaldomain of GPIb-IX complex. After centrifugation at 100,000 3 g for 30min, the lysates (200 ml) were incubated with 25 ml (50% (v/v)) MBP-conjugated control beads or 14-3-3z-conjugated beads at 4 °C for 1 h.The beads were then washed three times in a 1:1 mix of Hepes bufferand solubilization buffer. Bound proteins were extracted with SDS-PAGE sample buffer and analyzed by SDS-PAGE followed by Westernblotting with a monoclonal antibody against GPIba, WM23 (kindlyprovided by Dr. Michael C. Berndt, Baker Institute, Melbourne, Aus-tralia (39)). In some experiments, GPIb-IX was also detected using apolyclonal anti-peptide antibody against the C-terminal domain ofGPIba, anti-IbaC (14). Reactions with antibodies were visualized usingan enhanced chemiluminescence kit (Amersham Pharmacia Biotech),and Kodak X-Omat AR film. In some experiments, reactions werevisualized using SuperSignal chemiluminescence substrate (Pierce)and quantitated with a Bio-Rad PhosphorImager and chemilumines-cence-sensitive phosphor-screens that have a theoretical linear range of100–104. Binding of recombinant GPIba cytoplasmic domain fusionprotein (Mal-IbaC) to 14-3-3z was performed essentially as describedabove except purified Mal-IbaC fusion protein was used. The Mal-IbaCbinding was performed in the platelet lysate buffer described above orin 0.1 M sodium citrate, pH 5.6. Binding of Mal-IbaC protein to beadswas detected by Western blotting with a rabbit anti-peptide antibodyagainst the cytoplasmic domain of GPIba, anti-IbaC (14).

Binding of Recombinant c-Raf to 14-3-3z—Human recombinant c-RafcDNA (a gift from Dr. Michael Karin, University of California at SanDiego, La Jolla) was subcloned into a pmalC2 vector by three-fragmentligation (EcoRI, HindIII, and XbaI) and expressed as a fusion proteinwith maltose-binding protein. The purified recombinant c-Raf wasphosphorylated by incubation with protein kinase A catalytic subunit

(10 units/100 ml) (Sigma) and 1 mM ATP in 15 mM Hepes, 5 mM

magnesium acetate, 0.1 mM EGTA, 130 mM KCl, 1 mg/ml bovine serumalbumin, 1 mM dithiothreitol, pH 7.4, at 22 °C for 30 min. The PKA-treated c-Raf was incubated with 14-3-3z-conjugated beads at 4 °C for1 h. After three washes, bead-bound proteins were then analyzed bySDS-PAGE and immunoblotted with an antibody against c-Raf (SantaCruz Biotechnology).

Analysis of the Molecular Mass of 14-3-3z under Non-denaturingConditions—Purified MBP-14-3-3z fusion protein and 14-3-3 mutants(200 ml, ;1.5 mg/ml) were analyzed by gel filtration using a PharmaciaSuperdex 200 HR 10/30 column and a Pharmacia Explorer 10 highperformance liquid chromatography system at a flow rate of 0.5 ml/min.The column was equilibrated with 0.02 M Tris, 0.15 M NaCl, pH 7.4. Themolecular mass was determined by comparing with the elution volumesof the molecular mass standard proteins, IgG (150 kDa), bovine serumalbumin (67 kDa), and ovalbumin (43 kDa).

RESULTS

Truncation Mutagenesis of 14-3-3z—In order to identify thesequence within 14-3-3z that is responsible for its interactionwith the GPIb-IX complex, we made various 14-3-3z truncationmutants (Fig. 1). Mutants T1 to T6 were truncated from theC-terminal end of 14-3-3z. Mutants T7–T9 were truncated pro-gressively from the N-terminal end of the protein. T11-(136–209) encompasses helices G and H, and T12 contains helices Hand I. T13-(188–209) contains a single helix H that was impli-cated in tryptophan hydroxylase binding (34). T14-(202–231)containing helix I was generated when results obtained withT1–T13 indicated the location of the GPIba-binding site (seebelow). As 14-3-3z is composed of nine anti-parallel a-helices(A-I), truncation sites in all these mutants are located betweentwo neighboring a-helices as determined by the published crys-tal structure to avoid disruption of each of these helical struc-tures (Fig. 1). These mutant proteins were expressed as fusionproteins with maltose-binding protein. Equivalent amounts ofthe purified wild type and mutant 14-3-3z were conjugated tocyanogen bromide-activated Sepharose 4B as described previ-ously (38).

