(Received for publication, December 4, 1995; and in revised form, February 14, 1996)
From the
Previously, we have demonstrated that the cytoplasmic tyrosine
kinase p72 is coupled to the platelet Fc
receptor for IgG (Fc
RIIA) (Chacko, G. W., Duchemin, A. M.,
Coggeshall, K. M., Osborne, J. M., Brandt, J. T., and Anderson, C. L.
(1994) J. Biol. Chem. 269, 32435-32440). Further
analysis of the platelet activation by Fc
RIIA demonstrated that
Fc
RIIA is also inducibly coupled to the serine/threonine and lipid
kinase, phosphoinositide 3-kinase (PI 3-K). Activation of platelets
with anti-Fc
RIIA antibodies resulted in the noncovalent
association of PI 3-K with Fc
RIIA as well as an increase in
Fc
RIIA-associated PI 3-K activity. Binding of both p72
and PI 3-K to Fc
RIIA was reconstituted with synthetic
phosphopeptides corresponding to the sequence of the atypical
immunoreceptor tyrosine-based activation motif (ITAM) in the
cytoplasmic domain of Fc
RIIA. Our findings demonstrate that
coupling of both p72
and PI 3-K activities to
Fc
RIIA is regulated by tyrosine phosphorylation of the ITAM, and
we speculate that p72
might act as an adapter to
recruit PI 3-K to activated Fc
RIIA.
Human platelets express a single Fc receptor for IgG (FcR), (
)the low affinity receptor encoded by the Fc
RIIA
gene(1, 2, 3, 4) . Clustering of
Fc
RIIA with ligand or anti-Fc
RIIA antibodies induces platelet
activation characterized by a dramatic increase in tyrosine
phosphorylation of a number of platelet proteins, by size and shape
changes in platelets, by secretion of intracellular granule contents,
by increased adhesion to platelet-specific ligands, and by platelet
aggregation(5, 6, 7, 8) .
The
intracytoplasmic domain of FcRIIA contains a single copy of an
activation motif termed immunoreceptor tyrosine-based activation motif
(ITAM) that is loosely defined by the consensus sequence
YX
LX
YX
L,
where X is any amino acid(9) . ITAM sequences have
also been identified in signaling subunits of the T and B cell antigen
receptors, the high affinity Fc receptor for IgE (Fc
R), and other
Fc
R (10, 11, 12, 13, 14) .
Clustering of ITAM-containing receptors by cognate ligand or specific
antibodies has been shown to result in phosphorylation of two critical
tyrosine residues in the ITAM which converts the ITAM into a high
affinity binding site for members of the Syk/ZAP-70 family of tyrosine
kinases that interact with this motif through tandem src homology region 2 (SH2) domains. Phosphorylation of ITAMs appears
to be mediated by members of the Src family of tyrosine kinases, and
the importance of Src kinases in ITAM phosphorylation and Syk/ZAP-70
recruitment has been well
documented(15, 16, 17) . Thus, ITAM
phosphorylation initiated by receptor clustering and the subsequent
recruitment of SH2 domain-containing proteins provides one inducible
mechanism by which receptors lacking endogenous catalytic activity can
be coupled to effector molecules that are catalytically active.
The
ITAM integral to FcRIIA is unusual in that it contains an extended
spacer of 12 amino acids (18) between the two YXXL
pairs, while other members of the ITAM family contain spacer regions of
6-8 amino acids. Additionally, while ITAM-containing receptors
exist as multisubunit complexes comprising an extracellular ligand
binding unit that is noncovalently associated with intracellular
ITAM-containing signaling subunits(10) , Fc
RIIA is a
single-chain transmembrane polypeptide with an ITAM integral to its
primary structure. Mutational analysis of the tyrosine residues of the
Fc
RIIA-ITAM has shown that they are required for receptor tyrosine
phosphorylation and for phagocytosis mediated by
Fc
RIIA(19, 20, 21) , suggesting that the
Fc
RIIA-ITAM remains functional despite its divergence from the
consensus sequence. Results from another study suggest that the ITAM of
Fc
RIIA transduces a qualitatively different signal from the ITAM
of the
-chain subunit of the high affinity Fc
R, implying that
there are unique aspects to the Fc
RIIA-ITAM despite the common
functional features it shares with other members of the
group(22) . Analysis of the mechanism by which Fc
RIIA in
platelets transduces signal therefore provides insight into the
understanding of ITAM signaling from the perspective of this simple yet
unique model.
