(Received for publication, October 24, 1994; and in revised form, March 14, 1995)
From the
To investigate the function of the human Ras-related CDC42
GTP-binding protein (CDC42Hs) we studied its subcellular redistribution
in platelets stimulated by thrombin-receptor activating peptide (TRAP)
or ADP. In resting platelets CDC42Hs was detected exclusively in the
membrane skeleton (9.6 ± 1.5% of total) and the detergent
soluble fraction (90 ± 4%). When platelets were aggregated with
TRAP or ADP, CDC42Hs (10% of total) appeared in the cytoskeleton and
decreased in the membrane skeleton, whereas RhoGDI (guanine-nucleotide
dissociation inhibitor) and CDC42HsGAP (GTPase-activating protein)
remained exclusively in the detergent-soluble fraction. Upon prolonged
platelet stimulation CDC42Hs disappeared from the cytoskeleton and
reappeared in the membrane skeleton. Rac translocated to the
cytoskeleton with a similar time course as CDC42Hs. When platelets were
stimulated under conditions that precluded the activation of the
The cell division cycle proteins, CDC42, belong to the Rho
family of Ras-related GTP-binding proteins, which also includes RhoA,
-B, -C, and -G, Rac1 and -2, and TC10(1) . This group of
proteins has been implicated in the control of cytoskeletal
organization(2, 3) . Like other Ras-related proteins
that bind and hydrolyze GTP, CDC42 proteins are thought to cycle
between GTP-bound (active) and GDP-bound (inactive) states and are
regulated by GTPase-activating proteins and guanine nucleotide exchange
factors(4) . Furthermore, similar to several other small
GTP-binding proteins of the Ras superfamily, CDC42 proteins contain a
C-terminal, geranylgeranylated cysteine residue that is also
carboxymethylated(5) . These post-translational modifications
are surmised to promote important protein-membrane and protein-protein
interactions(6, 7, 8) .
The human CDC42
proteins (CDC42Hs) are the homologs of the yeast cell division cycle
protein, CDC42Sc, which has been implicated in bud site assembly during
the yeast cell cycle(9, 10, 11) . Mutations
in the CDC42Sc gene result in changes of cell shape and disruption of
actin filaments, suggesting a role in cytoskeletal
organization(9) . Two almost identical CDC42Hs cDNAs have been
cloned from placental and fetal brain
libraries(12, 13) . The two cDNA-predicted CDC42
proteins are identical except for an 8-amino acid segment at the C
terminus. The specific function of CDC42Hs proteins in mammalian cells
remains to be defined. It has recently been reported that spreading of
differentiating human monocytes is associated with a dramatic 30-fold
increase in membrane-associated CDC42Hs(14) .
CDC42Hs is
expressed in high concentrations in platelets (15) that can be
rapidly induced to change their shape, to aggregate, and to secrete the
contents of their granules. This series of functional responses is
associated with specific and profound changes of the
cytoskeleton(16, 17) . In a variety of cells
cytoskeletal changes and cell motility are induced through integrin
activation(18, 19, 20, 21) . In
circulating discoid platelets the
To obtain the membrane skeletal
fraction(28) , supernatants of detergent extracts were further
centrifuged at 100,000
Triton X-100-insoluble cytoskeletons
of control and activated platelet-rich plasma were prepared according
to the method of Carrol et al.(29) . Briefly,
platelet-rich plasma (2
To examine the proteolytic cleavage of actin-binding
protein and talin, platelet proteins were separated on a 6%
SDS-polyacrylamide gel electrophoresis slab gel, followed by staining
of the gel with 0.2% Coomassie Brilliant Blue, destaining, and drying.
Calpain activity was judged from the extent of degradation of
actin-binding protein and talin. To quantify actin in platelet
cytoskeletal preparations platelet proteins were separated by
SDS-polyacrylamide gel electrophoresis (12%) and stained with Coomassie
Blue, and the bands corresponding to actin were measured by laser
densitometry.
Figure 1:
Translocation of CDC42Hs to the
cytoskeleton during platelet aggregation. Aliquots of washed human
platelets (1.5 ml) were stimulated by TRAP (10 µM) for the
times indicated. Platelet aggregation (Agg %) was measured.
