Low Density Lipoprotein Phosphorylates the Focal
Adhesion-associated Kinase p125FAK in Human Platelets
Independent of Integrin
IIb
3*
Christian M.
Hackeng
§,
Marc W.
Pladet
,
Jan-Willem N.
Akkerman§¶, and
Herman J. M.
van Rijn
From the Departments of
Clinical Chemistry and
§ Haematology, University Hospital Utrecht, and
Institute for Biomembranes, Utrecht University, 3508 GA Utrecht, The
Netherlands
 |
ABSTRACT |
Low density lipoprotein (LDL) is known to
sensitize platelets to agonists via integrin mediated outside-in
signaling (Hackeng, C. M., Huigsloot, M., Pladet,
M. W., Nieuwenhuis, H. K., Rijn, H. J. M. v., and
Akkerman, J. W. N. (1999) Arterioscler. Thromb. Vasc.
Biol., in press). As outside in signaling is associated with
phosphorylation of p125FAK, the effect of LDL on
p125FAK phosphorylation in platelets was investigated. LDL
induced p125FAK phosphorylation in a dose- and time-
dependent manner. The phosphorylation was independent of ligand binding
to integrin
IIb
3 and aggregation, such in
contrast to
-thrombin-induced p125FAK phosphorylation,
that critically depended on platelet aggregation. Platelets from
patients with Glanzmann's thrombastenia showed the same LDL- induced
phos- phorylation of p125FAK as control platelets,
whereas
-thrombin completely failed to phosphorylate the kinase in
the patients platelets. LDL signaling to p125FAK was
independent of integrin
2
1, the Fc
RII
receptor, and the lysophosphatidic acid receptor and not affected by
inhibitors of cyclooxygenase, protein kinase C, ERK1/2 or
p38MAPK. Phosphorylation of p125FAK by LDL was
strongly inhibited by cyclic AMP. These observations indicate that LDL
is a unique platelet agonist, as it phosphorylates p125FAK
in platelet suspensions, under unstirred conditions and independent of
integrin
IIb
3.
 |
INTRODUCTION |
Focal adhesion kinase (p125FAK) is a nonreceptor
tyrosine kinase implicated in signaling pathways mediated by integrins,
G-protein coupled receptors, tyrosine kinase receptors, and the v-Src
and v-Crk oncoproteins (1). After cell activation, p125FAK
translocates to the cytoskeleton at focal adhesions (2-4), where it
serves as a docking site for signaling proteins (5, 6). The protein
contains six tyrosine phosphorylation sites. Autophosphorylation of
Tyr397 generates a high affinity binding site for the SH2
domain of Src family kinases. The association of Src subsequently
initiates phosphorylation of Tyr407,576,577, inducing
maximal kinase activity of p125FAK (7). Association of Src
also leads to the phosphorylation of Tyr925, thereby
creating a docking site for the adaptor protein Grb2 that is known to
mediate Ras activation by binding of the GDP/GTP exchange factor Sos,
linking p125FAK to the Ras/MAP kinase pathway (8). The
relevance of phosphorylation on Tyr861 is not entirely
clear, but it could serve as another site of p125FAK
interaction with Src-family kinases.
Phosphorylation of p125FAK in blood platelets is different
for platelets in suspension and platelets adherent to immobilized ligand. In platelet suspensions, p125FAK phosphorylation
only occurred under aggregating conditions (9). An antibody against
integrin
IIb
3, that blocked fibrinogen
binding and aggregation, totally abolished p125FAK
phosphorylation by
-thrombin and collagen in stirred suspensions. In
the absence of stirring,
-thrombin (9) or the
IIb
3-activating antibody LIBS6 (10)
failed to induce p125FAK-phosphorylation. The role for
IIb
3 in this signaling event was further
supported by platelets from patients with Glanzmann's thrombastenia,
that lack
IIb
3, in which neither
-thrombin nor collagen induced p125FAK phosphorylation
in stirred suspensions (9).
