(Received for publication, November 2, 1995 )
From the
The -form 14-3-3 protein (14-3-3
) regulates protein
kinases and interacts with several signaling molecules. We reported
previously that a platelet adhesion receptor, glycoprotein (GP) Ib-IX,
was associated with a 29-kDa protein with partial sequences identical
to 14-3-3
. In this study, the interaction between GPIb-IX and
recombinant 14-3-3
is reconstituted. Further, we show that the
14-3-3
binding site in GPIb is within a 15 residue sequence at the
C terminus of GPIb
, as indicated by antibody inhibition and direct
binding of 14-3-3
to synthetic GPIb
cytoplasmic domain
peptides. The 14-3-3
binds to recombinant wild type GPIb-IX but
not to the GPIb
mutants lacking C-terminal 5 or more residues,
suggesting that the C-terminal 5 residues of GPIb
are critical.
Similarity between the GPIb
C-terminal sequence and the
serine-rich regions of Raf and Bcr kinases suggests a possible
serine-rich recognition motif for the 14-3-3 protein.
The platelet membrane glycoprotein Ib
(GPIb)()-glycoprotein IX (GPIX) complex (GPIb-IX) plays an
important role in the initial platelet adhesion to injured vascular
wall under high shear flow conditions such as in arteries and
capillaries(1, 2) . By binding to the
subendothelium-bound von Willebrand factor, GPIb-IX not only mediates
the physical adherence of platelets to the site of vascular injury but
also initiates an activation signal that is transduced across the
membrane resulting in a series of biochemical events. GPIb-induced
intracellular biochemical changes include synthesis of thromboxane
A
, hydrolysis of phosphoinositide, activation of protein
kinase C and tyrosine kinases, elevation of cytoplasmic calcium level,
cytoskeleton reorganization, and exposure of ligand-binding function of
other adhesion receptors such as the integrin
(3, 4, 5, 6, 7, 8) .
In addition, GPIb
binds thrombin and is involved in the signaling
process of thrombin-induced platelet
activation(9, 10, 11) . The mechanism of
signal transduction via GPIb-IX has been unclear. In search for a
possible intermediate between GPIb-IX and the intracellular signaling
pathways, we have recently found that GPIb-IX is associated with a
29-kDa intracellular protein that is identical in partial amino acid
sequence to the
-form 14-3-3 protein (14-3-3
)(12) .
The 14-3-3 proteins are a family of highly conserved eukaryotic
proteins, which are distributed in a variety of
cells(13, 14) . Several functions have been attributed
to the 14-3-3 proteins. At the cellular level, the 14-3-3 proteins have
been implicated in the regulation of cell cycle and stimulation of
exocytosis(15, 16) . At the molecular level, the
reported functions of 14-3-3 proteins include the activation of Pseudomonas aeruginosa exoenzyme S(14) , phospholipase
A activity(17) , and regulation of protein kinase C
and the tyrosine and tryptophan
hydroxylases(18, 19, 20, 21, 22) .
The 14-3-3 proteins, including the
-form, interact with and
activate Raf protein kinase in both yeast and mammalian
cells(23, 24, 25, 26, 27) .
Raf phosphorylates and activates mitogen-activated protein kinase
kinase, which subsequently activates mitogen-activated protein kinase.
Mitogen-activated protein kinase may phosphorylate and activate several
signaling proteins, including a cytosolic phospholipase
A
(28) . Thus, it is possible that association of a
14-3-3 protein with GPIb-IX may serve as a signaling mechanism that
transduces adhesion-initiated platelet activation signals.
The
functional mechanism of the 14-3-3 protein is unclear. Evidence of
direct binding of the 14-3-3 proteins has been shown in Raf, Bcr
kinase, and protein kinase C, as well as middle T antigen and
tryptophan
hydroxylase(19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31) .
This suggests that binding to target proteins may be required for its
regulatory functions. However, it is not understood how the 14-3-3
proteins interact with different types of proteins. In this study, we
have identified the 14-3-3 binding site in GPIb. This is the first
identified short peptide sequence that binds the 14-3-3 protein.