Platelets were lysed with the buffer containing Triton X-100, and the
cytoskeletal (CSK), membrane skeletal (MSK), and
soluble (SUP) fractions were isolated. The proteins in each
fraction were separated by SDS-polyacrylamide gel electrophoresis and
immunoblotted using an anti-CDC42Hs antibody. The experiment is
representative of six different
experiments.
Figure 2:
Disappearance of CDC42Hs from the
cytoskeleton upon prolonged platelet aggregation. Aliquots (1.5 ml) of
washed human platelets were stimulated with TRAP (10 µM)
for the times indicated. For further details and definitions of
abbreviations see the legend to Fig. 1. The experiment is
representative of six different
experiments.
Figure 3:
Comparison of the electrophoretic
mobilities of the cytoskeletal and membrane skeletal CDC42Hs with the
processed and unprocessed forms of the protein. Proteins were
electrophoretically separated and immunoblotted with an anti-CDC42Hs
antibody. Lane1, recombinant CDC42Hs protein
expressed in E. coli (unprocessed); lane2,
membrane skeleton obtained from resting platelets; lane3, cytoskeleton obtained from aggregated platelets; lane4, lysate from human monocytic U-937 cells
(processed).
Figure 4:
Translocation of Rac to the cytoskeleton
during platelet aggregation. Aliquots of washed human platelets (1.5
ml) were stimulated with TRAP (10 µM) for the times
indicated. Platelets were lysed with the buffer containing Triton
X-100, and the cytoskeletal fractions were isolated as described under
``Experimental Procedures.'' Proteins in the cytoskeleton
were immunoblotted using an anti-Rac1/Rac2 antibody. The experiment is
representative of three different experiments. Agg, %,
platelet aggregation.
Figure 5:
RhoGDI remains in the detergent-soluble
fraction during platelet aggregation. Platelets were stimulated by TRAP
(10 µM) as described in the legend to Fig. 1.
Immunoblotting was done using an antibody against RhoGDI. This figure
is typical of three different experiments. Abbreviations are as defined
in the legend to Fig. 1.
Figure 6:
CDC42HsGAP does not associate with the
cytoskeleton during platelet aggregation. Platelets were stimulated by
TRAP (10 µM) for the times indicated. Immunoblotting was
done using chicken anti-CDC42HsGAP. For further details and definitions
of abbreviations see the legend to Fig. 1. The experiment is
representative of three different
experiments.
Figure 7:
Translocation of CDC42Hs to the
cytoskeleton in stimulated platelets is mediated by
Figure 8:
Effect of cytochalasins B and D on the
cytoskeletal association of CDC42Hs in platelets aggregated by TRAP.
Washed platelets were incubated with either Me
Figure 9:
Effect of aspirin on the cytoskeletal
association of CDC42Hs in the platelets aggregated by TRAP or ADP.
Platelet-rich plasma was incubated with either aspirin (1 mM) (lanes3-5 and 9) or ethanol (0.2%) (lanes2, 6-8, and 10) for 15
min at 37 °C and stimulated with either ADP (10 µM) (lanes3-8) or TRAP (10 µM) (lanes9-10). Platelet aggregation (Agg
%) was measured. Cytoskeletal fractions were isolated as
described, subjected to SDS-polyacrylamide gel electrophoresis, and
analyzed for CDC42Hs by immunoblotting. Lane2,
control without agonist addition; lane1, recombinant
CDC42Hs.
