From the Kimmel Cancer Institute and Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, the ¶ Istituto di Istologia ed Embriologia, Universita' di Bologna, Bologna, Italy, and the § Dipartimento di Morfologia ed Embriologia, Universita' degli Studi, Via Fossato di Mortara 56, 44100 Ferrara, Italy
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ABSTRACT |
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We have observed that aggregation of human
platelets, caused by activation of integrin
IIb
3 and its consequent binding of fibrinogen, stimulates a novel pathway for synthesis of
phosphatidylinositol 3,4bisphosphate, thereby activating protein
kinase B/Akt. Such synthesis depends upon both the generation of
phosphatidylinositol 3-phosphate (PtdIns3P), which is sensitive to
wortmannin (IC50 7 nM) and calpain inhibitors,
and the phosphorylation of PtdIns3P by PtdIns3P 4-kinase. We now report
that a recently characterized C2 domain-containing phosphoinositide
3-kinase isoform (HsC2-PI3K) is present in platelets and a leukemic
cell line (CHRF-288) derived from megakaryoblasts, and is likely to be
responsible for the stimulated synthesis of PtdIns3P observed in
platelets. HsC2-PI3K, identifiable by Western blotting and
immunoprecipitatable activity, is sensitive to wortmannin
(IC50 6-10 nM), requires Mg2+, and
shows strong preference for PtdIns over PtdIns4P or
phosphatidylinositol 4,5-bisphosphate as substrate. HsC2-PI3K is
activated severalfold when platelets aggregate in an
IIb
3-dependent manner or when platelet or CHRF-288 lysates are incubated with Ca2+.
Activation is prevented by calpain inhibitors. CHRF-288, which cannot
undergo activation of
IIb
3 and thereby
aggregate in response to platelet agonists, do not generate PtdIns3P or
activate HsC2-PI3K under conditions that stimulate other
phosphoinositide 3-kinases. HsC2-PI3K may thus be an important effector
for integrin-dependent signaling.
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INTRODUCTION |
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Activation of phosphoinositide 3-kinase
(PI3K)1 is an important cell
signaling event that has been linked causally to a variety of
physiologic changes, including proliferative responses to growth factors (1), differentiation (2), anti-apoptosis (3), cytoskeletal
rearrangements and integrin activation (4, 5), and integrin-mediated
cell motility and carcinoma invasion (6, 7). The second messengers
involved in these events are thought to be PtdInsP3 and/or
PtdIns(3,4)P2, which are capable of stimulating the
activity of protein kinases such as PKB/Akt (8), and some protein
kinase C isoforms (9-11), but may act in additional ways. Most of the
PI3Ks studied in these contexts are, or have been assumed to be, of the
"Type I" class (see Ref. 12 for review), where classification is
based upon structural homology, substrate specificity, and mode of
regulation. The Type I PI3Ks are heterodimers, containing 110-130-kDa
catalytic subunits associated with 50-85- or 101-kDa adaptor entities
that regulate localization and function. Type I PI3Ks phosphorylate
PtdIns, PtdIns4P, and PtdIns(4,5)P2, with preference for
PtdIns(4,5)P2, at the 3-OH site of the myoinositol ring
in vitro and have been found to be stimulated by numerous growth factors and heterotrimeric GTP-binding protein-coupled receptors. They can be activated, depending upon the subtype, cell, and
receptor that has been stimulated, by tyrosine phosphorylation, association with tyrosine-phosphorylated or proline-rich domains, small
GTPases, or subunits of heterotrimeric GTP-binding proteins (12). Type II PI3Ks are catalytic entities about twice the size of Type
I catalytic subunits, and it is unknown whether they, like Type I and
Type III, have adaptor proteins or whether their increased size
provides a built-in adaptor. They contain a defining C2 domain at their
C termini, as well as an N-terminal extension. Importantly, as well,
they cannot utilize, or utilize poorly, PtdIns(4,5)P2 as a
substrate, and their means of activation, or even whether they can be
activated in a signal-transduction setting, has been unknown. The
cloning and characterization of two new PI3Ks of the Type II class from
human cells have been described recently (13, 14). These PI3Ks have
been designated PI3KC2
(13) and HsC2-PI3K (14). PI3KC2
(190 kDa),
which has been expressed, is resistant to wortmannin (IC50
420 nM) and resembles (90% homology) mouse m-cpk (15) and
p170 (16). It preferentially phosphorylates PtdIns and PtdIns4P, but
can, albeit poorly, phosphorylate PtdIns(4,5)P2. HsC2-PI3K
is a closely related 185-kDa protein, except for its divergent 350 N-terminal residues, which encompass two proline-rich sequences
appropriate for interaction with src-homology 3 domains (14). As
described here, expressed HsC2-PI3K phosphorylates PtdIns in strong
preference to the other two known PI3K substrates, PtdIns4P and
PtdIns(4,5)P2, and is very sensitive to wortmannin (IC50 ~10 nM), in contrast to PI3KC2
.
