Regulation of endothelial cell myosin light chain kinase by
Rho, cortactin, and
p60src
Joe G. N.
Garcia1,
Alexander D.
Verin1,
Kane
Schaphorst1,
Rafat
Siddiqui2,
Carolyn E.
Patterson3,
Csilla
Csortos3, and
Viswanathan
Natarajan1
1 Division of Pulmonary and
Critical Care Medicine, Johns Hopkins University School of Medicine,
Baltimore, Maryland 21224;
3 Richard Roudebush Veterans
Affairs Medical Center, Indianapolis 46202; and
2 Methodist Research Institute of
Indiana, Indianapolis, Indiana 46206
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ABSTRACT |
Inflammatory
diseases of the lung are characterized by increases in
vascular permeability and enhanced leukocyte infiltration, reflecting
compromise of the endothelial cell (EC) barrier. We examined potential
molecular mechanisms that underlie these alterations and assessed the
effects of diperoxovanadate (DPV), a potent tyrosine kinase activator
and phosphatase inhibitor, on EC contractile events. Confocal
immunofluorescent microscopy confirmed dramatic increases in
stress-fiber formation and colocalization of EC myosin light chain
(MLC) kinase (MLCK) with the actin cytoskeleton, findings consistent
with activation of the endothelial contractile apparatus. DPV produced
significant time-dependent increases in MLC phosphorylation that were
significantly attenuated but not abolished by EC MLCK inhibition with
KT-5926. Pretreatment with the Rho GTPase-inhibitory C3 exotoxin completely abolished
DPV-induced MLC phosphorylation, consistent with Rho-mediated MLC
phosphatase inhibition and novel regulation of EC MLCK activity.
Immunoprecipitation of EC MLCK after DPV challenge revealed dramatic
time-dependent tyrosine phosphorylation of the kinase in association
with increased MLCK activity and a stable association of MLCK with the
p85 actin-binding protein cortactin and
p60src. Translocation of
immunoreactive cortactin from the cytosol to the cytoskeleton was noted
after DPV in concert with cortactin tyrosine phosphorylation. These
studies indicate that DPV activates the endothelial contractile
apparatus in a Rho GTPase-dependent fashion and suggests that
p60src-induced tyrosine
phosphorylation of MLCK and cortactin may be important features of
contractile complex assembly.
Src kinases; myosin phosphorylation; endothelial cell contraction; permeability
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INTRODUCTION |
THE ROLE of Ser/Thr phosphorylation in the development
of smooth muscle and nonmuscle contraction is well accepted (1, 5). Agonist-mediated increases in
Ca2+/calmodulin (CaM) availability
produces myosin light chain (MLC) kinase (MLCK)-mediated MLC
phosphorylation on Ser19 and
Thr18, resulting in actomyosin
cross-bridge cycling and tension development. Garcia and colleagues
(10, 13) have previously demonstrated that the
physiological consequences of increased MLC phosphorylation in cultured
vascular endothelium include endothelial cell (EC) contraction and the
formation of paracellular junctional gaps. This loss of integrity of
the semiselective EC barrier is now well known to facilitate the
development of two cardinal features of tissue inflammation: leukocyte
diapedesis and increases in vessel permeability (9, 10, 15, 18, 19,
28). To better understand the regulation of EC
contraction, we recently cloned a nonmuscle
Ca2+/CaM-dependent EC MLCK isoform
with a molecular mass (214 kDa) that is significantly
greater than the conventional smooth muscle MLCK isoforms (130-160
kDa) (12, 48, 49). The activity of this key effector is regulated not
only by Ca2+/CaM availability but
by Ser/Thr phosphorylation of the enzyme as well (10, 12, 14, 49).