We show in this study that a significant fraction of the
Ras-related GTP-binding protein CDC42Hs translocates to the
cytoskeleton in aggregating platelets. From comparisons with the
electrophoretic mobility of the post-translationally processed and the
unprocessed forms of CDC42Hs, we suggest that the prenylated form of
the protein associates with the cytoskeleton. Mutational analysis in
yeast suggested that the CDC42Sc (Saccharomycescerevisiae) protein, which is 80% identical to the human
CDC42Hs protein, is involved in cytoskeletal organization, but so far
no direct interaction with the cytoskeleton could be demonstrated. The
increase in cytoskeletal CDC42Hs during platelet aggregation was
accompanied by a decrease in membrane skeletal CDC42Hs. The calculated
decrease in soluble CDC42Hs (2-4%) was too small to be
detectable, since 90% of CDC42Hs was in the cytosolic fraction. Thus,
CDC42Hs might have translocated from the membrane skeleton and from the
cytosol to the cytoskeleton. Interestingly, two proteins, RhoGDI and
CDC42HsGAP, which interact with CDC42Hs were both not found in the
cytoskeleton of aggregated platelets. These regulatory proteins will
keep CDC42Hs in its inactive GDP-bound form; RhoGDI is known to form
stable complexes with the GDP-bound form of Rho proteins (47, 48) and inhibits GDP dissociation, and CDC42HsGAP
stimulates GTP hydrolysis (24, 49) . The fact that
both proteins were still present in the cytosol of aggregated platelets
indicates that CDC42Hs (if translocating from the cytosol to the
cytoskeleton) must have dissociated from RhoGDI and is also most likely
not accessible to inactivation by CDC42HsGAP. Thus, cytoskeletal
CDC42Hs might be present in its active, GTP-bound form. Recently,
GTP-bound CDC42Hs has been reported to interact specifically with the
protein-tyrosine kinase p120
We observed no
association of CDC42Hs with the cytoskeleton during platelet shape
change. This initial platelet response is characterized by a specific
reorganization of the cytoskeleton, stimulation of actin
polymerization, and increased content of actin, myosin, and
actin-binding protein in the cytoskeleton(17, 52) .
Hence it is unlikely that CDC42Hs plays a role in the rearrangement of
the cytoskeleton during this initial platelet response, which is also
associated with the activation of the integrin
The
finding that the GTPase Rac cotranslocated with CDC42Hs might indicate
a common target protein in the cytoskeleton. Both CDC42Hs and Rac bind
to and activate the serine/threonine kinase p65
It
has been shown that the binding of fibrinogen to the integrin
We observed that the cytoskeletal association of CDC42Hs
protein upon prolonged stimulation with TRAP was reversible. CDC42Hs
completely disappeared from the cytoskeleton after 12 min of
stimulation, whereas aggregation was decreased only by 35%. CDC42Hs
disappearance from the cytoskeleton was accompanied by a corresponding
increase in the membrane skeleton; hence, proteolysis of CDC42Hs as a
cause for the complete disappearance from the cytoskeleton could be
excluded. We also did not observe any stimulation of platelet calpain,
which is known to degrade cytoskeletal proteins such as talin and
actin-binding protein. We found that cytoskeletal actin content was
decreased upon prolonged agonist stimulation, which might be related to
the reversible association of CDC42Hs with the cytoskeleton.
In
summary, we have shown that CDC42Hs incorporation into the cytoskeleton
is a specific event that is mediated by
We thank John A. Glomset for helpful discussions, Alan
Hall for providing the CDC42HsGAP cDNA, Ulrike Korf for preparation of
the chicken anti-CDC42HsGAP antibody, and F. Haag for the skillful
photographic reproduction of the autoradiographs.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
integrin and platelet aggregation,
cytoskeletal association of CDC42Hs was abolished. Translocation of
CDC42Hs to the cytoskeleton but not aggregation was also prevented by
cytochalasins B or D or the protein tyrosine kinase inhibitor
genistein. Platelet secretion and thromboxane formation were not
required but facilitated the cytoskeletal association of CDC42Hs. The
results indicate that in platelets stimulated by TRAP or ADP, a
fraction of CDC42Hs translocates from the membrane skeleton to the
cytoskeleton. This process is reversible and is mediated by activation
of the
integrin and subsequent
actin polymerization and protein-tyrosine kinase stimulation. CDC42Hs
might be a new component of a signaling complex containing specific
cytoskeletal proteins and protein-tyrosine kinases that forms after
activation of the
integrin in
platelets.
integrin, which serves as fibrinogen receptor, is not available
for binding by fibrinogen. The
integrin has to be activated before fibrinogen can bind to it.