Mammalian Type III PI3K (HsVPS34) contains a catalytic subunit similar
in size to those of Type I and an associated protein of 150 kDa.
HsVPS34 is inhibited by nanomolar wortmannin, phosphorylates only
PtdIns lipid substrate, and, in contrast to Type I and Type II kinases,
is stimulated by Mn2+ versus Mg2+.
Type III PI3Ks, based upon findings with the yeast homologue, VPSp, are
thought to participate constitutively in secretory protein sorting to
vacuoles, rather than being stimulated in response to cellular
agonists.
We have observed (17, 18) that activation of platelet integrin
IIb
3, via its consequent
fibrinogen-dependent aggregation or clustering, stimulates
a novel pathway for the generation of PtdIns(3,4)P2 in
platelets that is a function of PtdIns3P production and phosphorylation
of PtdIns3P by a 4-kinase. No PtdInsP3 is generated during
this integrin-dependent event. In the present study, we
have examined the nature of the PI3K involved in the integrin-activated
pathway, which results in the activation of platelet PKB
/Akt (17,
18). Our data indicate that a PI3K of the Type II subclass, HsC2-PI3K,
is most likely to be responsible for the generation of PtdIns3P that
occurs.
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EXPERIMENTAL PROCEDURES |
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Most reagents were obtained from sources described (17, 18).
Calpain I and II inhibitors were purchased from Boehringer Mannheim.
HsC2-PI3K cDNA was assembled using a combination of reverse
transcription-polymerase chain reaction and standard cDNA screening, by using the sequence of HsC2-PI3K (14) cloned in the
mammalian expression vector pBKCMV for transient expression in HEK 293 cells. Its activity after immunoprecipitation was assayed as described
below. PI3K isoform-discriminant polyclonal antisera against the first
350 amino acid portion of HsC2-PI3K (14), expressed in
Escherichia coli as an N-terminally fused glutathione S-transferase protein, were raised in rabbits. These
antisera were used for all immunoprecipitations and Western blots
directed at HsC2-PI3K and do not detect or immunoprecipitate Type I
PI3Ks, HsVPS34, or PI3KC2. Antibodies to p85
/
subunits and
PI3KC2
were the generous gifts of Drs. Ivan Gout and Jan Domin
(Ludwig Institute, London). Antibody to HsVPS34 was prepared by Dr.
Volinia (19).
Activation of Platelets and CHRF-288 Labeled with [32P]Pi-- Washed human platelets were labeled to equilibrium with [32P]Pi as described (17, 18). CHRF-288 cells were washed with DMEM, incubated overnight with 0.1% bovine serum albumin/DMEM in the absence of serum, and incubated in low Pi-DMEM with [32P]Pi (1 mCi/ml), as reported (20). Labeled platelets were incubated for up to 14 min at 37 °C ± FIB, LIBS (17), SFLLRN (25 µM; 18), or PMA (200 nM; Ref. 18), while stirring. In some cases, different concentrations of calpain inhibitors (calpeptin, calpain I, or calpain II) or wortmannin were incubated with platelets prior to platelet activation (17). Labeled CHRF-288 were incubated up to 15 min ± SFLLRN and FIB, as for platelets. Incubations were terminated with CHCl3/MeOH/HCl, and lipids were extracted, resolved, deacylated, and quantitated by in-line 32P detection after HPLC, as described (17, 18).