A Binding Site for the GPIb-IX Complex Is Located betweenResidues 188 and 231 of 14-3-3z—We first examined the bind-ing of Triton X-100-solubilized platelet GPIb-IX to the abovedescribed Sepharose beads conjugated with 14-3-3 truncationmutants (T1–T13). MBP-conjugated beads were used as a neg-

FIG. 1. Construction of truncation mutants of 14-3-3z. The wildtype 14-3-3z contains 246 amino acid residues (41) and is composed of 9a-helices from A to I starting from the N terminus (18, 19). Each a-helixregion is represented by a box, and non-helical regions are shown aslines. Truncation mutants of 14-3-3 were constructed and expressed asdescribed under “Materials and Methods.” The truncation mutantsused in this study are as follows: T1, residues 1–109; T2, residues1–162; T3, residues 1–31; T4, residues 1–187; T5, residues 1–209; T6,residues 1–231; T7, residues 33–246; T8, residues 136–246; T9, resi-dues 188–246; T11, residues 136–209; T12, residues 188–231; T13,residues 188–209; T14, residues 202–231.

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ative control, and the wild type 14-3-3z-beads was used as apositive control. Bead-bound GPIb-IX was detected by Westernblotting with an anti-GPIba antibody, WM23, and quantitatedwith a Bio-Rad PhosphorImager and chemiluminescence-sen-sitive phosphor-screens. Fig. 2 shows that GPIb-IX binds towild type 14-3-3z- but not MBP-conjugated beads. Removal ofthe C-terminal tail of 14-3-3z by truncation at amino acidresidue 231 (mutant T6) did not negatively affect GPIb-IXbinding, suggesting that the C-terminal region between resi-dues 231 and 246 is not required. Further truncation at residue209 (mutant T5), which removes the helix I (the first a-helixfrom the C-terminal end), completely abolished GPIb-IX bind-ing (Fig. 2). Furthermore, none of the truncation mutants thatlack helix I bound to GPIb-IX (T5, T11, T13, Fig. 2; T1-T4, datanot shown). Thus, the helix I region encompassing residues209–231 of 14-3-3z appears to be required for binding to GPIb-IX. Truncation mutants T9-(188–246) and T12-(188–231) con-taining helix H and I interacted with GPIb-IX, indicating thatthe sequence between residues 188 and 231 of 14-3-3z (helicesH and I) contains a binding site for the GPIb-IX complex.Similar results were also obtained when bead-bound GPIb-IX

was detected with an antibody against the C-terminal region ofGPIba, anti-IbaC (data not shown).

High Affinity Interaction Requires Dimerized 14-3-3z andMore Than One Subunit of GPIb-IX—Fig. 2 also shows thattruncation mutants of 14-3-3z lacking the N-terminal domain(T7, T8, T9, and T12) binds significantly less GPIb-IX in com-parison with wild type 14-3-3z. Since similar amounts of pro-teins were conjugated to these beads (Fig. 2C), this resultindicates that GPIb-IX bound to these mutants with reducedaffinity. In particular, the mutant T7 lacking only 33 residuesin helices A and B (1–33) showed dramatically reduced bindingto GPIb-IX (7%) in comparison with wild type 14-3-3z (Fig. 2),suggesting that the N-terminal helices (A and B) are importantto the high affinity interaction between wild type 14-3-3z andGPIb-IX. However, this reduction in GPIb-IX binding affinity ispartially “rescued” by further truncations from the N terminusthat removed helices A–E (T8, 20% binding compared with wildtype 14-3-3z) or helices A–G (T9, 50% binding compared withwild type) (Fig. 2). It is not clear why these further truncationsrescued the loss of binding affinity. One of the possibilities isthat further truncations resulted in increased accessibility ofGPIb-IX to the binding site in helix I. Nevertheless, this resultsuggests that the N-terminal domain, while important for highaffinity, is not required for binding to GPIb-IX.