Platelets adherent to immobilized ligand show
IIb
3-dependent and
-independent p125FAK phosphorylation. Platelets bound to
fibrinogen show p125FAK phosphorylation via
IIb
3. Platelets from a patient with a
truncated cytoplasmic domain of the
3-subunit (11) and
Chinese hamster ovary cells transfected with truncated forms of the
3-subunit (12) bound to immobilized fibrinogen but
failed to induce phosphorylation of p125FAK. Expression of
a constitutively active mutant of
3 together with
IIb led to a slight degree of p125FAK
phosphorylation in suspended Chinese hamster ovary cells in the presence of fibrinogen. However, this phosphorylation was negligible compared with the same cells adherent to fibrinogen (13). Also
IIb
3-independent p125FAK
phosphorylation has been described. Collagen (14-16) and
immunoglobulins (15) immobilized on a surface induced
p125FAK phosphorylation in the presence of an
anti-
IIb
3 antibody and in Glanzmann's
platelets. Hence, in platelets p125FAK may play a central
role in signal transduction after
IIb
3
ligation or in platelet adhesion, thereby strengthening ligand-
receptor interaction and coordinating further signaling.
Low density lipoprotein
(LDL)1 is known to increase
the sensitivity of human platelets to different agonists (17-20), but
the intracellular mechanisms involved remain largely unknown. Among the
signal transducing elements that are activated by LDL are protein
kinase C (PKC) (21, 22), Ca2+ mobilization (20, 23, 24) and
phosphoinositide turnover (20, 21, 24, 25). The faster collagen-induced
secretion in LDL-treated platelets critically depends on ligand-induced outside-in signaling via integrin
IIb
3.
This is illustrated in
IIb
3-deficient
platelets, where pretreatment with LDL failed to increase dense granule
secretion in response to collagen (22). Similar results were obtained
when binding of released fibrinogen was blocked with the fibrinogen
-chain-derived peptide
400-411. As integrin
signaling involves receptor ligation and clustering, and concentration
of signaling elements in focal adhesions (reviewed in Refs. 5 and 6),
we set to explore the involvement of p125FAK in LDL-induced
sensitization of platelets.
 |
EXPERIMENTAL PROCEDURES |
Materials--
BSA (demineralized) was from Organon Teknika
(Eppelheim, Federal Republic of Germany), and Sepharose 2B and protein
A-Sepharose were from Pharmacia Biotech (Uppsalla, Sweden). Enhanced
chemiluminescence reagent (ECL) was from NEN Life Science Products.
Human
-thrombin, dibutyryl cyclic AMP, indomethacin, trypsin
inhibitor, and 1-oleoyl-L-
-lysophosphatidic acid (LPA)
were purchased from Sigma. GF109203X and N-octyl glucoside were from Boehringer Mannheim (Mannheim, Federal Republic of Germany), and reinforced nitrocellulose sheets were from Schleicher and Schuell
(Dassel, Germany). PD98059 was from Calbiochem and iloprost from
Schering (Berlin, Federal Republic of Germany). SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl) imidazole) was from Alexis (San Diego, CA).
Anti-phosphotyrosine mAb 4G10 was from Upstate Biotechnology (Bucks,
United Kingdom), anti-p125FAK mAb was from Transduction
Laboratories (Lexington, NY) and anti-p125FAK polyclonal
antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). The
antibody IV-3 against the Fc
RII receptor was purchased from Medarex
(Annandale, NY), and monoclonal antibody against the integrin
2- subunit (6F1) was a kind gift of Dr. Barry Coller, Mt. Sinai School of Medicine, New York. The fibrinogen-derived peptide
HHLGGAKQAGDV (
400-411) was kindly provided by the department of Biochemistry, University Utrecht. All other chemicals used were of analytical grade.