Activation of the
integrin is
regulated by specific signal transduction mechanisms and alterations of
the cytoskeleton initiated during platelet shape change (reviewed in (22) ). Subsequently, fibrinogen binds to the
integrin, leading to further
cytoskeletal rearrangements and platelet aggregation (reviewed in (23) ). Thus, platelets may represent an ideal model system to
study the function of CDC42Hs in mammalian cells. In this study we
demonstrate that CDC42Hs translocates from the membrane skeletal and
cytosolic fractions of the cell to the actin-rich cytoskeleton
following platelet aggregation. The CDC42Hs translocation is mediated
by activation of the
integrin and
is dependent on actin polymerization and protein-tyrosine kinase
activation.
Materials
Rabbit polyclonal antibodies were
raised against peptides corresponding to amino acids 167-183 and
17-28 of the sequences of CDC42Hs and RhoGDI,(
)respectively, and affinity-purified on columns
containing the respective peptides coupled to SulfoLink gel
(Pierce)(14) . Chicken polyclonal antibodies were raised
against a glutathione S-transferase fusion protein of
CDC42HsGAP(24) . The specificities of the antibodies against
CDC42Hs and CDC42HsGAP were tested on Western blots of pure recombinant
CDC42Hs, Rac1 or RhoA, or CDC42HsGAP. Pure recombinant proteins were
prepared by expression as glutathione S-transferase fusion
proteins in Escherichia coli and cleavage with thrombin (25) . Antibodies against Rac1, Rac2, and Rac1/Rac2 were from
Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal antibodies
PY20 and Z027 against phosphotyrosine were purchased from Zymed
Laboratories (San Francisco, CA). The horseradish peroxidase-conjugated
secondary antibodies were from Amersham Corp. Apyrase, ADP, RGDS,
acetylsalicylic acid, sodium orthovanadate, fetal bovine serum, Triton
X-100, leupeptin, pepstatin A, aprotinin, phenylmethylsulfonyl
fluoride, cytochalasin B, and cytochalasin D were from the Sigma. Tween
20 and the reagents for electrophoresis were obtained from Bio-Rad.
Genistein and daidzein were obtained from Biomol (Hamburg, Germany).
The chemiluminescence-based Western blot detection system ECL was from
Amersham Corp. The thrombin receptor-activating peptide (TRAP) (SFLLRN)
was custom synthesized by Dr. Arnold (Max Planck Institute,
Martinsried, Germany).
Preparations of Washed Platelets
Washed human
platelets were prepared as described previously(26) .
Platelet-rich plasma was obtained from 200 ml of freshly drawn human
blood, anticoagulated with 0.1 volume of 3.8% trisodium citrate and
centrifuged at 180 g for 20 min. Unless otherwise
stated, platelet-rich plasma was incubated with 1 mM acetylsalicylic acid for 15 min at 37 °C. Citric acid (9
mM) and EDTA (5 mM) were added, and platelets were
pelleted by centrifugation at 800
g for 15 min. They
were resuspended in 3 ml of washing buffer (20 mM Hepes, 138
mM NaCl, 2.9 mM KCl, 1 mM MgCl
,
0.36 mM NaH
PO
, and 1 mM EGTA,
supplemented with 5 mM glucose and 3 ADPase units/ml of
apyrase, pH 6.2) and diluted to 30 ml with the same buffer but
containing 0.6 ADPase units of apyrase/ml. Platelets were pelleted and
resuspended in 20 ml of the resuspension buffer (pH 7.4), which was the
same as the washing buffer but without EGTA and apyrase. The final
platelet concentration was adjusted to 1
10
/ml.
Platelet Activation and Aggregation
Aliquots (1.5
ml) of washed platelets were incubated with stirring (1800 rpm) at 37
°C in a LABOR aggregometer (Fresenius, Bad Homburg, Germany) for 1
min prior to the addition of TRAP (10 µM). CaCl (50 µM) was added 15 s before TRAP. Aggregation was
measured by the percentage change of the light transmission. In some
experiments, aggregation was prevented by (a) incubating the
platelets at 37 °C in the absence of stirring, (b)
preincubating platelets with EGTA (2 mM) for 20 min at 37
°C, or (c) preincubating platelets with RGDS (4
mM) for 1 min at 37 °C prior to stimulation with TRAP. In
other experiments, platelets were pretreated with cytochalasin B (20
µM), cytochalasin D (2 µM), or
Me
SO (0.2%, control) at 37 °C for 1 min prior to
exposure to TRAP. In further experiments, platelets were preincubated
with genistein (100 µM and 150 µM), daidzein
(100 µM and 150 µM), or Me
SO
(0.2%, control) for 2 min prior to the addition of TRAP.