Previous work from our laboratory, and from others,
has revealed that clustering of FcRIIA on platelets induces
receptor tyrosine phosphorylation and noncovalent association of the
tyrosine kinase p72
with tyrosine-phosphorylated
Fc
RIIA(2, 7) . These observations lend support to
a model of p72
coupling to Fc
RIIA induced
by tyrosine phosphorylation of the ITAM(1, 5) presumably by one or more of the five Src kinases expressed
in platelets(23) . p72
is a member of
the Syk/ZAP70 family of tyrosine kinases and has been shown to be
involved in signal generation by the ITAM-containing receptors, such as
the B cell antigen receptor), and the high affinity Fc receptors for
IgG and IgE(10) .
Activation of p72 in platelets is induced by several platelet agonists
including thrombin, collagen, and platelet activation
factor(24, 25, 26) . Engagement of the
fibrinogen receptor (the integrin
)
by fibrinogen in thrombin-activated platelets further up-regulates
p72
activity(8, 23, 27) . These
observations suggest that p72
plays a central
role in platelet activation and that regulation of its activity is
complex. Additionally, targeted gene disruption of the p72
gene in the mouse has been shown to result in perinatal
death due to massive hemorrhages(28, 29) , further
suggesting a critical role for p72
in platelet
function.
Experiments with ITAM-containing receptors have shown that
phosphoinositide 3-kinase (PI 3-K) is often activated in response to
receptor clustering(30, 31) . PI 3-K is a
serine/threonine and lipid kinase that exists as a heterodimer
comprising an 85-kDa regulatory subunit containing two SH2 domains and
one SH3 domain and a 110-kDa catalytic
subunit(32, 33) . The presence of the SH2 and SH3
domains in PI 3-K implies that noncovalent interactions with target
proteins take place, and considerable experimental evidence exists to
support this contention(31, 32) . PI 3-K is also
rapidly activated and translocated to the cytoskeleton when platelets
are stimulated with the potent agonist
thrombin(34, 35) . More recently, it has been shown
that the translocation of PI 3-K to the cytoskeleton is
integrin-dependent and that platelet aggregation induced by thrombin
through its G-protein-coupled receptor can be reversed by inhibitors of
PI 3-K activity (36, 37) suggesting that PI 3-K, like
p72, plays an important role in platelet
activation.
PI 3-K has been implicated in FcR function in
neutrophils, the monocytic cell line U937, and natural killer (NK)
cells suggesting a conserved role in Fc
R-mediated
signaling(38, 39, 40) . Fc receptor ligation
results in an increase in the specific activity of PI 3-K as well as an
increase in phosphotyrosine-associated PI 3-K activity in response to
Fc
R stimulation, although the specific mechanism of activation was
not determined. The understanding of how PI 3-K is activated on
phagocytes and NK cells is further complicated by the different
isoforms of Fc
R expressed on these cell types. Analysis of
Fc
RIIA activation in platelets which express only a single
Fc
R, provides specific information on the mechanism by which the
low affinity Fc
RIIA transduces signal and contributes to the
general paradigm describing how Fc
R activates PI 3-K.
In this
study we demonstrate that, upon platelet activation by FcRIIA
clustering, PI 3-K is coupled-associated with Fc
RIIA. We show that
noncovalent association of p72
and PI 3-K can be
reconstituted using synthetic phosphopeptides that correspond to the
sequence of the Fc
RIIA-ITAM. Interestingly, while
p72
association with the phosphorylated
Fc
RIIA-ITAM appears independent of the activation status of
platelets, the binding of PI 3-K to Fc
RIIA requires platelet
activation. These data showing that the presence of a phosphorylated
ITAM is sufficient to induce the binding of p72
but not PI 3-K to Fc
RIIA suggest that additional
modifications are required to promote association of the activated
Fc
RIIA complex with molecules that are downstream of the receptor
complex in the signaling cascade. We propose that PI 3-K binding to
Fc
RIIA may require an adapter molecule(s) that binds both
Fc
RIIA and PI 3-K and speculate that p72
is
an attractive candidate for this function.
Figure 4:
Analysis of FcRIIA function with
synthetic phosphopeptides. Peptides (P1 through P4)
were synthesized that overlap the sequence of the ITAM present in the
cytoplasmic tail of Fc
RIIA. Phosphotyrosine residues were
incorporated to allow representation of all four phosphorylation states
of the two tyrosine residues of the ITAM. Biotinylation of the
N-terminal glutamic acid residue facilitated coupling to a solid
support.