LDL Isolation--
Fresh, nonfrozen plasma from four healthy
subjects each containing less than 100 mg lipoprotein(a)/liter was
pooled, and LDL (density range 1.019-1.063 kg/liter) was isolated by
sequential flotation in a Beckman L-70 ultracentrifuge
(26). To prevent lipid modification and bacterial contamination, 0.25 mM PMSF, 0.2 mM thimerosal, 2 mM
NaN3, and 4 mM EDTA (final concentrations) were
present during the first run (20 h, 175,000 × g,
10 °C). Subsequent centrifugations (20 h, 175,000 × g, 10 °C) were carried out in the absence of additives
except for NaN3 and EDTA. LDL was filtered through a
0.45-µm filter (Millipore, Molsheim, France) and subsequently
dialyzed against 103 volumes of 150 mM NaCl
containing 1.5 mM NaN3 and 1 mM
EDTA. LDL was stored at 4 °C under nitrogen for not longer than 14 days and before each experiment dialyzed overnight against
104 volumes 150 mM NaCl. ApoB100 and
lipoprotein(a) concentrations were measured using the Behring
Nephelometer 100. As described previously (22), these LDL preparations
contained only minimal amounts of thiobarbituric acid-reactive
substances (0.20 ± 0.07 nmol/mg) and lipid peroxides (6.7 ± 1.9 nmol/mg). The concentrations of these oxidation markers were below
or within other reported values for native LDL (27-31). Lipoprotein(a)
concentrations (Apotech, Organon Technika, Rockville, MD) were below 14 mg/liter. LDL preparations were analyzed for possible contamination by
fibrinogen (Laurell technique), fibronectin (ELISA) or von Willebrand
Factor (ELISA); all concentrations were below detection limits, which
were <50 µg fibrinogen, <50 ng fibronectin, and < 5 ng von
Willebrand Factor per g of B100 protein.
The concentration of LDL was expressed as gram of apoB100
protein/liter.
Platelet Isolation--
Freshly drawn venous blood from healthy
volunteers was collected with informed consent into 0.1 volume of 130 mM trisodium citrate. The donors claimed not to have taken
any medication during 2 weeks prior to blood collection. Platelet-rich
plasma was prepared by centrifugation (200 × g for 15 min at 22 °C). Gel-filtered platelets were isolated by gel
filtration through Sepharose 2B equilibrated in Ca2+-free
Tyrode's solution (137 mM NaCl, 2.68 mM KCl,
0.42 mM NaH2PO4, 1.7 mM
MgCl2, and 11.9 mM NaHCO3, pH 7.25)
containing 0.2% BSA and 5 mM glucose. Platelets were
adjusted to a final count of 2 × 1011/liter.
Analysis of Phosphorylated and Total
p125FAK--
Platelets (108 cells) were
incubated with LDL or
-thrombin as indicated under "Results" and
thereafter mixed with ice-cold lysis buffer (1:10 v/v) containing 10%
Nonidet P-40, 5% N-octyl glucoside, 10 mM
Na3VO4, 20 mM PMSF, 200 µg/ml
trypsin inhibitor, 50 mM N-ethylmaleimide, 10 mM EDTA, 1% SDS, and 100 mM benzamidine in
Tyrode's solution. Tyrosine-phosphorylated proteins were precipitated using 1 µg of 4G10 and protein A-Sepharose (100 µl of a 1%
suspension of protein A-Sepharose in lysis buffer, previously blocked
in 1% BSA, 1 h, 22 °C) for 5 h at 4 °C. Precipitates
were washed five times with lysis buffer and taken up in sample buffer.
Proteins were separated by SDS-PAGE using a 7.5% gel and transferred
to a nitrocellulose membrane. Phosphorylated p125FAK was
visualized by incubation with anti-p125FAK mAb (0.25 µg/ml, 15 h, 4 °C), peroxidase-linked rabbit anti-mouse IgG
(1:10,000 v/v, 1 h, 4 °C), and enhanced chemiluminescence. In
some experiments, as indicated, samples (2 × 108
cells) were split in two equal aliquots and precipitated with polyclonal anti-p125FAK (1.5 µg) under the same
conditions. Phosphorylated p125FAK was visualized using
4G10 (0.5 µg/ml) and total p125FAK using
anti-p125FAK mAb. For semiquantitative determination of
p125FAK, the density of the bands was analyzed using Image
Quant software.