Isolation and Analysis of Detergent Lysates of
Platelets
Cytoskeletal fractions were prepared by using a
modification of the method described by Phillips et al.(27) . Briefly, platelet suspensions (1.5 ml) were lysed
before or at various intervals after agonist addition by adding equal
volumes of ice-cold 2 Triton lysis buffer (pH 7.5) containing
2% Triton X-100, 100 mM Tris-HCl, 10 mM EGTA, 10
mM EDTA, 2 mM sodium orthovanadate, 21 µM leupeptin, 2 mM phenylmethylsulfonyl fluoride, 20
µM pepstatin A, and 0.56 trypsin inhibitor unit/ml
aprotinin. Samples (3 ml) were vortexed for 10 s, left on ice for 30
min, and spun at 15,600
g for 15 min at 4 °C in a
Kontron analytical centrifuge (type ZK 401). The pellets were washed
once without resuspension in 1
Triton X-100 (1%) lysis buffer.
The pellets were resuspended in 300 µl of 1
lysis buffer
plus 75 µl of 5
sample buffer (250 mM Tris-HCl,
8.2 g/100 ml SDS, 40% (v/v) glycerol, 0.05 g/100 ml bromphenol blue, 5%
(v/v) 2-mercaptoethanol, and 5 mM sodium orthovanadate, pH
6.8) and heated at 95 °C for 10 min. For immunoblotting with
anti-Rac antibodies the cytoskeletal pellet was resuspended in 200
µl of 1
lysis buffer plus 50 µl of 5
sample
buffer, followed by boiling.
g for 2.5 h at 4 °C in a
Beckman ultracentrifuge (L5-50) with a 70.1 Ti rotor. The pellets
were resuspended as above. The Triton-soluble supernatant fractions
were mixed at a ratio of 4:1 with 5
sample buffer and boiled.
Thus, the final detergent-insoluble samples (both cytoskeleton and
membrane skeleton preparations) were concentrated 10 times relative to
the Triton-soluble fractions.
1.5 ml) was mixed before and at various
times after agonist addition with equal volumes of 2
Triton
X-100 lysis buffer (see above), vortexed and centrifuged at 10,000
g for 5 min. The supernatants were discarded. The
pellets were washed twice with 1
Triton X-100 lysis buffer. The
final pellets were resuspended in 300 µl of 1
Triton X-100
lysis buffer plus 75 µl of 5
sample buffer and heated at 95
°C for 10 min.
Protein Gel Electrophoresis and Immunoblotting of
CDC42Hs, Rac1/Rac2, CDC42HsGAP, RhoGDI, and
Phosphotyrosine
Platelet proteins were separated by overnight
vertical electrophoresis of samples on 1.5-mm-thick and 20-cm-long
SDS-polyacrylamide (12%) slab gels (Protean II xi system, Bio-Rad) at a
constant current of 13 mA/gel. Proteins were electrophoretically
transferred to Immobilon-P polyvinylidene difluoride membranes
(Millipore Corp., Bedford, MA) by using the Nova Blot semidry system
(Pharmacia Biotech. Inc., Bromma, Sweden) and the procedure recommended
by the manufacturer. In some experiments proteins were separated and
electroblotted using the Mini Protean II system (Bio-Rad). Blocking of
residual sites on the membranes was effected by incubating the blots
for 1 h at room temperature with 20% (v/v) fetal bovine serum in 20
mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.3% Tween 20.
The blots were incubated for 1 h with one of the following primary
antibodies: (a) polyclonal anti-CDC42Hs antibody (1:500), (b) monoclonal anti-phosphotyrosine antibody PY20 and Z027
(1:2000 dilution each), (c) polyclonal antibodies against Rac1
(1:150), Rac2 (1:150), or Rac1/Rac2 (1:500), (d) polyclonal
anti-RhoGDI antibody (1:500), or (e) polyclonal
anti-CDC42HsGAP antibody (1:2000). Following washing, the blots were
incubated for 45 min with peroxidase-labeled donkey anti-rabbit IgG
(1:2500) for anti-CDC42Hs and anti-GDI primary antibodies, sheep
anti-mouse IgG (1:15,000) for the primary antibody against
phosphotyrosine, or rabbit anti-chicken IgG (1:20,000) for
anti-CDC42HsGAP primary antibody. After three washes of the blot,
antibody binding was detected using the enhanced chemiluminescence
(ECL) system (Amersham Corp.). Bands corresponding to CDC42Hs and
RhoGDI were measured by laser densitometry (Ultroscan XL, Pharmacia
Biotech Inc.).