Figure 1:
Clustering of FcRIIA on platelets
induces a rapid and transient increase in Fc
RIIA-associated PI 3-K
activity. Platelets were activated by Fc
RIIA clustering and lysed
in 1% Triton X-100, and immune complex kinase assays to detect PI 3-K
activity were performed on PI 3-K or Fc
RIIA immunoadsorbates (see
``Experimental Procedures''). PI 3-K products were separated
by TLC and visualized by autoradiography.
P incorporated
in PI 3-K products was quantified by scraping TLC plates and counting
by liquid scintillation. Top panel, PI 3-K immunoadsorbates
from platelets were subjected to the immune kinase complex assay for PI
3-K, and lipid products were separated by TLC. Addition, in
vitro, of 100 nM wortmannin (lane 2) or of 0.1%
Triton X-100 (lane 3) completely inhibits detectable PI 3-K
activity (arrow) compared to untreated samples (lanes 1 and 5) or platelets pretreated with 1 µM genistein (lane 4). Other labeled lipid products are
insensitive to both Triton X-100 and wortmannin and are likely the
result of low levels of contaminating phospholipases and
phosphoinositide kinases. Bottom panel, Fc
RIIA
immunoadsorbates from resting and activated platelets show a rapid
increase in associated PI 3-K activity within 30 s of receptor
clustering. Absolute values of
P (as disintegrations/min)
incorporated due to PI 3-K activity are expressed as fold increase
above PI 3-K activity associated with Fc
RIIA immunoadsorbates of
unstimulated platelets which were normalized to an arbitrary value of
1. Control immunoadsorptions to ascertain nonspecific binding of PI 3-K
were performed with MOPC 141 (IgG2b) a monoclonal antibody of
irrelevant specificity from both resting and activated platelet
lysates. These results are derived from three independent experiments. Error bars indicate standard deviation from the
mean.
Figure 2:
Clustering of FcRIIA on platelets
induces a rapid and transient association of PI 3-K with activated
Fc
RIIA. Platelets were activated by Fc
RIIA clustering and
lysed in 1% Triton X-100 as described in Fig. 1.
Immunoadsorbates with anti-Fc
RIIA (lanes 3, 4,
and 5) or anti-PI 3-K antibodies (lanes 1 and 2) were separated by reducing SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose, and immunoblotted to
detect the p85 subunit of PI 3-K. An 85-kDa band is detected in
Fc
RIIA immunoadsorbates from lysates of platelets activated for 30
s by Fc
RIIA clustering (lane 4), is not detected in
Fc
RIIA immunoadsorbates from resting platelets (lane 5)
or in Fc
RIIA immunoadsorbates from lysates of platelets activated
for 1 min or greater by Fc
RIIA clustering (lane 3).
Control immunoadsorptions with anti-p85 antibodies demonstrate the
presence of PI 3-K in activated platelet lysates (lanes 1 and 2) and the position to which it migrates on the
gel.
Figure 3:
Inhibition of FcRIIA-mediated
platelet aggregation by wortmannin. Human platelets were isolated and
prepared for aggregometry as described (see ``Experimental
Procedures''). Platelets were incubated with varying doses of
wortmannin and then stimulated with thrombin (2 units/ml),
anti-Fc
RII monoclonal antibody IV.3 (IgG2b), and goat anti-mouse
(GAM), anti-Fc
RIIA monoclonal antibody CIKM5 (IgG1), or GAM alone.
CIKM5 is an anti-Fc
RII monoclonal antibody of the murine IgG1
subclass that clusters Fc
RII via a two-point interaction through
its Fab and Fc portions and does not require clustering with a
secondary antibody(31) . Maximal platelet aggregation was
measured on a Chronolog Dual Channel Lumi-Aggregometer (Model 560).
Results shown are the average of three independent experiments with
platelets from different donors. Error bars indicate standard
deviation from the mean.
Figure 5:
p72 binds the
doubly phosphorylated Fc
RIIA-ITAM. Synthetic peptides of sequence
corresponding to the Fc
RIIA-ITAM were coupled to beads and
incubated with lysates of Raji cells (see ``Experimental
Procedures''). Peptides coupled to beads were separated by
centrifugation and washed extensively with Triton X-100 lysis buffer.
Peptide-associated proteins were analyzed by SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose, and associated
p72
was detected by immunoblotting with a
specific anti-p72
antibody and visualized by
enhanced chemiluminescence. A 72-kDa band reactive in immunoblots with
anti-p72
antibody is seen in adsorbates from
Raji cell lysates of the doubly phosphorylated peptide (P4)
but not in adsorbates of the unphosphorylated peptide (P1) or
avidin-coated beads alone (Beads). These observations were
consistent for six independent experiments.
