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Human Signaling Protein 14-3-3z Interacts With Platelet Glycoprotein Ib
Subunits Iba and Ibb
By David C. Calverley, Terrance J. Kavanagh, and Gerald J. Roth
The initiation of primary hemostasis is mediated by interaction of the platelet glycoprotein Ib (GPIb) surface receptor
and its arterial subendothelial von Willebrand factor (vWF)
ligand. The intracellular signaling immediately following
GPIb receptor occupancy connecting the adhesive event to
platelet activation and aggregation has not been well characterized. The 14-3-3 proteins are a 27- to 30-kD ubiquitous
protein family with diverse biologic roles, including functional modulation of several prominent signaling proteins.
We used the yeast two-hybrid system and confocal microscopy to characterize the recently described interaction between GPIb and platelet 14-3-3z, and provide evidence for
the potential signaling role of this protein. Two-hybrid
interactions suggest that platelet 14-3-3z associates with the
cytoplasmic domain of GPIb subunits Iba and Ibb in transformed yeast cells. The 14-3-3 interaction with GPIbb may be
partly mediated through the latter’s phosphorylated serine
166 residue as its mutagenesis results in 20% to 40% reduced
interaction. There was 51% to 59% reduced interaction
between GPIb and three 14-3-3z deletion mutants compared
with full-length 14-3-3z, suggesting that either the Nterminal dimerization or membrane-binding domains or
more than one noncontiguous 14-3-3z element may be
required for optimal GPIb interaction. Confocal studies of
platelets and a megakaryocyte cell line provided additional
evidence for interaction of 14-3-3z with GPIba and GPIbb. We
also found that, similar to the signaling mediators phosphatidylinositol 3-kinase and Src, platelet cytoskeletal 14-3-3z
content is increased following vWF and ristocetin stimulation. We suggest that platelet 14-3-3z interacts with GPIba
and Ibb, that this interaction may be partly mediated through
phosphoserine recognition, and that 14-3-3z cytoskeletal
translocation may serve as a GPIb post–receptor occupancy
signaling event.
r 1998 by The American Society of Hematology.
P
mammalian cell types, plants, and yeast.7-13 The 14-3-3 family
has recently been implicated in the regulation of intracellular
signaling pathways through interaction with several oncogene
and proto-oncogene products. Association with and regulation
of proteins with key signaling roles such as Raf-1, protein
kinase C, and phosphatidylinositol 3-kinase by 14-3-3 has been
described.12,14,15 This group may have their kinase activity
altered by this binding depending on the experimental conditions used.
Recent affinity chromatography experiments using a monoclonal antibody to GPIba led to the coelution of a 29-kD protein
with GPIb.16 Amino acid sequencing of the protein led to its
identification as the z isoform of 14-3-3. In view of the
association of 14-3-3 with mediators that participate in known
early signaling events,14,15,17 and the protein’s membranebinding domain8,18 and phosphoserine site,11 one may speculate
that this protein participates in GPIb signaling following vWF
stimulation. The current study uses the yeast two-hybrid
expression system, confocal microscopy, and platelet compart-
LATELET ADHESION TO arterial subendothelium is an
integral component of thrombus formation and is mediated
by a shear-dependent interaction between the platelet glycoprotein (GP) Ib/V/IX receptor on the platelet surface and its ligand,
von Willebrand factor (vWF). Following this interaction, the
platelet undergoes characteristic morphologic and biochemical
changes associated with its transition from a resting state to an
activated state. This includes a conformational change of the
platelet surface GPIIb-IIIa integrin that facilitates its interaction
with ligands, including plasma fibrinogen, that lead to platelet
aggregation.
The platelet GPIb receptor consists of four polypeptide
chains with certain features in common.1 First, they are all
transmembrane proteins with extracellular and cytoplasmic
domains. The GPIba subunit is associated covalently with the
smaller GPIbb subunit, while GPIX and GPV are noncovalently
associated with the complex. Surface expression of GPIb
subunits is significantly reduced or absent in the congenital
bleeding disorder Bernard-Soulier syndrome, in which mutations involving three subunits have been described.2,3 Each
GPIb subunit contains one or more 24–amino acid tandem
repeats rich in leucine that have been demonstrated to participate in protein-protein interactions in other cells.1
Tyrosine phosphorylation of multiple platelet proteins is
demonstrable upon GPIb stimulation with vWF and ristocetin,
but the precise molecular signaling events leading to platelet
activation remain to be elucidated. Previous investigation has
demonstrated the translocation of platelet Src and phosphatidylinositol 3-kinase to cytoskeletal elements in response to vWF
stimulation, suggesting these enzymes play a role in the
cytoskeletal reorganization associated with platelet activation
following vWF-GPIb interaction.4
The 14-3-3 proteins are a 27- to 30-kD family originally
isolated from the brain, with at least 10 homologous mammalian isoforms now described.5-7 Current knowledge regarding
the functional domains of the 14-3-3 family is shown in Fig 1.