Immunoprecipitations--
Nonlabeled platelets or CHRF-288 cells
were incubated ± agonists/calpain inhibitors as above, and
incubations were terminated with ice-cold Triton lysis buffer
containing 100 µg/ml calpeptin (17) or RIPA buffer, containing 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5%
Na+ deoxycholate, 0.1% SDS, 2 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml
leupeptin, and calpeptin, pH 7.6. After lysates (platelet, CHRF-288, or
HEK293) were spun at 100,000 × g × 90 min
4 °C, HsC2-PI3K was immunoprecipitated from 450 µl of supernatants
with antibody and protein A/Sepharose. Immunoprecipitates were washed
once with Triton lysis buffer, then three times with HEPES (5 mM)/EDTA (2 mM), pH 7.4, and re-suspended to 80 µl in HEPES/EDTA for PI3K activity assays (40 µl/100 µl assay)
using PtdIns, PtdIns4P, or
PtdIns(4,5)P2 (200 µM) substrates
that had been suspended in HEPES/EDTA. In some cases, varied
concentrations of wortmannin in Me2SO/buffer vehicle were
prepared freshly and added to washed immunoprecipitates for 5 min at
37 °C prior to assay of activity, in comparison with the same amount
of Me2SO/buffer vehicle control. Assays contained
[-32P]ATP (40 µM; 1 mCi/ml),
MgCl2 (4 mM), or MnCl2 (4 mM), Tris (20 mM)/NaCl (100 mM)/EGTA (0.5 mM)/HEPES (3 mM)/EDTA
(1.2 mM), pH 7.5, and were run at 30 °C for 20 min.
Activity with respect to PtdIns/Mg2+ was determined to be
linear for 30 min. Lipids were extracted and resolved by HPLC as above.
The PI3K, p85/PI3K, was also immunoprecipitated from Triton-soluble
fractions using antibodies to the p85
/p85
subunits (5) for assay
of kinase activity under the above conditions. In other studies, Triton
lysates from unstimulated platelets or CHRF-288 cells, without
calpeptin or EGTA, were incubated at room temperature for various
periods up to 60 min ± calpeptin ± 2 mM Ca2+free. Incubations were terminated with 50 µg/ml calpeptin, 5 mM EGTA, pH 7.4, and samples chilled
and centrifugated. HsC2-PI3K was immunoprecipitated from supernatants
and PI3K activity assayed with PtdIns, as above.
Western Blotting-- Platelet and CHRF-288 Triton-soluble and insoluble ("CSK") fractions and HsC2-PI3K immunoprecipitates were dissolved in SDS-reducing buffer and proteins resolved by one-dimensional SDS-polyacrylamide gel electrophoresis on 7.5% gels, prior to transfer to nitrocellulose for Western blotting and enhanced chemiluminescence detection, as described previously (5). CSK from Triton lysates were suspended in buffer at 4-11 times their concentrations in Triton lysates before mixing with sample buffer, such that the same amount of CSK protein would be applied per lane.
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RESULTS AND DISCUSSION |
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Exposure of platelets to a variety of agonists, under conditions
that promoted IIb
3 + FIB-dependent aggregation, led to the activation of
HsC2-PI3K and, in 32P-labeled platelets, transient
accumulation of [32P]PtdIns3P (Fig.
1). Both effects were inhibited by
wortmannin (IC50 7 nM, for
[32P]PtdIns3P; IC50 6 nM for
HsC2-PI3K), in keeping with the wortmannin sensitivity of expressed
HsC2-PI3K (IC50 ~10 nM). As shown in Fig. 1A,
the increased activity of HsC2-PI3K in immunoprecipitates from
Triton-soluble fractions of platelets incubated with SFLLRN (directed
to the thrombin receptor) + FIB was sustained for up to 14 min and
slightly preceded the accumulation of [32P]PtdIns3P in
stimulated platelets. The transient accumulation of
[32P]PtdIns3P is probably attributable to the increased
activity of PtdIns3P 4-kinase under these conditions (17, 18). In the absence of FIB (not shown), or in the presence of maximally effective concentrations of calpeptin (IC50 1 µM) or
calpain I inhibitor (IC50 0.3 µM), the
increase in PtdIns3P levels and activation of HsC2-PI3K were abolished.
Calpain I inhibitor (90% inhibition at 1 µM) was more
effective than calpain II inhibitor (18% inhibition at 1 µM). Similarly, increases in HsC2-PI3K activity (Fig.