Aggregation-dependent Reversible Translocation of
CDC42Hs and Rac but not of RhoGDI and CDC42HsGAP to the Platelet
Cytoskeleton
In unstimulated platelets CDC42Hs was present in
both the membrane skeleton and the supernatant fraction but was absent
from the cytoskeleton (Fig. 1). Laser densitometric evaluations
of several (n = 5) immunoblots indicated that 90
± 4% of total CDC42Hs was present in the supernatant fraction
whereas 9.6 ± 1.5% (mean ± S.D.) was in the membrane
skeleton. Upon platelet stimulation with 10 µM TRAP the
protein appeared in the cytoskeleton. The degree of cytoskeletal
association of CDC42Hs correlated with the extent of platelet
aggregation (Fig. 1); at 50% aggregation the CDC42Hs protein
band was barely detectable, and at 95% aggregation the association was
maximal. At maximal aggregation about 10% of total CDC42Hs translocated
to the cytoskeleton, whereas its decrease in the membrane skeleton
amounted to 6-8% of total CDC42Hs. No significant change of
CDC42Hs during platelet aggregation was observed in the soluble
fraction; the calculated decrease was 2-4% of total CDC42Hs.
The association of CDC42Hs with the platelet cytoskeleton was
reversible; on prolonged activation with TRAP CDC42Hs gradually
decreased and completely disappeared from the cytoskeleton after 12 min
and reappeared in the membrane skeleton (Fig. 2) while platelet
aggregation was reduced only by 35% (data not shown). These changes
were paralleled by changes in the distribution of actin that decreased
in the cytoskeleton and concomitantly increased in the membrane
skeleton and the soluble fraction.
We observed in the membrane
skeleton and the soluble fraction an additional protein band recognized
by the anti-CDC42Hs antibody that showed a slightly lower
electrophoretic mobility than the major CDC42Hs band. The major band
most likely represents the prenylated form of CDC42Hs because it
exactly comigrated with the CDC42Hs band of nucleated human U-937
cells, which are considered to contain almost exclusively the
prenylated form of proteins. The additional upper band might represent
the unprocessed CDC42Hs because it comigrated with the recombinant
CDC42Hs expressed in E. coli that lacks post-translational
modifications (Fig. 3). Interestingly, this putative unprocessed
CDC42Hs did not translocate to the cytoskeleton upon platelet
aggregation.
Rac GTPases are the closest homologs of CDC42Hs within
the Ras family, and at least one Rac isoform, Rac1, seems to be present
in platelets (30) . Using antibodies specific for Rac1 and Rac2
both proteins were found in human platelets, but more Rac1 than Rac2
immunoreactivity was detected (data not shown). Interestingly, Rac (12%
of total) translocated to the cytoskeleton in aggregating platelets
with a time course similar to CDC42Hs (Fig. 4).
RhoGDI, which
forms stable complexes with CDC42Hs, Rho, and Rac proteins in the
cytosol of cells(31, 32) , was exclusively confined to
the soluble fraction. Upon platelet aggregation RhoGDI did not
translocate to the cytoskeleton (Fig. 5). The GAP specific for
CDC42Hs also did not associate with the cytoskeleton in the aggregated
platelets. However, we observed a faint appearance of this protein in
the membrane skeletal fraction of the platelets aggregated for 90 s (Fig. 6).