They exist as homodimers and heterodimers in vivo and have
been found to exhibit a wide array of biologic functions in many
Blood, Vol 91, No 4 (February 15), 1998: pp 1295-1303
From the Medical and Research Services, Seattle Veterans’ Affairs
Medical Center, Seattle; and the Division of Hematology, Department
of Medicine, and Department of Environmental Health, University of
Washington, Seattle, WA.
Submitted May 5, 1997; accepted September 29, 1997.
Supported by Grants No. HL39947 and ES07033 from the National
Institutes of Health, a Merit Review Award from the Department of
Veterans Affairs, and the Medical Research Council of Canada.
Presented in part at the 1996 38th Annual Meeting of the American
Society of Hematology, Orlando, FL (December 7-10, 1996).
Address reprint requests to Gerald J. Roth, MD, Medical Service,
Seattle VA Medical Center (111), 1660 S Columbian Way, Seattle, WA
98108.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9104-0005$3.00/0
1295
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1296
CALVERLEY, KAVANAGH, AND ROTH
Fig 1. 14-3-3z structural features and deletion mutants used in
yeast 2-hybrid cotransformations with platelet GPIb subunits. Known
and putative domains include a 26-residue N-terminal dimerization
domain (DD),8,9 a domain with homology to the C terminus of the
annexin protein family that acts as a protein kinase C (PKC) inhibitor
(AHD),10 and a putative C-terminal PKC-inhibitory domain that a
recent mutagenesis investigation was unable to more precisely
localize (PKC ID).8 A C-terminal phosphoserine (residue 185*) has also
been characterized.11
ment immunoblot studies to document and quantify the interaction of 14-3-3z with cytoplasmic domains of the GPIb subunits,
and begins to characterize its potential role in GPIb signaling.
MATERIALS AND METHODS
Yeast two-hybrid constructs and transformation. The cytoplasmic
domains of platelet GPIba and GPIbb were each polymerase chain
reaction (PCR)-amplified from platelet cDNA generated by platelet
RNA reverse transcription. Platelet RNA was isolated from the therapeutic pheresis material of a patient with essential thrombocythemia. The
sense primers each contained EcoRI sites and the antisense primers
contained SalI sites to facilitate directional cloning of the restriction
enzyme–digested PCR products into the yeast expression vectors
pGBT9 and pGAD424 (Clontech Laboratories Inc, Palo Alto, CA). This
resulted in the in-frame fusion of each cytoplasmic domain to the 38 end
of either the GAL4 (1-147) DNA-binding domain (pGBT9) or the
GAL4 (768-881) activation domain. Similar PCR and cloning procedures led to construction of yeast expression vectors containing
full-length platelet 14-3-3z.19
Construction of the GPIbb (ser166ala) substitution mutant was
achieved with the site-directed mutagenesis technique of Michael.20
PCR amplification of the Ibb cytoplasmic domain used the original two
wild-type primers along with a third sense primer incorporating the
ser166ala mutation and an HpaI site. The latter facilitated colony
screening following transformation into Escherichia coli without altering the amino acid sequence of the resultant protein other than the
desired substitution. PCR was performed in the presence of a thermostable DNA ligase (Taq ligase; Sigma Inc, St Louis, MO) to permit 58 to
38 ligation of the wild-type sense strand to the first base of the
mutagenic sense primer. The 14-3-3 deletion constructs (Fig 1) were
prepared similarly to the wild-type constructs using primers with EcoRI
(sense) and SalI (antisense) restriction site adaptors. All wild-type and
mutant DNA sequences were confirmed by automated sequencing
technology.
Transformations of pairwise vector combinations into yeast strain
SFY526 (MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901,
leu2-3, 112, canr, gal4-542, gal80-538, URA3 :: GAL1-lacZ; Clontech)
were made as described by Bartel et al.21 After verification of the
strain’s nutritional phenotype (auxotrophic for tryptophan and leucine),
early log-phase (OD600 5 0.2 to 0.3) cultures were grown for 3 hours at
30°C in YPD medium before transformation of 100 µL with 100 ng of
each construct. Following agitation in 40% polyethylene glycol/100mmol/L lithium acetate at 30°C for 30 minutes, addition of 10%
dimethyl sulfoxide, and heat shock at 42°C for 15 minutes, the
transformants were plated on selective media at 30°C until 2-mm
colonies were visible.