1B) and [32P]PtdIns3P (Fig. 1C) in
response to PMA (activating protein kinase C) + FIB or LIBS (directly
activating
IIb
3) + FIB were blocked by
calpain inhibition and did not occur in the absence of FIB. Both PMA
and SFLLRN are known to activate p85/PI3K, whether or not FIB is
present, and thereby contribute to "inside-out" signaling leading
to the sustained activation of
IIb
3 (5).
SFLLRN also activates another Type I enzyme, PI3K
, which is
dependent upon
subunits of GTP-binding proteins, and apparently
is not involved in signaling leading to
IIb
3 activation (5). LIBS by-passes such
a pathway, promoting the FIB binding conformation of
IIb
3 by interaction with the
3 subunit (21) in a wortmannin-insensitive manner,
i.e. without the prerequisite of p85/PI3K activation or stimulated accumulation of 3-OH-phosphorylated phosphoinositides (5,
17). Once FIB binds to
IIb
3 and
aggregation occurs, however, "outside-in" signaling that promotes
calpain activation is triggered, leading to formation of PtdIns3P and
PtdIns(3,4)P2 (17, 18). Calpain, a
Ca2+-dependent thiol protease, is known to be
activated intracellularly when FIB binds to
IIb
3 and aggregates platelets that have
been stimulated in the presence of Ca2+ (22). To simulate
these calpain-activating conditions, in the absence of integrin
activation, unstimulated platelet lysates were incubated at room
temperature in the presence of millimolar Ca2+, and the
activity of immunoprecipitated HsC2-PI3K was then assayed. It was found
that activity rose transiently (2.49 ± 0.12-fold after 10 min,
4.08 ± 0.18-fold after 20 min, and 3.24 ± 0.17-fold after
60 min) and that the increase was blocked by omitting Ca2+
or by including calpain inhibitors.
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The increased 3-kinase activity in HsC2-PI3K immunoprecipitates from
stimulated platelets was specific for PtdIns as a substrate (Fig.
2). As would be expected from the
substrate specificity of 293 cell-expressed HsC2-PI3K, which exhibited
a greater than 10 times preference for PtdIns (not shown),
immunoprecipitated HsC2-PI3K from resting platelets strongly favored
PtdIns ([32P]PtdIns3P = 155,122 ± 6968 dpm)
over PtdIns4P ([32P]PtdIns(3,4)P2 = 316 ± 55 dpm) or PtdIns(4,5)P2
([32P]PtdInsP3 = 573 ± 41 dpm), and
activity with respect to PtdIns was selectively increased by platelet
stimulation. Thus, the increased PI3K activity seen with respect to
PtdIns is not attributable to co-precipitating Type I kinase activity
from activated platelets (5). Furthermore, the marked sensitivity of
the activity to wortmannin and selectivity of HsC2-PI3K antibody argue
against PI3KC2 (13) being co-precipitated, despite the fact that
PI3KC2
is present in platelets and CHRF-288
cells.2 Finally, unlike the
case for both expressed and native HsVPS34 (also present in platelets
and CHRF-288 cells2), which shows enhanced activity with
Mn2+ versus Mg2+ as a
co-factor (19), immunoprecipitated HsC2-PI3K was less than 10%
as active when Mn2+ was substituted for Mg2+.
Thus, the stimulated PI3K activity of HsC2-PI3K immunoprecipitates is
not due to contaminating or co-precipitating Type III (HsVPS34) activity. Additionally, such other PI3Ks were not immunologically detectable in HsC2-PI3K immunoprecipitates (not shown).
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As we have reported (20, 23), CHRF-288, a leukemic cell line derived
from a platelet precursor cell, the megakaryoblast (24), can be
stimulated by a variety of physiological agonists to accumulate
PtdInsP3 and PtdIns(3,4)P2. These increases are sensitive to wortmannin, and CHRF-288 cells contain p85/PI3K and PI3K, Type I enzymes whose activities in lysates can be stimulated by guanosine 5'-O-(thiotriphosphate) and
subunits of
GTP-binding proteins, respectively. Despite displaying apparently
normal
IIb
3 at their surface, however,
CHRF-288 cells cannot undergo the activation of this integrin that
leads to the binding of FIB (25) and aggregation. We found that
CHRF-288 cells, after incubation with SFLLRN + FIB (or PMA + FIB, not
shown) accumulated no [32P]PtdIns3P, whereas
[32P]PtdInsP3 and
[32P]PtdIns(3,4)P2 were formed rapidly (Fig.