Furthermore, we report a similarity between this 14-3-3 protein binding
sequence and a short segment from each of the serine-rich 14-3-3
protein binding regions of Raf and Bcr kinases, suggesting a possible
serine-rich recognition motif.
Figure 1:
Binding of the platelet GPIb to a
recombinant 14-3-3 column. Washed platelets (2
10
/ml) were solubilized and lysates were allowed to pass
through a Sepharose 4B column conjugated with the recombinant
MBP-14-3-3
fusion protein (14-3-3), or a control column (MBP). After extensive washing, the bound proteins were eluted
with a 0.15 M to 1 M NaCl gradient. The proteins
eluted at 1 M NaCl were analyzed by SDS-PAGE, followed by
silver staining and immunoblotting with an anti-GPIb
antibody
(WM23) or a control antibody against the platelet glycoprotein IIb
(PMI-1).
Figure 2:
Binding of the C-terminal domain of the
GPIb-IX complex to the recombinant 14-3-3. A, washed
platelets were solubilized in the presence of 1 mM CaCl
but in the absence of calpain inhibitors allowing calpain to
cleave GPIb and ABP, releasing ABP and the extracellular region of
GPIb
from the C-terminal domain of the GPIb-IX complex.
Solubilized platelets were then allowed to pass through the recombinant
14-3-3
-conjugated Sepharose column or a control column. After
extensive washing, bound proteins were eluted with a salt gradient of
0.15-1.0 M NaCl. Eluates were analyzed by SDS-PAGE,
followed by immunoblotting with an anti-peptide antibody against the
C-terminal region of GPIb
cytoplasmic domain (anti-Ib
C). B, a schematic of GPIb-IX, indicating the protease-sensitive
region of GPIb
.
Figure 3:
Inhibition of 14-3-3 binding to
GPIb-IX by anti-GPIb cytoplasmic domain antibodies. A,
platelet lysates (200 µl) were first incubated in the absence (None) or in the presence of a preimmune rabbit serum (preimmune), anti-Ib
C antibody (against the C-terminal 15
residue sequence of GPIb
) or anti-Ib
C antibody (against the
C-terminal 14 residue sequence of GPIb
) at 4 °C for 30 min.
The lysates were then further incubated with 25 µl (50%; v/v) of
MBP-conjugated beads (MBP) or the 14-3-3
-conjugated
beads. B, platelet lysates (200 µl) were first incubated
with a preimmune rabbit serum (preimmune), anti-Ib
C or
anti-Ib
C antibodies at 4 °C for 30 min, and then with protein
A-conjugated beads at 4 °C for 2 h. The beads from both A and B were then washed three times and analyzed by
SDS-PAGE, followed by immunoblotting with an anti-GPIb monoclonal
antibody WM23.
Figure 4:
Binding of I-labeled
14-3-3
to synthetic peptides. Microtiter wells were coated with
synthetic peptides DLLSTVSIRYSGHSL corresponding to C-terminal 15
residues of GPIb
(Ib
C) or TDPLVAERAGTDES corresponding to
C-terminal 14 residues of GPIb
(Ib
C). Various concentrations
of
I-labeled recombinant 14-3-3
protein or a
truncated 14-3-3
-MBP fusion protein (1433T3, as negative control)
were added to the microtiter wells and incubated at 22 °C for 2 h.
Bound proteins were estimated by
-counting. Data shown are the
mean value of triplicate samples ± standard deviation. Closed circles, 14-3-3
binding to Ib
C peptide; open circles, 14-3-3
binding to Ib
C peptide; open square, 1433T3 binding to Ib
C
peptide.