Liquid culture and colony filter-lift b-galactosidase assays. For
quantitative b-galactosidase assays, a single yeast colony was innoculated into 5 mL SD medium without tryptophan and leucine and agitated
overnight at 30°C, and 2 mL was added to 8 mL YPD liquid and grown
until the OD600 was 0.5 to 0.7. Then, 1.5 mL was processed for
quantitation of b-galactosidase activity expressed in units using ONPG
substrate and taken to represent the strength of interaction between the
two fusion proteins.21-24 Colony filter-lifts were performed on transformants by submersion of nitrocellulose transfer membranes lifted from
each colony plate into liquid nitrogen (Clontech). This was followed by
incubation at 30°C for up to 8 hours over Whatman (Maidstone, UK)
filter paper presoaked in Z buffer (60 mmol/L Na2 HPO4 · 7H2O, 40
mmol/L NaH2PO4H2O, 10 mmol/L KCl, and 1 mmol/L MgSO4, pH 7.0)
with 0.27% b-mercaptoethanol and 1.67% X-gal in 100-mm petri
dishes.
Platelet and human erythroleukemia cell confocal microscopy studies. Human erythroleukemia (HEL) cells were grown at 37°C in a 5%
CO2 atmosphere in RPMI 1640 medium (Bio-Whittaker Inc, Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine serum,
2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 100 U/mL penicillin,
0.1 mg/mL streptomycin, 2.5 µg/mL amphotericin B, and 2.05 µg/mL
desoxycholate (Fungizone; Life Technologies Inc, Grand Island, NY).
To separate the platelets, platelet-rich plasma was centrifuged twice
(1,000g for 10 minutes) followed by suspension of the platelet pellet in
35 mL washing buffer (10 mmol/L Tris, pH 7.0, 150 mmol/L NaCl, 1
mmol/L EDTA, and 7 mmol/L theophylline). Following centrifugation
at 100g for 10 minutes, the suspended platelets were pelleted at 1,000g
for 10 minutes and the concentration was determined in 2 mL Tyrode’s
buffer.25
Rabbit anti-human GPIba polyclonal antibody,26 mouse monoclonal
antibody Beb1 (anti-human GPIX), and mouse monoclonal antibody
C-34 (anti-human Iba) were used in confocal microscopy studies. The
latter two were characterized by our laboratory and others in association
with the Fifth International Workshop on Leukocyte Differentiation
Antigens.27 Mouse anti-CD62P was obtained from Becton Dickinson
Immunocytometry Systems (San Jose, CA). Gi27 (mouse anti-human
GPIbb) was a gift from Dr Sentot Santoso of Universitat Giessen
(Giessen, Germany). Rabbit polyclonal anti-human 14-3-3z was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Fluoresceinisothiocyanate (FITC)-conjugated goat F(ab8)2 anti-rabbit antibody was
obtained from Southern Biotechnology Associates Inc (Birmingham,
AL), and CY3-conjugated goat anti-mouse antibody was obtained from
Jackson Laboratories (West Grove, PA).