Patients--
Four unrelated patients with Glanzmann's
thrombastenia (MAV, CPW, LYON1, and LYON2) were studied. The diagnosis
was made on the basis of a markedly prolonged bleeding time (Simplate,
>30 min; normal, <8 min) and a severe reduction in platelet
IIb
3. Patients MAV and CPW have been
described previously (22). On a fluorescence-activated cell sorter,
0.3% and 0.4%
IIb
3-positive platelets
were detected, respectively. Platelets from patients LYON1 and LYON2
(32) were obtained with the kind assistance of Dr. Claude Negrier,
Hospital Edouard Herriot, Lyon, France.
 |
RESULTS |
LDL Induces p125FAK Phosphorylation in a Dose- and
Time-dependent Manner--
Fig.
1 illustrates that LDL induced a
dose-dependent increase in phosphotyrosine content of
p125FAK, in unstirred platelet suspensions. When
precipitation was performed with an anti-phosphotyrosine antibody
followed by SDS-PAGE, significant p125FAK phosphorylation
was observed at an LDL concentration of 0.5 g/liter apoB100 (Fig. 1,
A and B). Inversely, when precipitation was
performed with an anti-p125FAK antibody, and blots were
probed for phosphotyrosine containing proteins, similar results were
obtained (Fig. 1C). Samples from Fig. 1C revealed
equal amounts of p125FAK protein in each lane when the blot
was probed with anti-p125FAK (Fig. 1D).

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Fig. 1.
Activation of p125FAK
by different concentrations of LDL. A,
platelets were incubated with LDL (10 min, 37 °C) at the indicated
concentrations. After lysis, phosphotyrosine-containing proteins were
precipitated with anti-phosphotyrosine mAb 4G10. Identification of
p125FAK was performed by SDS-PAGE and Western blotting
using a mAb against p125FAK, followed by ECL. The blot
shown is representative for four separate experiments. B,
densitometric analysis of tyrosine-phosphorylated p125FAK
was performed using Image Quant software. Data were corrected for
background intensities without LDL and were expressed as percentage of
the intensity at 1 g/liter LDL (100%; open symbol). Data
are means ± S.D., n 3; p < 0.05 for all
concentrations at 0.5 g/liter and more. C, platelets were
incubated with the indicated LDL concentrations (10 min, 37 °C).
After lysis, total p125FAK was precipitated with
anti-p125FAK polyclonal antibody. Identification of
tyrosine-phosphorylated proteins was performed by SDS-PAGE and Western
blotting using anti-phosphotyrosine mAb 4G10. The blot shown is
representative for three separate experiments. D,
p125FAK was precipitated from lysates described in the
legend to C, and total p125FAK was detected with
anti-p125FAK mAb, and bands were identified by ECL.
|
|
A rise in phosphorylation of p125FAK after LDL incubation
was observed as soon as 10 s after addition of LDL; activation was maximal at about 2 min. This maximum was sustained until 30 min (Fig.
2, A and B) and did
not decline until 90 min after stimulation (not shown).

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Fig. 2.
LDL activates p125FAK in a
time-dependent manner. A, platelets were
incubated with LDL (1.0 g/liter, 37 °C) for the indicated time
periods. Tyrosine-phosphorylated proteins were precipitated using 4G10
mAb, and p125FAK was identified as indicated in the legend
to Fig. 1A. B, densitometric analysis of
tyrosine-phosphorylated p125FAK. For each time curve, the
p125FAK band intensity after 10-min incubation with LDL was
set at 100% (open symbol). Data were corrected for
background intensities without LDL and expressed as means ± S.D.,
n 3; p < 0.05 for all incubations at 30 s
and more.
|
|
LDL-induced p125FAK Phosphorylation Is Independent of
Integrin
IIb
3--
P125FAK
phosphorylation in platelet suspensions stimulated with
-thrombin
and collagen is known to depend on ligand binding to integrin
IIb
3 and aggregation (9, 10, 33). In
concert with these reports,
-thrombin (1 unit/ml) failed to induce
p125FAK phosphorylation in unstirred platelet suspensions,
where aggregation was absent (Fig.