3A), a pattern similar to the
"pre-integrin" accumulation of 3-OH-phosphorylated
phosphoinositides in stimulated platelets (18). Furthermore, although
much immunoprecipitatable HsC2-PI3K activity was present in Triton or
RIPA lysates of CHRF-288 cells, no increased activity was observed when
CHRF-288 were activated with SFLLRN + FIB or PMA + FIB (Fig.
3B). In contrast, lysates of CHRF-288 incubated in the
presence of Ca2+ showed a time-dependent
increase in immunoprecipitatable HsC2-PI3K activity (2.3 ± 0.2-fold in 10 min, 3.6 ± 0.1-fold in 20 min, and 4.2 ± 0.4-fold in 60 min), which was prevented by omitting Ca2+
or including calpain inhibitor. This indicated that HsC2-PI3K of
CHRF-288 cells could be activated in an apparently
calpain-dependent manner if the requirement for activated
integrin were bypassed. Thus, failure to activate
IIb
3 is associated in intact CHRF-288 with failure to activate HsC2-PI3K and stimulate accumulation of
PtdIns3P.
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Western blotting of CSK and Triton-soluble (TS) fractions of platelets (Fig. 4) with antibody to HsC2-PI3K revealed a protein band at about 200 kDa in both fractions. This band in the Triton-soluble fraction could be decreased by immunoprecipitation with HsC2-PI3K antibody (PS), and a corresponding band was found for the Western blot of the immunoprecipitate (IP). The migration of the band did not shift detectably for blots from activated platelets (SFLLRN) or from platelet lysates incubated with Ca2+.
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In conclusion, our data indicate that HsC2-PI3K is activated in
platelets in a manner dependent upon the reorganization of integrin
IIb
3, FIB binding and aggregation, and,
most probably, calpain I activation. Given its strong preference for
Ptd-Ins as a substrate, its susceptibility to wortmannin, and its
activation under the same conditions required for the accumulation of
PtdIns3P in intact cells, HsC2-PI3K is the most reasonable choice for
the enzyme responsible for generating PtdIns3P in stimulated platelets. It is possible that HsC2-PI3K is a substrate for calpain I and thereby
activated by it, but, at this point, other mechanisms involving other
calpain targets are equally likely to contribute to the stimulation
observed in vivo. Inasmuch as the antibody used for
immunoprecipitation and identification of resting and activated
HsC2-PI3K is directed to the N-terminal 350-amino acid region of
HsC2-PI3K, and the catalytic domain lies approximately between amino
acids 1037 and 1320 (14), any activating cleavage target for calpain
would have to be C-terminal to these regions. If the cleavage site were
in the C2 domain of HsC2-PI3K, for example, a decrease in size might
not be easily detectable under our conditions. Further studies with
tagged, expressed HsC2-PI3K and purified calpain I may be needed to
identify possible activating cleavage sites for HsC2-PI3K in stimulated
platelets.
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ACKNOWLEDGEMENTS |
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We thank Dr. Mark Ginsberg for generously contributing LIBS antibody, Drew Likens for artwork, and the Blood Bank at the Cardeza Foundation for blood drawing facilities.
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FOOTNOTES |
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* This work was supported by NHLBI Grant HL38622 (to S. E. R.), NATO Grant RSG 950672 (to S. E. R.), and by AIRC (to S. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The first two authors contributed equally to this work.
To whom correspondence should be addressed. Fax:
215-923-7145.
1
The abbreviations used are: PI3K,
phosphoinositide 3-kinase; PtdIns, phosphatidylinositol
(locants of other phosphates on the myoinositol ring are indicated);
PtdInsP3, PtdIns(3,4,5)P3; FIB, fibrinogen;
LIBS, anti-ligand-induced binding site 6 antibody Fab fraction; CSK,
cytoskeleton; PKB/Akt, protein kinase B related to AKR mouse T cell
lymphoma-derived oncogenic product; SFLLRN, peptide; PMA, -phorbol
myristate acetate; DMEM, Dulbecco's modified Eagle's medium; HPLC,
high performance liquid chromatography.
2 J. Zhang, S. Volinia, J. Domin, and S. E. Rittenhouse, manuscript in preparation.
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REFERENCES |
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