Platelets and HEL cells assessed for 14-3-3 and GPIb colocalization
were aliquotted to 30 3 106 platelets/mL or 5 3 106 cells/mL
phosphate-buffered saline (PBS) or PBS with 1% BME if subsequently
labeled with anti-Ibb. After 15 minutes, each platelet sample was
cytospun onto a slide for the rest of the labeling procedure while HEL
cells were centrifuged (130g for 10 minutes) following each of the
remaining steps and then resuspended for the next step. The PBS
incubation step was followed by suspension of platelets and HEL cells
in 90% ethanol in PBS for 45 minutes.28 After washing in PBS, they
were incubated for 30 minutes with 1:10 to 1:100 dilutions of two
primary antibodies. After washing twice, they were then incubated for
30 minutes with 1:10 to 1:100 dilutions of fluorochrome-conjugated
secondary antibodies. Following this, the platelets and HEL cells were
washed twice and then suspended in 50 µL PBS. For confocal
microscopy, 20 µL antibody-labeled suspended HEL cells were mounted
onto a glass slide to which a glass cover slip was applied separated by an
adhesive tape spacer to prevent crush artifact. Platelet and HEL cell
slides were visualized using an ACAS Ultima Laser Cytometer
(Meridian Instruments, Okemos, MI). Slides were scanned in dual-color
confocal mode (pinhole 5 225 µm for platelets and 400 µm for HEL
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PLATELET GLYCOPROTEIN IB AND 14-3-3
cells) using a 1003 oil-immersion objective (numeric aperture 5 1.3),
a step size of 0.1 µm/pixel for platelets and 0.2 µm for HEL cells, and a
field size of 360 3 360 pixels (platelets) or 180 3 180 or 270 3 270
pixels (HEL cells). FITC fluorescence was detected in PMT1 with a
530/30-nm band-pass filter, and CY3 fluorescence was detected after
separation with a dichroic beam splitter (560-nm short-pass filter) in
PMT2 with a 580/30-nm band-pass filter. Dual-color digital images
were displayed on a DASY 9000 Image analysis workstation (Meridian)
using the manufacturer’s software, and were saved as tag interchange
file format (TIFF) files. Similarly, after using a threshold for background fluorescence, pixel histograms were generated from these
images and displayed using the manufacturer’s software and printed
with a Sony Mavigraph video printer (Sony Corp, Tokyo, Japan).
Platelet 14-3-3 translocation studies. To study the relative amount
of 14-3-3z present in platelet compartments during the resting state or
following stimulation with known agonists, fresh platelets were first
isolated from whole blood as already described. After suspension in
Tyrode’s buffer, 108 platelets were aggregated for 5 minutes at 37°C
following addition of either (1) 10 µg/mL vWF isolated from human
cryoprecipitate,29 (2) 1 mg/mL ristocetin (American Biochemical and
Pharmaceutical Corp, New York, NY), (3) both 10 µg/mL vWF and 1
mg/mL ristocetin, (4) 1 U/mL thrombin (Sigma), (5) 20 µmol/L
adenosine diphosphate (ADP), or (6) 500 µg/mL arachidonic acid
(Bio/Data Corp, Hatboro, PA). This was followed by sample incubation
in platelet lysis buffer (10 mmol/L EGTA, 2% Triton X-100, 1 mmol/L
PMSF, 100 mmol/L benzamidine, 4 mg/mL leupeptin, 100 mmol/L Tris
hydrochloride, pH 7.4, and 2 mmol/L sodium orthovanadate) on a
rocker at 4°C for 1 hour. Samples were then fractionated into Triton
X-100–soluble and –insoluble (cytoskeletal) components by 10,000g
centrifugation for 5 minutes. This supernatant was in turn centrifuged at
100,000g for 3 hours at 4°C to separate the membrane skeletal
component from platelet cytosol.30 Pellet samples, after washing twice
in PBS/0.05% Tween 20, along with 50 µL of each supernatant solution
were then resuspended in sodium dodecyl sulfate (SDS) protein loading
buffer, boiled for 2 minutes, electrophoresed on a 10% SDSpolyacrylamide gel,31 electrotransferred to a 0.45-µm nitrocellulose
membrane (Millipore Corp, Bedford, MA), and immunoblotted with
anti–14-3-3 antibody C-16 or K-19 (Santa Cruz Biotechnology) followed by 32.5 µCi 125I-labeled Staphylococcal protein A (New England
Nuclear, Boston, MA). Autoradiograph bands were subsequently analyzed quantitatively using area integration on an ImageQuant densitometer (Molecular Dynamics, Sunnyvale, CA).
RESULTS
Yeast two-hybrid assays. We were interested in characterizing the interaction of the signaling protein 14-3-3z with the
cytoplasmic domains of the GPIb a and b subunits. Our aims
were to determine to which of the GPIb subunits 14-3-3 would
bind; to determine what role, if any, was played by a previously
documented phosphoserine residue on GPIbb32; and to map the
GPIb binding domain on 14-3-3.