Similar to the involvement of both EC MLCK and myosin-associated
phosphatase activities in determining the extent of MLC phosphorylation in endothelium, the steady-state level of phosphotyrosine in most cellular proteins is a dynamic balance between the relative catalytic activities of intracellular protein tyrosine kinases and phosphatases. Although the precise role of tyrosine phosphorylation in the
development of smooth muscle tension and force is incompletely
understood (3, 6-8, 51), in prior work, Shi et al. (45) described the capacity of a tyrosine kinase inhibitor, genistein, to attenuate thrombin-induced tyrosine kinase activity,
Ca2+ transients, MLC
phosphorylation, and EC barrier dysfunction, suggesting a role for
tyrosine kinase activity in EC barrier regulation. Unfortunately,
neither the tyrosine kinase involved nor the relevant targets in this
model were precisely identified, although indirect evidence suggested
that a genistein-sensitive Src family tyrosine kinase may
regulate EC MLCK activity (45). The speculation that EC MLCK activity
may be regulated by tyrosine phosphorylation was supported by companion
studies that employed vanadate, an inhibitor of tyrosine phosphatases,
to explore the contribution of tyrosine phosphorylation to EC barrier
regulation (16). Vanadate treatment directly increased MLC
phosphorylation in the absence of a rise in cytosolic
Ca2+ and also directly perturbed
EC barrier function (16). In the present study, we have extended these
observations by examination of the participation of protein tyrosine
phosphorylation in the activation of the EC actomyosin cytoskeleton.
For these studies, we utilized the cell-permeable tyrosine kinase
activator and tyrosine phosphatase inhibitor diperoxovanadate (DPV),
which has been identified as the major peroxovanadium compound
generated when equimolar amounts of
H2O2
and sodium ortho- or metavanadate are mixed at a neutral pH (4, 17, 21,
37). Our results indicate that DPV evokes significant EC MLCK
phosphotyrosine accumulation, increased EC MLCK activity, and EC
contraction. These events appear to involve the activation of
p60src, which exists in stable
association with EC MLCK and the EC cytoskeleton and catalyzes the
phosphorylation of EC MLCK as well as the p85 actin-binding protein
cortactin. Furthermore, Rho GTPases appear to be major
participants in determining the final level of DPV-induced MLC
phosphorylation and contraction. Together, these data indicate the
potential participation of Rho GTPases,
p60src, and cortactin in the
assembly of a functional MLCK complex and in EC contractile regulation.
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METHODS |
Reagents. Bovine EC cultures were
maintained in DME (GIBCO BRL, Chagrin Falls, OH) supplemented with 20%
(vol/vol) colostrum-free bovine serum (Irvine Scientific, Santa Ana,
CA), 15 µg/ml of EC growth supplement (Collaborative Research,
Bedford, MA), a 1% antibiotic-antimycotic solution (10,000 U/ml of
penicillin, 10 µg/ml of streptomycin, and 25 µg/ml of amphotericin
B; KC Biologicals, Lenexa, KS), and 0.1 mM nonessential amino acids
(GIBCO BRL). Unless specified, reagents were obtained from Sigma (St.
Louis, MO). Phosphate-buffered saline (PBS) and Hanks' balanced salt solution without phenol red were purchased from GIBCO BRL (Grand Island, NY). Sodium orthovanadate
(Na3VO4;
vanadate) and hydrogen peroxide
(H2O2)
were obtained from Fisher Scientific (Fair Lawn, NJ).
C3 exotoxin derived from
Clostridium difficile was purchased from List (Campbell, CA). DPV was prepared as previously described (30). Polyacrylamide gradient 4-15% ready-to-use gels were
purchased from Bio-Rad (Hercules, CA).
Bovine and human pulmonary EC
cultures. Bovine pulmonary arterial ECs were obtained
frozen at 16 passages from American Type Culture Collection (Manassas,
VA), utilized at passages
19-24, and
cultured in complete medium (9). Human pulmonary microvascular endothelium was purchased from Clonetics (San Diego, CA) and utilized at passages
3-8 (48). The EC
cultures were maintained at 37°C in a humidified atmosphere of 5%
CO2-95% air and grew to
contact-inhibited monolayers, with typical cobblestone morphology.
Cells from each primary flask were detached with 0.05% trypsin,
resuspended in fresh culture medium, and passaged into 60-mm dishes for
immunoprecipitation studies, tyrosine protein phosphorylation
determination, or MLC phosphorylation studies.