3A). Under these conditions,
prevention of fibrinogen binding to integrin
IIb
3 by the fibrinogen
-chain-derived dodecapeptide (
400-411) (34) did
not have any effect. Under stirred conditions,
-thrombin induced a
strong phosphorylation of p125FAK. The phosphorylation was
completely abolished by
400-411, which inhibits
IIb
3 occupancy by fibrinogen that is
released by
-thrombin-stimulated platelets, which is in agreement
with an earlier report (9). Again, LDL induced p125FAK
phosphorylation in the absence (Fig. 3) and presence (not shown) of
stirring. Surprisingly, p125FAK phosphorylation by LDL was
insensitive to the presence of
400-411 (Fig.
3A), although under these conditions LDL induced a slight but significant
-granule secretion (5-10% P-selectin (CD62P) expression compared with 20 µM thrombin receptor
activating peptide (SFLLRN)) (22). Thus, LDL induced
p125FAK phosphorylation independent of ligand binding to
integrin
IIb
3 and aggregation. To
investigate whether the phosphorylation of p125FAK by LDL
involved ligand-independent signaling via
IIb
3, or completely bypassed the
integrin, the experiments were repeated with platelets from patients
with Glanzmann's thrombastenia that are deficient in
IIb
3. Fig. 3B shows the
phosphorylation of p125FAK by LDL in the absence and
presence of
400-411 in Glanzmann's platelets, which
was the same as seen in normal subjects. In stirred suspensions of
Glanzmann's platelets,
-thrombin completely failed to induce
p125FAK phosphorylation, both in the absence or presence of
400-411. From these experiments we conclude that LDL is
a unique platelet agonist, as it initiates phosphorylation of
p125FAK in unstirred platelet suspensions, independent of
integrin
IIb
3.

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Fig. 3.
LDL-induced phosphorylation of
p125FAK is independent of platelet integrin
IIb 3. A, platelets were
incubated with vehicle or 400-411 (200 µM, 2 min) and subsequently incubated with LDL (1 g/liter, 10 min, 37 °C) or -thrombin (1 unit/ml, 2 min,
37 °C), under nonstirred or stirred (900 rpm) conditions as
indicated. Phosphotyrosine-containing p125FAK was
identified using 4G10 as a precipitating antibody. The results are
representative for seven similar experiments with LDL and three
experiments with -thrombin. B, platelets from patients
with Glanzmann's thrombastenia were treated as in A. The
shown blot is representative for results from four different
patients.
|
|
Characterization of Signaling Pathways Involved in LDL-induced
p125FAK Phosphorylation--
To investigate whether
LDL-induced p125FAK phosphorylation involved the same
signaling mechanisms as seen in surface-activated platelets,
incubations were performed with agents that interfere with signal
processing at the level of surface receptors (Fig. 4A) or intracellular signaling
routes (Fig. 4B). As already shown in Fig. 3A,
400-411 did not affect p125FAK
phosphorylation. Earlier work has shown that collagen-induced p125FAK phosphorylation was mediated by integrin
2
1 and was inhibited by the antibody 6F1
against this integrin (16). p125FAK phosphorylation by
immobilized IgG was inhibited by anti Fc
RII antibody IV-3 (15). None
of these antibodies affected LDL-induced p125FAK
phosphorylation. Also inhibition of thromboxane A2
(TxA2)-formation by indomethacin had no effect. As it was
reported that p125FAK phosphorylation is regulated via PKC
in platelets adherent to immobilized fibrinogen (35) or collagen (15),
the studies were repeated in the presence of the PKC inhibitor
GF109203X, but p125FAK phosphorylation remained the same.
Also the mitogen-activated protein kinase (MAPK) ERK1/2
(p44/42MAPK) has been implicated in integrin-mediated
signaling (36, 37). Therefore, we tested the effect of the inhibitor
PD98059 of MEK, the upstream activator of ERK1/2. The stress-activated
p38 mitogen-activated protein kinase (p38MAPK) has been
implicated in actin reorganization and cell migration in endothelial
cells, both processes that are accompanied by focal adhesion formation
(38). Inhibition of p38MAPK was achieved using SB203580
(39). Both inhibitors did not change LDL-induced p125FAK
phosphorylation. In contrast, a strong inhibition was observed in the
presence of agents that increase cyclic AMP (cAMP). PGI2 (prostacyclin), its stable analogue iloprost and the cell-permeable cAMP analogue dibutyryl cAMP (Bt2cAMP) all strongly
decreased p125FAK phosphorylation induced by LDL.