Previously reported crystal structural analysis of 14-3-3z hasrevealed that the N-terminal helices (A and B) are involved inthe formation of 14-3-3z dimers (18, 19). Thus, it is possiblethat the reduced affinity for GPIb-IX is caused by disruption ofdimerization. To examine this possibility, we determined themolecular mass of the MBP-14-3-3z fusion proteins by gel fil-tration chromatography under non-denaturing conditions. The14-3-3z monomer has a molecular mass of ;30 kDa (40). MBPhas a molecular mass of ;40 kDa. The molecular mass for therecombinant MBP-14-3-3 fusion protein is ;70 kDa as ana-lyzed by SDS-PAGE. We found that the MBP-wild type 14-3-3z

fusion protein has a molecular mass of ;160 kDa as deter-mined by gel filtration chromatography (Fig. 3A). Thus, therecombinant wild type 14-3-3z indeed exists as a dimer eventhough its N terminus is fused to MBP. In contrast, the fusionproteins encoding 14-3-3 mutants (T7, T9, and T12) lacking theN-terminal domain showed a molecular mass of approximately70, 55, and 45 kDa by gel filtration (Fig. 3A), indicating thatthey are monomers. Thus, the N-terminal domain deletiondisrupted dimerization of 14-3-3z. Taken together, these re-sults suggest that a dimeric structure of 14-3-3z is required forhigh affinity binding to GPIb-IX.

Previous studies suggest that 14-3-3 interaction withGPIb-IX may involve two binding sites in GPIb-IX located inthe C-terminal domain of GPIba and GPIbb, respectively (14–16). To examine whether the high affinity binding of dimerized14-3-3z to GPIb-IX may involve interaction with the cytoplas-mic domains of both GPIba and GPIbb, we compared binding of14-3-3z to the recombinant GPIba cytoplasmic (C-terminal)domain alone and to the C-terminal domain of the GPIb-IXcomplex (composed of the C-terminal domain of GPIba, GPIbb

and GPIX (6, 14)) (Fig. 4). As we reported previously (14), theC-terminal domain of GPIb-IX maintained the high affinity14-3-3z binding function (Fig. 4). In contrast, the recombinantcytoplasmic domain of GPIba bound much more weakly to14-3-3z, indicating that in addition to GPIba, other subunits ofthe GPIb-IX complex (probably GPIbb) are involved in highaffinity binding to 14-3-3z dimer.

Helix I of 14-3-3z (Residues 202–231) Contains a Binding Sitefor the GPIba Cytoplasmic Domain—We showed previouslythat the binding of the GPIb-IX complex to 14-3-3z is dependenton the interaction between 14-3-3z and the C-terminal se-

FIG. 2. Binding of the GPIb-IX complex to wild type 14-3-3z andtruncation mutants. Binding of the GPIb-IX complex to 14-3-3z wasperformed as reported previously (see “Materials and Methods”) (14).Triton X-100-solubilized platelet lysates were incubated with Sepha-rose beads conjugated with equivalent amounts of maltose-bindingprotein (MBP) as negative control, wild type 14-3-3z (WT), or truncationmutants T5-(1–209), T6-(1–231), T7-(33–246), T8-(136–246), T9-(188–246), T11-(136–209), T12-(188–231), and T13-(188–209). After threewashes, bead-bound GPIb-IX complex was separated by SDS-PAGEand detected by Western blotting with the monoclonal antibody againstGPIba, WM23. Relative quantity of GPIb on the same gel was esti-mated using a Bio-Rad PhosphorImager GS525 and chemilumines-cence-sensitive phosphor screen. Results from three separate assays(mean 6 S.D.) are shown in A. The picture of a representative Westernblot is shown in B. The position of GPIba is shown by the arrow. Thelower molecular weight band is a proteolytic fragment of GPIba (45).GPIb-IX binding to mutants T1-(1–109), T2-(1–162), T3-(1–31), andT4-(1–187) were also examined, but no specific binding was detected. Toestimate the amounts of various 14-3-3z mutant MBP fusion proteinsconjugated to Sepharose beads, the beads were incubated with a rabbitantibody against MBP at 4 °C for 1 h. After washing, the bead-boundantibody was analyzed by SDS-PAGE and Western blotting with anti-rabbit IgG and enhanced chemiluminescence (C).