MLC phosphorylation in intact
endothelium. EC monolayers grown in 60-mm tissue dishes
were analyzed for MLC phosphorylation by urea PAGE as previously
described by Garcia et al. (10), followed by Western immunoblotting
with specific anti-MLC antibodies. The blot was scanned on a Bio-Rad
densitometer, and the percent MLC phosphorylation was determined by
dividing the total of the phosphorylated and nonphosphorylated areas.
This method takes advantage of the fact that the mono- and
diphosphorylated forms of MLC migrate more rapidly than
nonphosphorylated MLC and are independent of sample loading.
Stoichiometry (in mol/mol) was calculated with the formula
[P1 + 2(P2)]/(U + P1 + P2), where U is the percent
unphosphorylated EC MLC, P1 is the
percent monophosphorylated MLC, and
P2 is the percent diphosphorylated
MLC. The diphosphorylated MLC is multiplied by a factor of 2 to reflect
the presence of two phosphate groups per light chain. The percentage of
light chain phosphorylation was calculated by adding the densitometric values of each phosphorylation state for each isoform, i.e.,
unphosphorylated, monophosphorylated, and diphosphorylated.
Western immunoblotting. Total protein
extracts or immunoprecipitates solubilized in sample buffer were
separated by 4-15% gradient SDS-PAGE (Bio-Rad), transferred to
nitrocellulose (18 h at 30 V), and reacted with either antibodies to
MLCK (D119) or anti-phosphotyrosine antibodies (Upstate
Biotechnology) as previously described (14, 16, 49).
Immunoreactive proteins were detected with an enhanced
chemiluminescence detection system according to the manufacturer's
instructions (Amersham).
MLCK immunoprecipitation. For
immunoprecipitation under nondenaturing conditions, confluent ECs from
60-mm dishes were rinsed with medium 199 and two times with PBS, then
lysed for 20 min on ice with 300 µl of Nonidet P-40 (NP-40) lysis
buffer (1% NP-40, 20 mM MOPS, pH 7.0, 25 mM
MgCl2, 10% glycerol, and 0.5 mM
EGTA) containing protease inhibitors [40 µg/ml of aprotinin, 18 µg/ml of
N-tosyl-L-phenylalanine
chloromethyl ketone, 6 µg/ml of
N
-p-tosyl-L-lysine chloromethyl ketone (TLCK), and 0.5 mM
phenylmethylsulfonyl fluoride] (12, 16). The lysate was scraped
and microcentrifuged for 5 min at 4°C, and the supernatant was used
for immunoprecipitation. Each sample was diluted with 700 µl of
washing buffer (0.1% NP-40, 50 mM MOPS, pH 7.0, 25 mM
MgCl2, and 1 mM EDTA) and
incubated overnight with 2 µl of anti-MLCK antibodies D119 at 4°C
followed by incubation for 1 h at 4°C with 30 µl of 10%
Pansorbin suspension [Formalin-hardened and heat-killed Cowan 1 strain Staphylococcus aureus cells
purchased from Calbiochem (La Jolla, CA)]. The immunoprecipitated complex was harvested by microcentrifugation, washed three times with
washing buffer, and used for MLCK activity measurement (see Determination of MLCK
activity) or resuspended in 200 µl of
Laemmli sample buffer and heat treated at 110°C for 5 min.
Immunoprecipitated proteins were separated from the Pansorbin beads by
microcentrifugation for 1 min and subjected to Western immunoblotting
analysis with specific antibodies to contractile proteins.