PCR-amplified cDNA fragments encoding the entire cytoplasmic domains of GPIba and GPIbb along with a GPIbb
sequence in which serine 166 had been mutated to alanine were
fused in-frame to either the DNA-binding domain or activation
domain of the yeast plasmids pGBT9 or pGAD424, respectively. cDNAs encoding deletion mutants along with wild-type
14-3-3z (Fig 1) were similarly cloned into the same yeast
expression vectors. Fusion protein expression of transformed
unpaired GPIb and 14-3-3 constructs by themselves did not
cause transactivation of the lacZ GAL4 reporter gene present in
the yeast host strain SFY526, as determined by colony filter-lift
assays. These vectors were next transformed into yeast in
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pairwise combinations along with three pairwise positive and
four negative control transformations as follows: wild-type
full-length GAL4 gene in pGBT9 alone; murine p53(72-390)/
SV40 large T antigen(84-708); GPIba/GPIbb; pGBT9/pGAD424;
murine p53(72-390)/pGAD424; pGBT9/SV40 large T antigen(84708); and human lamin C(66-230)/SV40 large T antigen(84-708)
(Clontech). To further exclude nonspecific interactions, each
vector was also cotransformed into yeast with other two-hybrid
vectors encoding irrelevant proteins not expected to interact
with the GPIb or 14-3-3 constructs (Figs 2 and 3).
Following incubation at 30°C for 3 to 4 days, individual
colonies were grown overnight in selective SD medium, and
b-galactosidase activity was quantitatively determined using
ONPG substrate.21 In this system, the extent of expression of the
reporter gene lacZ can be interpreted as an indication of the
strength of interaction between the two fusion proteins.21-24
Figure 2 shows the wild-type 14-3-3/GPIb fusion protein
quantitative interactions along with five of eight negative
control combinations. The findings suggest 14-3-3z interacts
with both the GPIb a and b cytoplasmic domains, with
significantly increased b-gal activity evident in both of these
interactions versus the eight negative control combinations (Fig
2 and data not shown). Furthermore, when ser166 on GPIbb is
replaced with alanine, its interaction is reduced 20% to 40%
(mean, 27%) with 14-3-3z.
Figure 3 shows results for b-galactosidase quantitative assays
when the Ibb cytoplasmic domain is cotransformed into yeast
with full-length 14-3-3z and the 14-3-3z deletion mutants
outlined in Fig 1. These findings along with the findings from
qualitative X-gal assays (Fig 4) suggest that both Iba and Ibb
interact optimally with full-length 14-3-3 compared with any of
the deletion mutants studied. Assays of Ibb/14-3-3z colonies
from each of the three 14-3-3z deletion mutant transformations
showed a reduction in reporter gene activity of 51% to 59%
compared with the analysis of Ibb/14-3-3z full-length colonies.
Interactions between the cytoplasmic domain of Iba and the
14-3-3z deletion mutants were too low to quantify using an
ONPG substrate, and were barely appreciable using the more
sensitive X-gal colony filter-lift assay (Fig 4). Thus, we were
unable to map the GPIb binding domain on 14-3-3 using the
yeast two-hybrid assay, perhaps due to the need for at least two
noncontiguous regions of 14-3-3z for optimal interaction with
GPIb and/or the need for 14-3-3z to be dimerized or membraneassociated.
Confocal microscopy studies. After demonstrating the interaction of 14-3-3z with GPIba and GPIbb through the yeast
two-hybrid system, we were next interested in documenting
these relationships in vivo using human platelets and HEL cells,
a tumor cell line that manifests several megakaryocyte and
platelet antigens including GPIb.33 Following membrane permeabilization, we proceeded to colabel platelets and cells with
anti–14-3-3z and either anti-human GPIba or anti-GPIbb
antibodies followed by fluorochrome-conjugated secondary
antibody combinations. Samples were also colabeled with
primary and secondary antibody combinations to either 14-3-3
and CD62P, Iba and CD62P (negative controls), or GPIba and
GPIX (positive control). Cytospun slides (platelets) or HEL
cells in suspension were then visualized by confocal microscopy. Our objective was to determine if each of the protein pairs
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1298
CALVERLEY, KAVANAGH, AND ROTH
Fig 2. 14-3-3z binds to the cytoplasmic domains
of GPIba and GPIbb in yeast. Yeast were cotransformed with fusion proteins composed of GAL 4
modular domains hybridized to either GPIb, 14-3-3z,
or control proteins. Data bars represent the mean 6
SE of 5 separate experiments using analysis of 36
independent colonies and 3 spectrophotometric readings per colony at different concentrations. Five
representative negative control transformations
among the 8 performed are shown.
colocalized and associated with each other to within the limits
of resolution of a single pixel (,200 nm). Colocalization of
FITC (green) and Cy3 (red) is evidenced by a yellow appearance (Fig 5C to E) and by corresponding pixel histograms that
demonstrate a linear correlation in antibody expression for each
fluorescence intensity level34 (Fig 5G to I). Absence of colocalization is suggested by a red and green appearance (Fig 5A and
B) and by a pixel histogram exhibiting a diffuse pattern34 (Fig
5F). These data support the yeast two-hybrid evidence that
14-3-3z interacts with GPIba and GPIbb present in platelets
and HEL cells.