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quence of the GPIba cytoplasmic domain (14). It is thus possi-ble that the above identified GPIb-IX-binding site of 14-3-3zcontains a binding site for GPIba. To examine this possibility,we tested the binding of recombinant GPIba C-terminal do-main MBP fusion protein (Mal-IbaC) to wild type 14-3-3z andvarious 14-3-3 mutants. Fig. 5 shows that wild type Mal-IbaCprotein specifically binds to the 14-3-3z-conjugated beads. Re-moval of the C-terminal tail of 14-3-3z by truncation at residue231 (mutant T6) did not negatively affect Mal-IbaC binding,whereas truncation at residue 209 (mutant T5) to remove helixI abolished Mal-IbaC binding. In addition, binding of othertruncation mutants that lack the helix I to Mal-IbaC was alsodisrupted. Thus, the same helix I region (residues 209–231) of14-3-3z that is required for GPIb-IX binding is also required for14-3-3z binding to the GPIba cytoplasmic domain. In contrastto the results obtained with the GPIb-IX complex, however, thebinding of Mal-IbaC to the monomeric truncation mutants T7,T8, T9, and T12 (lacking the N-terminal dimerization site) wasnot significantly reduced but rather a little stronger than thedimeric wild type 14-3-3z. This suggests that the dimeric struc-ture of 14-3-3z is not required for binding to the recombinantGPIba cytoplasmic domain. It is not clear why Mal-IbaC boundto the small truncation mutants stronger than to wild type14-3-3z. It is unlikely that this is caused by the variation in theamounts of bead-conjugated proteins as the wild type andmutant 14-3-3z-MBP fusion protein bound identical amounts of

anti-MBP antibody (see Fig. 2C). It is possible that truncationscaused increased exposure of the ligand-binding site in thehelix I of 14-3-3z to the recombinant GPIba cytoplasmicdomain.

To verify further that helix I is indeed a binding site for theGPIba cytoplasmic domain, we made an additional 14-3-3z

FIG. 3. Disruption of 14-3-3z dimerization by N-terminal dele-tion. Molecular mass of the wild type 14-3-3z-MBP fusion protein andthe deletion mutants T7, T9, and T12 were analyzed by gel filtrationchromatography (A) as described under “Materials and Methods” andby SDS-PAGE (B). The elution volume for wild type 14-3-3z (WT1433)on a Superdex 200HR 10/30 column is similar to that of rabbit IgG,indicating that it has a molecular mass of ;160 kDa, in comparisonwith the molecular mass determined by SDS-PAGE (;70 kDa). Thus,wild type 14-3-3z exists as a dimer under non-denaturing conditions.Elution volume of mutant T7 lacking the N-terminal helices A and B(residues 1–32) indicated that its molecular mass is ;70 kDa undernon-denaturing conditions (A), similar to its molecular mass deter-mined by SDS-PAGE (67 kDa) (B). Similarly, molecular mass of T9 andthat of T12 as determined by gel filtration are comparable to thatdetermined by SDS-PAGE, indicating that the mutants lacking theN-terminal domain exist as monomers. Shown at the bottom of A is thechromatograph of the molecular mass standard proteins IgG (150 kDa),bovine serum albumin (67 kDa), and ovalbumin (45 kDa). FIG. 4. Comparison between 14-3-3z binding to the C-terminal

domain of GPIb-IX complex and to the recombinant C-terminaldomain of GPIba. Platelets were solubilized as described previously(14) to allow proteolysis at the protease-sensitive region of GPIba, thusgenerating the C-terminal fragment of GPIba complexed with GPIbband GPIX (see C). The lysates were then incubated with 14-3-3z-conju-gated beads or control MBP-conjugated beads for 1 h. Comparableamounts of purified recombinant GPIba cytoplasmic domain MBP fu-sion protein were also incubated with 14-3-3z beads (14-3-3) or controlbeads (MBP) under identical conditions. The beads were washed threetimes. Bound proteins were eluted with SDS-PAGE sample buffer,separated by SDS-PAGE, and detected by Western blotting with anantibody against the C terminus of GPIba (anti-IbaC). A, a typicalpicture of Western blots showing that comparable amounts of GPIb-IXC-terminal domain or recombinant GPIba cytoplasmic domain wereincubated with 14-3-3z beads (added), but 14-3-3z-bound recombinantGPIba cytoplasmic domain (Mal-IbaC) was reduced in comparison withthe C-terminal domain of GPIb-IX complex (Ib-IX). B, the Western blotwas scanned, and optical density of each band from the same blot wasquantitated with NIH Image software. Note that the amount of boundGPIb-IX C-terminal domain (Ib-IX) is approximately 3 times that of therecombinant GPIba cytoplasmic domain (Mal-IbaC) when comparableamounts were incubated with 14-3-3z-conjugated beads. C, a schematicof the GPIb-IX complex. Locations of 14-3-3z-binding sites in bothGPIba and GPIbb as well as the protease-sensitive region are indicatedby arrows.