Figure 5:
Binding of 14-3-3 to the recombinant
wild type and mutant GPIb-IX. A, wild type GPIb-IX and GPIb-IX
mutants
559 expressed in CHO cells were solubilized and 250 µl
of lysates were incubated in the absence or in the presence of 50
µl rabbit anti-GPIb
cytoplasmic domain serum
(+Anti-Ib
C) at 4 °C for 30 min. Cell lysates
were further incubated with MBP-conjugated Sepharose beads (MBP) or the 14-3-3
-conjugated beads(14-3-3) at 4 °C
for 1 h. Bead-bound proteins were solubilized in SDS-PAGE sample buffer
and analyzed by SDS-PAGE, followed by Western blot with an anti-GPIb
monoclonal antibody, WM23. Cell lysates (Lysate) expressing
wild type GPIb-IX,
559, or CHO cells were also directly analyzed
by SDS-PAGE and Western blotted with WM23 to visualize the quantity of
GPIb-IX in each or these cell lines. B, the cell lysates
expressing wild type GPIb-IX, or
591 and
605 GPIb-IX mutants
were solubilized and incubated with control beads (MBP) or the
14-3-3
-conjugated beads(14-3-3) at 4 °C for 1 h, and bound
proteins were analyzed by SDS-PAGE and Western blotting with WM23. In
addition, cell lysates (Lysate) were directly separated by
SDS-PAGE and Western blotted with WM23. C, a schematic of the
cytoplasmic domain of GPIb
indicating locations of the C-terminal
ends of the truncated GPIb
mutants and the 14-3-3
binding
site.
Figure 6:
A,
alignment of the C-terminal region of GPIb (residues
582-610) with segments of Raf (residues 221-252) and Bcr
(residues 327-359) kinases from the serine-rich regions reported
as critical for the binding of the 14-3-3 protein. Identical residues
are shaded. B, helical wheel analysis of the
sequences from A. Note that the serine residues are clustered
in one side of the helices in all the three
sequences.
In this study, we have reconstituted the binding of the
14-3-3 protein to an important platelet adhesion receptor,
GPIb-IX, and identified the 14-3-3
binding sequence at the C
terminus of GPIb
cytoplasmic domain. The identification of a short
peptide sequence that binds the 14-3-3 protein provides insight into
the structural basis required for the 14-3-3 protein recognition. The
similarity between the 14-3-3 protein binding sequence in GPIb
and
the segments from the 14-3-3 protein binding region of the Bcr and Raf
kinases suggests a possible serine-rich recognition motif in the
ligands of the 14-3-3 protein. In addition, characterization of the
interaction of the 14-3-3
with the cytoplasmic domain of a
membrane receptor may help to understand the roles of the 14-3-3
protein in the receptor-mediated signaling pathways.
Reconstitution
of the binding between the recombinant 14-3-3 and GPIb-IX (Fig. 1) confirmed the identity of the previously reported
GPIb-IX-associated 29-kDa protein (12) as 14-3-3
. In
platelets, 14-3-3
was first purified and cloned as a platelet
intracellular phospholipase A
(17, 43) ,
although this function of 14-3-3
was recently disputed by other
groups(44) . The 14-3-3
is relatively abundant in
platelets and has been shown to be both associated with the plasma
membrane and present in the cytosol(43) . Purified 14-3-3
,
however, does not bind to phospholipid vesicles (45) . Thus,
association with the cytoplasmic domain of GPIb-IX may account for at
least part of the membrane-associated fraction of this protein.
The
location of the 14-3-3 binding site on GPIb-IX is indicated by
following data. 1) The proteolytically generated C-terminal domain of
GPIb-IX bound to the 14-3-3
column; 2) an anti-peptide antibody
against the C-terminal 15 residues of GPIb
inhibited the binding
of 14-3-3
; 3)
I-labeled 14-3-3
directly bound
to this C-terminal 15 residue peptide; and 4) mutagenesis that
truncated 5 or more residues from C terminus of GPIb
abolished
14-3-3
binding. These data suggest that 14-3-3
binds to the
cytoplasmic domain GPIb
in the region between Asp
and Leu
, in which the C-terminal 5 residues
(Ser
-Gly-His-Ser-Leu
) are critical. A
significant feature of this 14-3-3
binding sequence is the
presence of a serine every 3-4 residues (XXXSXXSXXXSXXSX) (Fig. 6). Thus, if this sequence were to form an
-helix in
the intact protein, the serine residues may form a cluster in one face
of the helix (Fig. 6). Indeed, from the recently resolved
structure of the 14-3-3 protein, a ligand that fits into the ligand
binding groove of the 14-3-3 protein was predicted to be an amphipathic
helix(46, 47) . Furthermore, the serine-rich regions
in Bcr and Raf kinases appear to be critical for the 14-3-3 protein
binding, and serine residues from a 30-residue segment from each of the
serine-rich regions of Bcr and Raf kinases are well aligned with serine
residues from the 14-3-3 binding sequence of GPIb
(Fig. 6).