To provide statistical evidence for these conclusions, we
calculated the degree to which colocalization occurred in each
of the colabeled samples. This was determined by calculating
the coefficient of variation ([CV] the standard deviation divided
by the mean) for the ratio of red to green pixels for individual
cells. These CVs were averaged (4 to 15 cells per sample) for
Fig 3. GPIbb binding to 14-3-3z deletion mutants
is reduced compared with the interaction with fulllength 14-3-3z. The binary protein interactions along
with the further controls shown in Fig 2 and 3
additional positive and 4 negative control transformations not shown were studied, and quantitative
b-galactosidase assays were performed. Deletion
mutant activity was reduced 51% to 59% v fulllength 14-3-3. Data bars represent the mean 6 SE of
5 separate experiments using analysis of 37 independent colonies with 3 spectrophotometric readings
per colony at different concentrations.
each image and analyzed for differences between them using a
two-sample one-tailed t test assuming unequal variances. Thus,
for example, in one experiment the colocalization between
GPIba/GPIX was found not to significantly differ versus
GPIbb/14-3-3 (P 5 .23), while that between GPIbb/14-3-3 and
CD62P/14-3-3 did (P 5 .05). Lower average HEL cell CVs
from three experiments confirmed the increased colocalization
of GPIba and GPIbb with 14-3-3, compared with significantly
less colocalization exhibited by GPIba and 14-3-3 with CD62P
(data not shown). Hence, the confocal photomicrographs, pixel
histograms, and statistical analysis suggest that colocalization
between 14-3-3z and both GPIba and GPIbb cytoplasmic
domains occurs in platelets and HEL cells as suggested by the
yeast two-hybrid experiments.
Platelet 14-3-3z translocation studies. Having used the
yeast two-hybrid system and confocal microscopy studies to
obtain structural data concerning the interaction of 14-3-3z and
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PLATELET GLYCOPROTEIN IB AND 14-3-3
1299
Fig 4. b-Galactosidase colony
filter-lift assays of GPIb/14-3-3z
yeast 2-hybrid transformations.
Reporter gene activity is reflected
by expression of blue (dark) colonies, most notable with the combination of GPIbb and full-length
14-3-3z, followed by GPIbb and
deletion mutant 14-3-3 (1-123)
and GPIba and full-length 14-3-3z.
The X-gal substrate used in these
assays is considered more sensitive to b-galactosidase activity
than the ONPG used in the quantitative assays in Figs 2 and 3.
platelet GPIb, we were interested in determining whether
14-3-3z translocated between the platelet cytosol and either the
platelet cytoskeleton or membrane skeleton in response to
stimulation with known agonists.30 Translocation to the cytoskeletal component in response to surface receptor occupancy is a
property of many signaling proteins; these events typically lead
to cytoskeletal reorganization, which in turn promotes events
such as shape change, locomotion, and activation.4,35
Following isolation from whole blood, platelets were stimulated with either vWF alone, ristocetin alone (controls), both
vWF and ristocetin, thrombin, ADP, or arachidonic acid. They
were then subjected to aggregometry, lysed, and separated by
low- and high-speed centrifugation into cytosolic, membrane
skeletal, and cytoskeletal components, which were in turn
subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with one of two anti–14-3-3z antibodies. Immunoblot
band densitometry determinations from these experiments
showed a 4.9-fold increase of cytoskeletal 14-3-3z in vWF/
ristocetin-stimulated platelets compared with unstimulated platelets, and a 2.9-fold increase compared with thrombin-stimulated
platelets (Table 1). Additional studies showed a similar or
reduced amount of cytoskeletal 14-3-3z in response to ADP and
arachidonic acid stimulation, respectively, compared with thrombin stimulation (data not shown).
DISCUSSION
Intracellular signaling that takes place immediately following
GPIb receptor occupancy connecting the adhesive event to
platelet activation and aggregation has not been well characterized. In the present study, we used yeast two-hybrid experiments
and confocal microscopy to further characterize the interaction
of platelet GPIb with an isoform of the 14-3-3 protein family.