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Fig. 4.
LDL-induced p125FAK
phosphorylation is not affected by a wide variety of platelet
inhibitors. A, platelets were incubated with vehicle,
400-411 (200 µM, 2 min) or antibody (2 mg/liter, 30 min, 37 °C). Data were analyzed as indicated in the
legend to Fig. 1B. Data were corrected for background
intensities without LDL and expressed as means ± S.D., n 3. B, platelets were incubated with vehicle, indomethacin
(30 µM, 15 min), GF109203X (5 µM, 1 min),
PD98059 (20 µM, 10 min), SB203580 (10 µM,
15 min), PGI2 (10 ng/ml, 2 min), iloprost (2 µM, 15 min), or Bt2cAMP (250 µM, 10 min) before addition of LDL (1.0 g/liter, 10 min).
Phosphotyrosine-containing p125FAK was identified using
4G10 as a precipitating antibody. Data were analyzed as indicated in
the legend to Fig. 1B. Data were corrected for background
intensities without LDL and expressed as means ± S.D., n 3. C, platelets were incubated with vehicle, LDL (1 g/liter), or LPA at the indicated concentrations and time periods.
Phosphotyrosine-containing p125FAK was identified using
4G10 as a precipitating antibody.
|
|
Taken together, these data show that LDL-induced phosphorylation of
p125FAK occurred upstream of or independent from signal
transduction through TxA2 formation, PKC, ERK1/2, and
p38MAPK. However, the strong inhibition by agents that
raise cAMP reflects negative regulation by cAMP-dependent processes.
Previous studies have shown that LPA induces phosphorylation of
p125FAK in Swiss 3T3 cells (40) and rat hepatoma cells
(41). As activated platelets release LPA in vitro (42) and
possibly in vivo, we investigated whether the activating
properties of LDL were caused by contamination with LPA. In a
concentration that induces cell activation (1 µmol/liter, Ref. 43)
and 10-fold higher, LPA did not initiate the phosphorylation of
p125FAK.
 |
DISCUSSION |
The present findings show that LDL triggers the phosphorylation of
p125FAK in platelet suspensions independent of integrin
IIb
3 and aggregation. This is in sharp
contrast to phosphorylation by
-thrombin, collagen, and
costimulation of epinephrine with ADP (9) or LIBS6 Fab fragments (33)
in cell suspension, which requires ligand binding to
IIb
3 and platelet-platelet contact. The
requirement for ligand-induced outside-in signaling is illustrated by
the absence of p125FAK phosphorylation under conditions
that prevent platelet spreading (10, 11) (reviewed in Ref. 5). LDL
induces p125FAK phosphorylation within seconds after
stimulation, reaching a maximum at physiological concentrations of LDL
(0.26-1.23 g of apoB100/liter) (44). LDL signaling to
p125FAK is not dependent on TxA2 formation or
activation of PKC, p38MAPK, and ERK1/2. These observations
indicate that p125FAK phosphorylation occurs directly
downstream of the LDL receptor.
P125FAK is phosphorylated independent of
IIb
3 when platelets adhere to immobilized
collagen via integrin
2
1 (14-16) or to immobilized IgG via the Fc
RII receptor (15). It is therefore possible that the LDL particle acts as an activating surface that phosphorylates p125FAK by clustering of membrane receptors.
However, antibodies against integrin
2
1
and Fc
RII, known to block further signal generation to
p125FAK, had no effect. Additionally, LPA did not induce
p125FAK phosphorylation. Another candidate for LDL-induced
p125FAK phosphorylation is the collagen receptor
glycoprotein (GP)VI. Collagen signaling through this glycoprotein leads
to phosphorylation of p125FAK (45) and is inhibited by cAMP
(46). However, GPVI-mediated p125FAK phosphorylation is not
observed in Glanzmann's platelets or in the presence of the RGDS
peptide, such in contrast to the effect of LDL. The nature of the cAMP
sensitivity of p125FAK phosphorylation by LDL remains to be
elucidated. A rise in cAMP leads to activation of protein kinase A,
that in turn can activate vasodilator-stimulated protein (VASP), a
50-kDa protein that localizes to focal adhesions and regulates actin
dynamics (47). Phosphorylation of VASP correlates with a decrease in
IIb
3 activation and aggregation (48).