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truncation mutant, T14-(202–231), containing only helix I.This mutant strongly bound to Mal-IbaC (Fig. 5B), indicatingthat the 30-residue helix I-(202–231) region of 14-3-3z is suffi-cient for binding to the GPIba cytoplasmic domain.

PKA-dependent c-Raf Binding to 14-3-3z Requires Helix G(Residues 162–187) but Not Helix I (Residues 209–231)—Todetermine whether the GPIba-binding site of 14-3-3z is alsorequired for the binding of other 14-3-3 ligands, we examinedPKA-dependent binding of c-Raf (a 14-3-3 ligand with the RSX-pSXP binding motif (32)) to the 14-3-3z truncation mutants.Fig. 6A shows that, in the absence of PKA pretreatment, c-Raf-MBP fusion protein bound weakly to 14-3-3z-beads, compara-ble with control MBP-conjugated beads. After PKA-pretreat-ment, however, c-Raf bound to 14-3-3z strongly. This bindingwas not affected by truncation of 14-3-3z that removed helix I(T5) or both helices H and I (T4) from the C terminus, indicat-ing that the GPIba-binding site in helix I is not required for theinteraction of 14-3-3z with c-Raf (Fig. 6B). However, furthertruncation from the C terminus to residue 162 to remove helixG (T2) abolished specific c-Raf binding. The truncation mutantT8 lacking the N-terminal helices A–E still bound to c-Raf, butthe mutant T9, which contains C-terminal helices H and I butlacks helices A–G, lost c-Raf binding capacity (Fig. 6B). Thus,binding of the RSXpSXP motif-containing ligand c-Raf to 14-3-3z requires helix G but not helix I.

DISCUSSION

In this study, we have further characterized the interactionbetween GPIb-IX and 14-3-3z by analyzing the GPIb-IX bind-ing function of a series of truncation mutants of 14-3-3z. Weshow that a binding site for the GPIba cytoplasmic domain islocated in the helix I of 14-3-3z encompassing residues 202–231in the C-terminal domain (Fig. 5). This binding site is alsorequired for 14-3-3z binding to intact GPIb-IX complex (Fig. 2).The GPIba-binding site is distinct from the binding sites forRSXpSXP motif-dependent 14-3-3 ligands, as we show thatPKA-dependent binding of c-Raf to 14-3-3z requires helix G but

not helix I (Fig. 6). Previous studies also showed that theRSXpSXP-containing ligand tryptophan hydroxylase bound toa fragment of 14-3-3h containing helices G and H but not I (34).Furthermore, crystal structure data (35) and mutagenesisstudies (41) suggest that phosphorylation-dependent binding ofRSXpSXP motif containing ligands may involve the interactionof phosphoserine (pS) with Lys-49 and Arg-56 in helix C andArg-127 and Tyr-128 in helix E, which are not required forbinding to GPIba cytoplasmic domain (Fig. 5). In fact, thepresence of the helices C–E is inhibitory to the interactionbetween GPIb-IX and monomeric 14-3-3z mutants (Fig. 2).Thus, our data suggest that different types of 14-3-3 ligandspreferentially interact with 14-3-3 at different sites in the largeligand binding groove surrounded by helices C, E, G, and I (18,19, 35). It is likely that GPIba represents a different class of the14-3-3 ligands which preferentially recognize the binding sitein the helix I of 14-3-3 proteins, whereas RSXpSXP-like ligandspreferentially interact with helix G. Andrews et al. (15) alignedseveral 14-3-3-binding proteins that have sequences similar tothe GPIba binding sequence (including Cdc25a, Cdc25b, c-Raf,and c-Cbl). A striking feature in these 14-3-3 ligands is thepresence of an HSL tripeptide sequence. It would be interestingto investigate further whether this HSL motif is responsible forthe binding of this class of 14-3-3 ligands to helix I of 14-3-3.Interestingly, while our manuscript was in revision, Petosa etal. (42) reported crystal structural data indicating that anunphosphorylated non-RSXpSXP peptide ligand of 14-3-3z(WLDLE) obtained by screening a phage display library bindsby amphipathic interaction to sites within the ligand bindinggroove overlapping with but distinct from c-Raf PSXpSXP pep-tide-binding sites, supporting the notion that different ligandsmay interact with different sites in the groove. Our data pro-vide first evidence that the interaction with the helix I region isboth required and sufficient for 14-3-3z binding to a physiolog-