This suggests that clusters of serine residues in a helical structure
may be a common recognition motif important for the binding of the
14-3-3 protein.
The location of the 14-3-3 binding site in the
cytoplasmic domain of GPIb
suggests that the binding of
14-3-3
to GPIb-IX is likely to occur in intact platelets, and thus
may be of relevance to the functions of the cytoplasmic domain of
GPIb-IX. The cytoplasmic domain of GPIb
is known to bind to the
cytoskeletal protein ABP(48) . ABP, however, is not required
for the binding of the 14-3-3 protein, as co-immunoprecipitation of the
14-3-3
with GPIb-IX was not inhibited by the treatment of cell
lysates with DNase I and N-ethylmaleimide, which disrupted the
interaction between GPIb and ABP(12) . Furthermore, in the
present study, we show that lysis of platelets under conditions in
which calpain was active and the ABP-GPIb-IX interaction disrupted, did
not prevent the interaction of the C-terminal domain of GPIb-IX with
14-3-3
(Fig. 2). Moreover, 14-3-3
bound to GPIb-IX
from an ABP-deficient cell line (not shown). Conversely, the 14-3-3
protein is not required for the ABP-GPIb interaction, as the GPIb
mutant that was truncated at residue 605 retained its capacity to
interact with ABP,
yet lost its 14-3-3
binding
capacity. The ABP binding site is located in the central region
(Thr
-Phe
) of the GPIb
cytoplasmic domain(48) ,
while the C-terminal
region contains the 14-3-3
binding site (Fig. 4). Binding
of ABP to the cytoplasmic domain of GPIb
links GPIb-IX to the
membrane skeleton framework(40) . It is interesting to
speculate that the adjacent location of the binding sites of the
membrane skeleton protein, ABP, and a kinase regulator, 14-3-3
,
within the GPIb
cytoplasmic domain may be important in the
shear-dependent signaling transduction through GPIb-IX. For example, it
is possible that mechanical force generated by immobilized von
Willebrand factor binding to N-terminal domain of GPIb
under high
shear stress may change the conformation of the C terminus of GPIb
by leverage of the membrane skeleton and thus regulate the binding or
signaling functions of the 14-3-3
.
It has been unclear how the
14-3-3 proteins regulate their target proteins. A recent report
suggests that by binding to Raf kinase, the 14-3-3 protein may prevent
its inactivation by protein phosphatase (49) . It is possible
that 14-3-3 protein may prevent the inactivation of Raf by interaction
with phosphorylated serine residues in the serine-clustered region.
Similarly, it is possible that by binding to the C-terminal serine-rich
region of the cytoplasmic domain of GPIb, the 14-3-3
may
regulate the function of the GPIb
cytoplasmic domain by preventing
serine (or phosphoserine) residues from being modified. Similarity
between the GPIb
C-terminal region and a segment from the 14-3-3
protein binding region of both Raf and Bcr also suggests a possibility
that GPIb
may compete with kinases for the 14-3-3 protein binding
sites in the membrane compartment and thus regulate kinase activity.
Alternatively, as the membrane translocation is a mechanism of the Raf
and protein kinase C activation, it is also possible that the dimeric
14-3-3
may be involved in the translocation of the protein kinases
to the membrane(50) . Although phospholipid association of some
isoforms of the 14-3-3 protein family has been indicated, 14-3-3
does not appear to associate with phospholipid vesicles(45) .
Thus, a possible mechanism is that by binding to the cytoplasmic domain
of GPIb
, the 14-3-3
may mediate translocation of the protein
kinases to cytoplasmic face of the membrane and to the cytoplasmic
domain of this membrane receptor in order to relay signals. If this
were to be the case, there may also be membrane receptors in other cell
types that interact with the 14-3-3 proteins.