Figure 6:
p72 from both
activated and resting platelet lysates binds the doubly phosphorylated
Fc
RIIA-ITAM. Lysates of resting and activated platelets were
incubated with Fc
RIIA-ITAM peptides and processed to detect
p72
association as described (Fig. 4).
p72
is detected in P4 adsorbates of both resting
and activated platelets (lanes 1 and 3) but is not
detected in P1 adsorbates of either resting or activated platelets. The
p72
band detected comigrates with
p72
immunoadorbed with anti-p72
antibody (lane 7). p72
was
not detected in control immunoadsorptions with anti-PLC
and
anti-p85 antibodies (lanes 5 and 6).
Figure 7:
PI 3-K from activated but not resting
platelet lysates binds the doubly phosphorylated FcRIIA-ITAM. Top panel, platelets were activated by Fc
RIIA clustering (lanes 2, 4, and 6) or by thrombin
stimulation (lane 1). Lysates of resting and activated
platelets were incubated with Fc
RIIA-ITAM peptides (P4 and P1),
anti-phosphotyrosine antibody 4G10 (lanes 6 and 7),
anti-p85 antibody (lane 8), or p72
antibody (lane 9) and processed to detect
p72
association as described (Fig. 5). A, activated; R, resting; T,
thrombin-activated. PI 3-K is detected in P4 adsorbates from activated (lane 2) platelets but not from resting (lane 3)
platelets. There is no detectable PI 3-K in P1 adsorbates (lanes 4 and 5), 4G10, or p72
adsorbates (lanes 6, 7, and 9). Bottom panel,
stripping and reprobing the filter with anti-p72
antibodies indicates the presence of p72
in P4 adsorbates from both resting (lane 3) and
activated platelet lysates (lane 1, thrombin; lane 2,
Fc
RIIA) but not in P1 adsorbates from either resting or activated
platelets (lanes 4 and 5). p72
is detected in anti-phosphotyrosine immunoadsorbates from
platelets activated by Fc
RIIA clustering (lane 4) but not
from resting platelets. p72
detected in
association with peptide P4 comigrates with p72
immunoadsorbed with anti-p72
antibodies
from platelets.
Previously, we demonstrated functional coupling of the low
affinity receptor for IgG on platelets to the tyrosine kinase
p72. In this report we show that Fc
RIIA is
functionally coupled as well to a serine/threonine and lipid kinase.
Others have described p72
activation during platelet
activation by several agonists including thrombin which stimulates a
G-protein coupled receptor. Thrombin as well activates PI 3-K, although
the precise mechanisms by which these events take place are not clearly
understood. However, as our experiments show, in platelets activated by
the ITAM containing Fc
RIIA, the recruitment of p72
and PI 3-K to the phosphorylated ITAM occurs concurrently with
increased catalytic activity of both p72
and PI 3-K.
Therefore, it would appear that the activation of p72
and
PI 3-K may be a conserved phenomenon in platelet activation induced by
stimulation of membrane receptors.
The kinetics of association with
FcRIIA of both p72
and PI 3-K are rapid, occurring
within seconds of Fc
RIIA clustering and are consistent with
reports of tyrosine phosphorylation and PI 3-K activation in platelets
treated with thrombin. In thrombin-treated platelets, the activation of
PI 3-K, resulting in multiple phosphatidylinositol products, is
manifest over time by two major peaks of PI 3-K activity, the first due
to the generation of PtdIns-3,4,5-P
, and the second due to
the generation of PtdIns-4,5-P
(24, 25) .
We, as well, observed two temporal peaks of platelet PI 3-K activity
after Fc
RIIA clustering, although we were not able to characterize
the actual products of PI 3-K activity since we used an in vitro assay. Although there is correspondence between PI 3-K activation
by Fc
RIIA clustering and that induced by thrombin a valid
comparison of our results with those of other workers awaits a more
detailed study.
Recently, it has been proposed that the
PtdIns-3,4,5-P, a catalytic product of PI 3-K, may serve to
dissociate SH2 domains from their phosphotyrosine targets(53) .
The transient nature of the association between PI 3-K and Fc
RIIA
that we observed is very consistent with this hypothesis. It is
conceivable, therefore, that phosphorylation of the Fc
RIIA-ITAM
results in PI 3-K recruitment and activation which causes dissociation
of PI 3-K from the ITAM complex by the products of PI 3-K.
Subsequently, PI 3-K may undergo translocation to the cytoskeleton as
reported.
Although the precise nature of the molecular interaction
between FcRIIA and PI 3-K is not clear, we speculate that an
adapter molecule could mediate the association of PI 3-K with the
phosphotyrosines of the ITAM in the Fc
RIIA molecule.