While their exact function remains elusive, the 14-3-3 protein
family shows a diverse array of associations and biologic
activities related to signaling and cell-cycle progression.7,12
We have shown that 14-3-3z will interact in vivo with both
the GP Iba and Ibb subunit cytoplasmic domains; we are unable
to determine the preference, if any, of 14-3-3 for either subunit
with these methods. This interaction is modestly reduced when
serine 166 on GPIbb is replaced with alanine. We have also
demonstrated that GPIb interacts optimally with full-length
14-3-3z, while showing an approximately equally reduced
interaction with each of three deletion mutants. Confocal
microscopy studies of platelets and HEL cells have provided
additional characterization of the 14-3-3 interaction with GPIb
receptor subunits. In this respect, the positive confocal data
support the yeast two-hybrid findings but do not conclusively
prove them, because of the imaging system’s limits of resolution. Negative confocal data, on the other hand, would have
refuted the findings. Finally, platelet 14-3-3 translocation studies in stimulated and resting platelets have provided evidence
that 14-3-3z may translocate from the cytosolic component to
the cytoskeleton in response to vWF and ristocetin. A similar
agonist-induced cytoskeletal relocalization of platelet molecules with signaling roles such as Src, Syk, and FAK has been
observed.30,35 Our methods were unable to detect any appreciable amount of agonist-induced translocation to or from the
membrane skeleton.
Using affinity chromatography, a 29-kD protein found to be
14-3-3z was recently coeluted with GPIb-IX by Du et al,16 who
later showed that the 14-3-3 binding site on GPIba resides
within a 15 residue C-terminal sequence.36 Binding was not
contingent upon the presence of actin-binding protein and was
abolished by removing the C-terminal five residues of GPIba,
suggesting their critical role in the association.16,36 Our results
also suggest that 14-3-3 will associate with the cytoplasmic
domain of GPIba in vivo along with GPIbb (Figs 2, 4, and 5).
Du’s group provided some evidence that 14-3-3 may not
interact with GPIbb.36 Our study differs from theirs with respect
to the methods used to address this question (yeast two-hybrid
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CALVERLEY, KAVANAGH, AND ROTH
Fig 5. Colocalization of GPIb subunits and 14-3-3z in HEL cells (A to
E) and platelets (F to I). Permeabilized platelets and HEL cells were
colabeled with antibody pairs. HEL cells were labeled with either (A)
anti-GPIba/FITC (green) and anti-CD62P/CY-3 (red) (negative control),
(B) anti–14-3-3z/FITC and anti-CD62P/CY-3 (negative control), (C) antiGPIba/FITC and anti-GPIX/CY-3 (positive control), (D) anti-GPIbb/CY-3
and anti–14-3-3z/FITC, or (E) anti-GPIba/CY-3 and anti–14-3-3z/FITC.
Inset: detector 1, FITC fluorescence intensity; detector 2, CY-3 fluorescence intensity. Yellow fluorescence suggests that the 2 antibodies
colocalize to within approximately 200 nm. A and C to E field size, 36 3
36 mm; B field size, 54 3 54 mm. Representative platelet pixel histograms for each antibody pair are shown.
studies and confocal microscopy v peptide inhibition and
synthetic peptide binding studies). In addition, this group’s
synthetic peptides and anti-GPIbb immunogens did not incorporate the cytoplasmic domain GPIbb phosphoserine residue.
Recent investigation has provided evidence that phosphoserine
recognition may play an important role in 14-3-3 interaction
with other proteins.37,38
Since GPIba contains the receptor’s ligand binding site and
interacts with the membrane skeleton and GPIbb contains a
potentially signal-transducing phosphoserine residue in its
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PLATELET GLYCOPROTEIN IB AND 14-3-3
1301
Fig 5. (cont’d). (F to I) and reflect colocalization of GPIX/Iba (G), GPIbb/14-3-3z (H), and GPIba/14-3-3z (I). Note the approximately equal
contribution from each fluorochrome-conjugated secondary antibody at different fluorescence intensities in G (positive control), H, and I. This
suggests that each computer pixel is detecting an equal amount of both antibodies present, which implies their respective epitopes are present
in equal amounts (colocalized) in the 3-dimensional volume scanned by the microscope. This contrasts with F (1 of 2 negative controls), in which
a diffuse pattern reflecting noncolocalization is seen. Three experiments with HEL cells and 2 with platelets were performed.