The Redistribution of CDC42Hs Is Mediated by the
Activation of the
Platelet aggregation requires fibrinogen binding to the
membrane glycoprotein IIb-IIIa complex ( Integrin and
Is Dependent on Actin Polymerization and Protein-Tyrosine Kinase
Activity
integrin), which is activated during platelet activation and
shape change. To test the involvement of fibrinogen receptors in the
cytoskeletal association of CDC42Hs we stimulated platelets (a) in the absence of stirring, which prevents platelet
aggregation but allows fibrinogen binding to the
integrin, (b) after
preincubating platelets with EGTA at 37 °C to dissociate the
integrin, or (c) in the
presence of RGDS to inhibit fibrinogen binding. Fig. 7shows
that these conditions, which completely inhibited platelet aggregation
(data not shown), prevented cytoskeletal association of CDC42Hs. We
conclude from these data that the conformational change of the
integrin evoked by fibrinogen
binding is required but not sufficient for the translocation of CDC42Hs
to the cytoskeleton. Subsequent signaling events of the activated
fibrinogen receptor are necessary.
integrin activation. Washed human
platelets were exposed to TRAP (10 µM) for 90 s under the
following conditions: without stirring (lanes2 and 3), with stirring (lanes8 and 9),
after preincubation with EGTA (2 mM) for 20 min at 37 °C (lanes4 and 5), and after preincubation
with RGDS (4 mM) for 1 min at 37 °C (lanes6 and 7). EGTA and RGDS pretreatment completely inhibited
platelet aggregation (data not shown). Platelets were lysed with Triton
lysis buffer. Cytoskeleton and soluble fractions were isolated and
immunoblotted with an antibody against CDC42Hs as described. Lane1, recombinant CDC42Hs protein; lanes2, 4, 6, and 8, soluble
fractions; lanes3, 5, 7, and 9, cytoskeletal fractions. The experiment is representative of
three different experiments.
Platelet aggregation is
associated with an increase in actin polymerization and translocation
of the integrin as well as
regulatory proteins, such as the small GTP-binding proteins Rap1B (33) and Rap2B (34) , phosphatidylinositol
3-kinase(35, 36) , and pp60
(35, 37, 38) to the actin-rich
cytoskeleton. We asked next whether the increase of actin
polymerization is required for
-mediated association of CDC42Hs
with the cytoskeleton. When cells were pretreated with either
cytochalasin B or cytochalasin D, both inhibitors of actin
polymerization, before addition of TRAP, no suppression of platelet
aggregation was seen (Fig. 8A), despite an effective
reduction of actin in the cytoskeleton (Fig. 8B, bottom). The increase of actin in the cytoskeleton from 42.7%
in the resting platelets to 81.0% in TRAP-aggregated platelets was
reduced to 22.6 and 26.9% after treatment with cytochalasins B and D,
respectively. Cytochalasin treatment completely abolished the
cytoskeletal association of CDC42Hs with retention of the protein in
the membrane skeletal fractions (Fig. 8B, top). These results indicate that stimulation of actin
polymerization in aggregated platelets is required for the cytoskeletal
association of CDC42Hs.
SO (DMSO) (0.2%, control), cytochalasin B (20
µM), or cytochalasin D (2 µM) at 37 °C
for 1 min, followed by exposure to TRAP (10 µM). A, aggregation tracings of Me
SO- and cytochalasin (Cyt)-pretreated platelets stimulated by TRAP (arrow). B, soluble (SUP), membrane skeletal (MSK), and cytoskeletal (CSK) fractions were
subjected to electrophoresis and immunoblotting using an anti-CDC42Hs
antibody. The upperpart shows the laser
densitometric quantitation of CDC42Hs in the different fractions. The lowerpart shows the laser densitometric measurement
of actin in the Coomassie Blue-stained gel. Data are mean ± S.D. (n = 3).
Because it has been reported that
stimulation of protein tyrosine phosphorylation in platelets occurs
subsequently to the activation of the integrin (39, 40, 41) and the major
nonreceptor protein-tyrosine kinase,
pp60
, associates with the
cytoskeleton under similar conditions as does the CDC42Hs
protein(36, 37, 42, 43) , we asked
next whether inhibition of tyrosine kinase activity might affect the
translocation of CDC42Hs. Pretreatment of platelets with the
protein-tyrosine kinase inhibitor genistein (100 and 150
µM) inhibited the cytoskeletal association of CDC42Hs by
about 95% compared with the Me
SO-treated control platelets
aggregated to the same extent (Table 1). Pretreatment with
genistein effectively reduced the tyrosine phosphorylation of several
platelet proteins compared with Me
SO- or daidzein-treated
samples (data not shown), as observed
previously(44, 45, 46) . These observations
indicate a role for protein-tyrosine kinase activity in aggregated
platelets in inducing cytoskeletal association of CDC42Hs.