Significantly, the consensus YXXM-described binding site for
the SH2 domain of PI 3-K is not present in the cytoplasmic domain of
Fc
RIIA but it is represented three times in the sequence of
p72
(54, 55, 56) , although none
of these sites has yet been shown to be phosphorylated. Our observation
that PI 3-K associates with Fc
RIIA when both Fc
RIIA and
p72
are tyrosine-phosphorylated but not when only
Fc
RIIA is phosphorylated would suggest that PI 3-K could bind
phosphorylated p72
which in turn could bind
phosphorylated Fc
RIIA. While PI 3-K and p72
are
reported to be associated in thrombin-activated platelets(52) ,
conditions under which Fc
RIIA is not phosphorylated, it is not
clear whether PI 3-K binding to p72
is enhanced by the
simultaneous association of p72
with phosphorylated
Fc
RIIA. It is likely that phosphotyrosine-mediated interactions
are the primary binding determinant in the molecular interactions
between Fc
RIIA and PI 3-K. While the role of other interactions
such as those mediated by SH3 domains and proline-rich regions may
indeed contribute to the association of Fc
RIIA and PI 3-K, such
interactions have been shown to be of considerable lower affinity and
are unlikely to have been maintained under the relatively harsh
conditions of detergent lysis and washes employed in this study (see
``Experimental Procedures''). Therefore, the contribution, if
any, of the SH3 domain of PI 3-K to the association with Fc
RIIA
and PI 3-K has yet to be clarified.
Our reconstitution experiments
with synthetic peptides unequivocally demonstrate that p72 binds the doubly phosphorylated Fc
RIIA-ITAM with high
affinity relative to the affinity for unphosphorylated
Fc
RIIA-ITAM. This observation is consistent with the general
paradigm of ITAM-SH2 interactions despite, in the case of Fc
RIIA,
the variance from the consensus ITAM sequence. We have observed in
preliminary experiments that single phosphorylated ITAMs bind
p72
with intermediate affinity suggesting that engagement
of both phosphorylated tyrosines by the SH2 domains of p72
confers maximal binding affinity but that binding occurs even
when only one tyrosine residue is phosphorylated. It is not clear
whether both or only one of the SH2 domains of p72
is
involved in the interaction with the Fc
RIIA-ITAM. Having
identified the structural elements in Fc
RIIA that allow
p72
association, we are now in a position to identify the
specific structural elements in p72
that interact with
the phosphorylated Fc
RIIA-ITAM.
In this study, we have focused
on the proximal effects of FcRIIA clustering and the catalytic
molecules that are recruited to its cytoplasmic domain. It is not clear
whether the binding of PI 3-K to Fc
RIIA is direct or indirect and
experiments with purified recombinant PI 3-K are ongoing. Nevertheless,
our data lend support to a speculative model of Fc
RIIA activation
in which receptor clustering initiates a concatenation of events that
begins with tyrosine phosphorylation of the ITAM. The phosphorylated
ITAM would then be bound by p72
which would in turn be
phosphorylated at YXXM sites to then induce PI 3-K binding. We
observe that the stoichiometry of association of PI 3-K with
Fc
RIIA, apparently low, is consistent with a model of multiple
interactions where the extent of association of downstream molecules is
proportional to the level of tyrosine phosphorylation on specific sites
of the adapter molecules. Precedence for such models exists. In the
case of the T cell antigen receptor, it has been proposed (57) that recruitment of the tyrosine kinase ZAP-70 to a
phosphorylated ITAM results phosphorylation of ZAP-70 at multiple
tyrosine residues providing multiple docking sites for other
SH2-containing proteins. The observation that the protein-tyrosine
phosphatase Syp acts as an adapter to link the G62-SOS complex to the
PDGF receptor is another instance where a cytoplasmic enzyme binds to
an activated receptor and also serves to recruit another element of the
signaling cascade(58) .
To test our model, it is necessary
to map the sites at which p72 is tyrosine-phosphorylated
and to determine whether PI 3-K binds such sites in the context of the
anchoring of p72
to tyrosine-phosphorylated Fc
RIIA
or whether additional molecules are required for PI 3-K recruitment and
binding to the activated Fc
RIIA complex. Identification of the
kinases that are responsible for phosphorylating the tyrosine residues
of the Fc
RIIA-ITAM would then present us with an initial sketch of
the proximal effects of Fc
RIIA clustering in platelets.
Addendum-While this manuscript was in preparation,
Yanaga et al.(59) also demonstrated binding of
p72 to a synthetic phosphopeptide corresponding
to the sequence of the Fc
RIIA-ITAM.