intracellular domain, it is not unreasonable to speculate that
14-3-3z may indeed interact with both subunits. In this respect,
dimerization of 14-3-3 not only may serve to potentially link
receptors and signaling proteins but may also bridge important
functional elements within a multiple subunit complex like
GPIb.39
The reduction in interaction between 14-3-3 and GPIbb when
Table 1. Anti-14-3-3 Immunoblot Densitometry:
Fold Changes in Platelet Lysate 14-3-3 Binding Over Resting
State in Same Compartment
Agonist
Compartment
vWF
Ristocetin
vWF/
Ristocetin
Thrombin
None
Cytoskeleton 1.42 6 0.53 1.96 6 1.03 4.93 6 1.01 1.71 6 0.41 1.0
Cytosol
1.32 6 0.33 1.22 6 0.66 0.87 6 0.12 1.00 6 0.19 1.0
Results are the mean 6 SE of 7 experiments.
serine 166 is replaced by alanine is supported by the observation
that phosphoserine recognition may play an important role in
the former’s interaction with other proteins.37,38,40,41 It is thus
reasonable to speculate that the platelet GPIb interaction with
14-3-3 may be mediated, in part, by the phosphoserinecontaining cytoplasmic domain of GPIbb. Other groups have
suggested that 14-3-3 may recognize clusters of serine residues
in an amphipathic helical structure appropriate for its ligandbinding groove recently characterized through crystallography
studies.36,42,43 Such a serine-rich cluster exists at the C-terminal
region (residues 596 to 610) of GPIba where interaction with
14-3-3 has been recently characterized.36 Our yeast two-hybrid
and confocal microscopy data provide evidence for both of the
14-3-3 recognition motif theories by demonstrating 14-3-3
interactions with GPIba and GPIbb.
Our yeast two-hybrid experiments suggest that GPIb interacts
optimally with full-length 14-3-3z, with 51% to 59% reduced
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1302
CALVERLEY, KAVANAGH, AND ROTH
interaction demonstrable between GPIb and deletion mutants of
14-3-3z (Figs 1 and 3). Using these techniques, we were
therefore unable to map a specific GPIb binding site on 14-3-3z.
These results may be attributable to the dimerization domain
involving the N-terminal 26–amino acid residues, suggesting
that 14-3-3 may need to homodimerize or heterodimerize for
optimal activity.8,9,13,44,45 The 14-3-3 proteins have been found
at high concentrations on synaptic plasma membranes, and
another study suggests the N-terminal region is responsible for
this membrane binding.8,18 Optimal GPIb–14-3-3 interaction
may thus be facilitated by binding of the latter to the platelet
membrane. A third possible contributing cause to these results
rests with the fact that GPIb–14-3-3 interaction may be
mediated by at least two distinct and noncontiguous regions of
14-3-3. In this respect, the C-terminal phosphoserine 185 of
14-3-3 is surrounded by a consensus sequence for cyclindependent kinases, and amino acids 171 to 213 are required for
interaction with phosphorylated tryptophan hydroxylase, the
rate-limiting enzyme of serotonin synthesis.11,46 Therefore, for
example, if both the N-terminal dimerization domain and
C-terminal residues 171 to 213 were required for optimal interaction, only the full-length 14-3-3 species would be capable of
maximal interaction with GPIb, as our findings suggest.
The translocation of 14-3-3z to the platelet cytoskeleton upon
vWF and ristocetin stimulation is suggestive evidence that this
protein participates in GPIb signaling (Table 1). Similar fractionation procedures have been used to demonstrate the cytoskeletal
translocation of membrane skeleton and selective signaling
molecules, such as the tyrosine kinases Src and FAK, upon
platelet surface receptor binding.4,30,35,47 vWF stimulation and
aggregation of platelets is capable of inducing cytoskeletal
translocation and activation of both the lipid kinase phosphatidylinositol 3-kinase and Src independently of other agonists.4
These events were not observed in Bernard-Soulier platelets.
Recent investigation has demonstrated an association of 143-3t and phosphatidylinositol 3-kinase with additional evidence
showing that it may act as a negative regulator of this enzyme
by directly binding to its catalytic subunit.15 It could be
hypothesized that 14-3-3 may interact with platelet GPIb under
resting conditions and thereby exert a similar negative regulatory effect on phosphatidylinositol 3-kinase activity.
ACKNOWLEDGMENT
We are grateful to Chao-Yang Li and Kin Ritchie for helpful
discussion, to Susan Danner and Tanya Hill for excellent technical
assistance, and to Sally Swedine for help with the graphics.
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1998 91: 1295-1303
Human Signaling Protein 14-3-3ζ Interacts With Platelet Glycoprotein Ib
Subunits Ib α and Ibβ
David C. Calverley, Terrance J. Kavanagh and Gerald J. Roth
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