Thus, cAMP might inhibit LDL signaling to p125FAK via VASP
by preventing cytoskeleton rearrangements.
It has been reported that phosphorylation of p125FAK on
Tyr397 induces Src activity, leading to phosphorylation of
Tyr407,576,577, inducing maximal kinase activity of the
protein (7), but the importance of p125FAK activity remains
unclear. P125FAK knockout mice were not viable, and
embryonic cells of these mice had a reduced mobility. Surprisingly, the
number of focal adhesions in these mice was increased. From these
observations, it was proposed that p125FAK regulates focal
contact turnover (49), rather than their formation.
Two major signaling mechanisms downstream of p125FAK are:
(i) p130CAS associates to one of the two C-terminal
proline-rich regions of p125FAK. This association results
in phosphorylation of p130CAS and subsequent binding of Crk
via its SH2 domain (reviewed in Refs. 5 and 6). Subsequently, Crk can
associate with the GDP/GTP exchange factors Sos (for Ras) or C3G (for
Rap1) (50). (ii) The GTPase regulator associated with FAK (Graf)
associates directly to p125FAK via the proline-rich region
of residues Pro875-Arg880 in an SH3
domain-dependent manner. Activation of Graf was induced by
phosphorylation mediated by ERK1/2 and was shown to stimulate the
GTPase activity of CDC42 and Rho, but not Ras or Rac (51). The
implications of CDC42 and Rho activity for stress fiber and focal
adhesion assembly (52) led to the proposition that activation of Graf
might down-regulate these CDC42 and Rho-mediated cytoskeletal changes
(1).
Recently, we observed that LDL induced activation of the small GTPases
Rap1 and Ral in platelets.2
Activation of Rap1 and Ral critically depend on Ca2+ (53,
54), and Ral is a putative effector molecule of Rap1 (55). In turn, Ral
is thought to be an upstream regulator of a member of the Rac/Rho
family, CDC42. This small GTPase is involved in the rearrangement of
the cytoskeleton. The strong phosphorylation of p125FAK
triggered by LDL described in the present report might therefore initiate these two pathways: (i) activation of Rap1 and Ral and CDC42
via C3G and (ii) cytoskeleton regulation via Ral-activated Rho,
controlled by Graf.
In conclusion, via these two separate mechanisms, LDL might be
controlling cytoskeleton rearrangements, thereby targeting several
signal transducing proteins to an appropriate site of action, leading
to an increased sensitivity to platelet agonists such as
-thrombin
and collagen.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Claude Negrier, Hospital Edouard
Herriot, Lyon, France, for his assistance in the studies on two
patients with Glanzmann's thrombastenia and Eric Litjens and
José Donath for their support.
 |
FOOTNOTES |
*
This work was supported by the University Hospital Utrecht
and the Netherlands Thrombosis Foundation.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.
¶
To whom correspondence should be addressed: Dept. of
Haematology, University Hospital Utrecht, P. O. Box 85500, 3508 GA
Utrecht, The Netherlands. Tel.: 31-302506512; Fax: 31-302511893;
E-mail: j.w.n.akkerman{at}lab.azu.nl.
2
C. M. Hackeng et al., submitted
for publication.
 |
ABBREVIATIONS |
The abbreviations used are:
LDL, low density
lipoprotein;
PKC, protein kinase C;
BSA, bovine serum albumin;
LPA, 1-oleoyl-L-
-lysophosphatidic acid;
mAb, monoclonal
antibody;
PMSF, phenylmethylsulfonyl fluoride;
ELISA, enzyme-linked
immunosorbent assay;
PAGE, polyacrylamide gel electrophoresis;
TxA2, thromboxane A2;
MAPK, mitogen-activated protein kinase;
PGI2, prostaglandin
I2;
Bt2cAMP, dibutyryl cyclic AMP;
GP, glycoprotein;
VASP, vasodilator-stimulated protein..
 |
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