We
further studied whether secretion is also required for the association
of CDC42Hs with the cytoskeleton. Aggregation induced by a strong
agonist like TRAP is accompanied by the secretion of the contents of
platelet granules and dense bodies.
granules contain
various adhesion molecules (fibrinogen, fibronectin, and von Willebrand
factor) that bind to the
integrin
(for review, see (22) ). Platelet-rich plasma, pretreated with
the cyclooxygenase inhibitor aspirin, was stimulated with ADP. Under
these conditions strong aggregation occurs without secretion from
granules and dense bodies. We observed the translocation of CDC42Hs to
the cytoskeleton under these conditions, indicating that secretion was
not necessary for that effect (Fig. 9). However, ADP caused a
smaller percentage of CDC42Hs to translocate to the cytoskeleton than
TRAP (compare lanes5 and 8 with lanes9 and 10 in Fig. 9). Also aspirin-treated
platelets that were aggregated by ADP or TRAP contained less CDC42Hs in
the cytoskeleton than nontreated cells at 95% aggregation. The results
indicate that secretion and prostaglandin endoperoxides/thromboxane
A
are not necessary, but enhance the association of CDC42Hs
with the cytoskeleton.
and the
serine/threonine protein kinase
p65
(50, 51) .
. Platelet aggregation is
associated with further specific changes in the
cytoskeleton(27) , most importantly the translocation of the
integrin
from the membrane
skeleton to the
cytoskeleton(27, 53, 54, 55) .
Activation of the integrin
has
been shown to cause other components of the membrane skeleton to
undergo increased association with the cytoskeleton. To these
components belong the cytoskeletal proteins talin and vinculin (36, 56) and the protein-tyrosine kinases
pp60
(36, 37, 56) and
pp62
(56) . The integrin
-dependent translocation of CDC42Hs
from the membrane skeleton to the cytoskeleton shown in this study
indicates that CDC42Hs belongs to this complex of specific signaling
molecules that might regulate integrin-actin interaction.
(51) and the p85 subunit of phosphatidylinositol
3-kinase(57) . The latter enzyme also translocates to the
cytoskeleton of activated platelets (35, 36) and at
this location might be activated by the CDC42Hs or Rac proteins.
results in increased tyrosine
phosphorylation of several proteins in stimulated
platelets(39, 40, 41, 58, 59, 60, 61) .
We observed that the cytoskeletal association of CDC42Hs in aggregated
platelets could be prevented by inhibition of actin polymerization with
cytochalasin B and D and inhibition of protein-tyrosine kinase
activity. Cytochalasin D has been reported to inhibit translocation of
pp60
to the cytoskeleton (42) and to reduce tyrosine phosphorylation of several platelet
proteins(42, 61, 62) . Therefore,
protein-tyrosine phosphorylation appears to occur subsequently to actin
polymerization and pp60
translocation in aggregated platelets. Our results showing
that both cytochalasin and genistein blocked CDC42Hs association with
the cytoskeleton indicate that (a) actin polymerization and (b) protein-tyrosine kinase activity are required for this
process. It is possible that a protein-tyrosine kinase translocates to
the actin cytoskeleton and phosphorylates itself or a cytoskeletal
protein, thereby providing docking sites for CDC42Hs. A tyrosine kinase
(p120
) that binds to GTP-bound CDC42Hs and has
some homology to the focal adhesion kinase was detected recently (50) . The possibility that CDC42Hs could be
tyrosine-phosphorylated was raised previously(63) , but we did
not find tyrosine-phosphorylated CDC42Hs in aggregated platelets (data
not shown).
integrin activation and is dependent on both actin polymerization
and tyrosine kinase activity. Platelet secretion and thromboxane
formation are not required but facilitate cytoskeletal CDC42Hs
association. CDC42Hs might play a role in the reorganization of the
platelet cytoskeleton observed during platelet aggregation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.