FIG. 5. Binding of recombinant GPIba cytoplasmic domain towild type and mutant 14-3-3z. A, purified recombinant GPIba cyto-plasmic domain MBP fusion protein (Mal-IbaC) was incubated withSepharose beads conjugated with equivalent amounts of maltose-bind-ing protein (MBP), wild type 14-3-3z (WT), truncation mutants T5-(1–209), T6-(1–231), T7-(33–246), T8-(136–246), T9-(188–246), T11-(136–209), T12-(188–231), and T13-(188–209). After three washes, boundMal-IbaC was eluted by boiling in SDS-PAGE sample buffer and de-tected by Western blotting with a rabbit anti-peptide antibody, anti-IbaC, against the sequence DLLSTVSIRYSGHSL corresponding to theC terminus of GPIba cytoplasmic domain. Mutants T1-(1–109), T2-(1–162), T3-(1–31), and T4-(1–187) were also examined, but no specificbinding to Mal-IbaC was detected (data not shown). B, binding ofMal-IbaC to the 14-3-3z truncation mutant T14-(202–231) containing asingle a-helix (helix I). The assay was performed as described in A.

FIG. 6. Phosphorylation-dependent binding of c-Raf to wildtype and mutant 14-3-3z. A, recombinant human c-Raf was expressedas a fusion protein with MBP. Purified recombinant c-Raf (100 mg/ml)was pretreated in the absence or presence of 10 units/100 ml proteinkinase A catalytic subunit and 1 mM ATP for 30 min at 22 °C and thenallowed to incubate with Sepharose beads conjugated with equivalentamounts of MBP or wild type 14-3-3z. After washing, the bound c-Rafwas eluted in SDS-PAGE sample buffer, separated by SDS-PAGE, anddetected by Western blotting with a rabbit antibody against c-Raf. B,binding of PKA-treated c-Raf to beads conjugated with wild type 14-3-3z(WT) and the truncation mutants T2-(1–162), T3-(1–31), T4-(1–187),T5-(1–209), T6-(1–231), T7-(33–246), T8-(136–246), and T9-(188–246).In all cases, c-Raf was pretreated with PKA as described in A.

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ical non-RSXpSXP ligand of 14-3-3z, GPIba.We show that the GPIb-IX complex has a higher affinity for

14-3-3z than the recombinant GPIba cytoplasmic domain (Fig.4), suggesting that other subunits of GPIb-IX complex in addi-tion to GPIba may be involved in the interaction with 14-3-3z.This result is consistent with the previous findings that highaffinity binding to 14-3-3z involves both GPIba (14, 15) andGPIbb which contains a phosphorylation-dependent 14-3-3z-binding site (15, 16). Wild type 14-3-3z exists as a homodimervia the interaction between the N-terminal domains (helices Aand B) of each monomer (18, 19). Thus, we investigated thepossibility that high affinity interaction involves simultaneousbinding of GPIba and GPIbb to a 14-3-3 dimer. We show thatthe 14-3-3z mutants lacking the dimerization site in helices Aand B bound to the GPIb-IX complex with significantly reducedaffinity compared with the dimeric wild type 14-3-3z (Fig. 2).Gel filtration chromatography confirmed that deletion of heli-ces A and B disrupted dimerization of 14-3-3z (Fig. 3), suggest-ing that the high affinity binding of 14-3-3z to GPIb-IX requires14-3-3z dimerization. Furthermore, since the binding of therecombinant GPIba cytoplasmic domain and the phosphoryl-ated c-Raf to these monomeric 14-3-3z mutants was not de-creased compared with dimeric wild type 14-3-3z (Figs. 5 and6), the reduced binding of GPIb-IX is unlikely to result from thedisruption of ligand-binding sites in these monomeric mutants.Thus, 14-3-3z binding to GPIb-IX appears to involve the inter-action of 14-3-3z dimer to GPIba and an additional binding sitein other GPIb-IX subunits. As GPIbb contains a PKA-catalyzedphosphorylation-dependent binding site for 14-3-3z, it is likelythat high affinity binding of intact GPIb-IX involves interactionof GPIba and GPIbb to each monomer of the 14-3-3z dimer. AsGPIbb phosphorylation has been found to be dynamically reg-ulated in platelets by increase in intracellular c-AMP level (36,37, 43), this dual site binding mechanism may enable the14-3-3-GPIb-IX interaction to be dynamically regulated inplatelets and thus may participate in the GPIb-IX-coupled in-tracellular signaling and cytoskeleton regulation.

We show that helix G of 14-3-3z is critical for binding to theRSXpSXP motif containing ligand c-Raf (Fig. 6). The publishedcrystal structural data, however, indicate that the critical phos-phoserine in RSXpSXP motif is in the proximity of the residuesLys-49, Arg-56, Arg-127, and Tyr-128 in helices C and E, sug-gesting that helix G may not be the recognition site for thephosphoserine in the RSXpSXP motif. While our manuscript isunder revision, Petosa et al. (42) and Wang et al. (44) reportedcrystal structure of 14-3-3z bound to a RSXpSXP peptide de-rived from c-Raf, suggesting a hydrophobic surface lining theligand binding groove is proximal to the ligand peptide. Thishydrophobic surface includes residues Leu-172, Val-176, Leu-216, Leu-220, and Leu-227. Among these residues, Leu-172 andVal-176 are located in helix G. Replacement of Leu-172 orVal-176 with negatively charged residues abolished the bind-ing of c-Raf suggesting that these hydrophobic residues arecritical for the c-Raf binding. Thus, it is possible that RSXpSXPligands may bind to helix G by hydrophobic interaction. Ashelix G is required for interaction with phosphorylated c-Raf,we suggest that interaction of the phosphoserine in the ligandpeptide with residues in helices C and E is not sufficient tosupport the binding of c-Raf. Furthermore, as we show thatmutant T8 lacking helices C and E binds to phosphorylatedc-Raf, these helices are not required for interaction with c-Raf.Thus, it is possible that the interaction between phosphoserinein the RSXpSXP motif and helices C and E serves to regulatethe ligand interaction with the helix G (c-Raf) and helix I(GPIba) regions in the wild type 14-3-3z.

Interestingly, Wang et al. (44) showed that point mutations

that replace the hydrophobic residues Leu-216, Leu-220, andLeu-227 in the helix I with negatively charged residue asparticacid also significantly reduced 14-3-3z binding to c-Raf. How-ever, we show that PKA-phosphorylated c-Raf bound to 14-3-3zmutant lacking helix I (Fig. 6). Our data suggest that, while itis possible that helix I is involved in the interaction betweenc-Raf and 14-3-3z, this region is not required for this interac-tion. A major difference between Wang et al. (44) and our datais that the recombinant c-Raf used in our assays is in vitrophosphorylated by PKA which is known to phosphorylate theRSXpSXP motif (32), whereas Wang et al. (44) used the yeasttwo-hybrid system, in vitro translated protein, or cell lysateswith unknown phosphorylation status. Furthermore, bindingof c-Raf to 14-3-3z in their assays did not appear to be influ-enced by stimulation of c-Raf phosphorylation (44), which issimilar to GPIb-IX binding to 14-3-3z (13, 14). Thus, it ispossible that in the assay by Wang et al. (44), c-Raf binding to14-3-3z may involve a GPIba-like motif in addition to the RSX-pSXP motifs. An HSL sequence similar to the 14-3-3z bindingsequence of GPIba was indeed found in c-Raf (15). Thus, wespeculate that c-Raf binding to dimeric 14-3-3z in vivo may besimilar to GPIb-IX binding, involving a dual binding sitemechanism.

Acknowledgments—We thank Drs. Randal Skidgel and Richard Yefor critical reading of this manuscript, and Drs. Michael Berndt, MarkGinsberg and Jerry Ware for providing reagents.

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Minyi Gu and Xiaoping Du and Distinct from the c-Raf-binding SiteαGlycoprotein Ib

-Form 14-3-3 Protein Recognizing the PlateletζA Novel Ligand-binding Site in the

doi: 10.1074/jbc.273.50.334651998, 273:33465-33471.J. Biol. Chem. 

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