For immunoprecipitation under denaturing conditions, confluent EC
monolayers in 60-mm tissue culture dishes were rinsed twice with 2 ml
of medium, further rinsed with 2 ml of PBS, and scraped into 100 µl
of SDS-denaturing stop solution (PBS, pH 7.4, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 10 mM sodium pyrophosphate, 0.2 mM orthovanadate,
1% SDS, and 14 mM
-mercaptoethanol) (49, 50). The homogenate was
prepared by passing the cell suspension several times through a
16-gauge needle. Homogenates were heat treated at 110°C for 5 min,
diluted 1:10 with 900 µl of PBS, and incubated with 50 µl of 10%
Pansorbin for 30 min at room temperature. Samples were clarified by
microcentrifugation (5 min; Eppendorf), and the supernatants were
incubated with 10 µl of anti-MLCK antibodies (60 min at room
temperature or overnight at 4°C), then with 50 µl of 10%
Pansorbin suspension for 60 min at room temperature. Immunocomplexes
were pelleted by microcentrifugation for 5 min, washed three times with
1 ml of PBS, separated from Pansorbin by microcentrifugation, and
subjected to SDS-electrophoresis (16, 27). After electrophoresis, the
proteins were transferred to nitrocellulose membranes, and signals were
detected by immunostaining with anti-phosphotyrosine antibodies.
Determination of MLCK activity. Kinase
activity present in nondenaturing MLCK immunoprecipitates was
determined as described by Garcia et al. (12), Gilbert-McClain et al.
(16), and Hefletz et al. (17). Immunocomplexes were resuspended in 110 µl of 50 mM MOPS, pH 7.4, 10 mM magnesium acetate, 1 mg/ml of BSA,
and 8 mM
-mercaptoethanol and preincubated with and without the
specific MLCK inhibitor KT-5926 (10 µM) for 15 min at 25°C.
Kinase activity in MLCK immunoprecipitates was measured with
baculovirus-expressed and His-Tag purified smooth muscle MLC (1 mg/ml)
as a substrate in a buffer consisting of 50 mM MOPS, pH 7.4, 10 mM
magnesium acetate, 1 mg/ml of BSA, 1 µM CaM, 0.1 mM
[
-32P]ATP (1 Ci/mmol), and 0.3 mM CaCl2 for 30 min at 25°C. The kinase reaction was stopped by pipetting aliquots
onto Whatman P81 filters and immediately rinsing with ice-cold 10%
TCA, 2% (wt/vol) sodium pyrophosphate, and 95% ethanol.
Finally, filters were rinsed in ethyl ether, dried, and counted by
liquid scintillation counting. Specific MLCK activity was defined in
our assay conditions as the total kinase activity in nondenaturing MLCK
immunoprecipitates sensitive to the specific MLCK inhibitor KT-5926
(12).
EC detergent fractionation. Confluent
EC monolayers were partitioned into subcellular fractions with a
protocol that is based on the detergent extractability of cell proteins
under increasingly stringent conditions (39). Briefly,
agonist-challenged cells grown to confluence in 100-mm dishes were
rinsed with ice-cold PBS to remove nonadherent cells and then incubated
for 10 min at 4°C in 1,500 µl of buffer
A [0.01% digitonin, 10 mM PIPES, 300 mM sucrose,
100 mM NaCl, 3 mM MgCl2, 5 mM
EDTA, and a protease inhibitor cocktail (0.5 mM phenylmethylsulfonyl
fluoride, 2 mM benzamidine, 1 mM TLCK, and 25 µg/ml of leupeptin), pH
6.8]. The supernatant was harvested (cytosolic
extract A), and adherent cell ghosts
were next washed with 3 ml of buffer A
and incubated for 20 min at 4°C in buffer
B consisting of 0.5% (vol/vol) Triton X-100, 10 mM
PIPES, pH 7.4, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 3 mM EDTA, and a protease
inhibitor cocktail. The extract was harvested (Triton X-100-soluble
extract B), and residual cellular material was next washed with 3 ml of buffer
B and then scraped into 450 µl of
buffer C (1% Tween 40, 0.5%
deoxycholate, 10 mM PIPES, pH 7.4, 10 mM NaCl, 1 mM
MgCl2, and a protease inhibitor cocktail). The cellular material was suspended by probe sonication on
ice three times for 10 s and extracted with constant rotation at
4°C for 20 min. The insoluble elements were collected by
centrifugation, and the supernatant was harvested (Triton
X-100-insoluble extract C). The
remaining insoluble cytoskeleton was washed with
buffer C and suspended in 1%
(wt/vol) SDS-20 mM Tris · HCl, pH 6.8, by probe sonication and rapidly boiled (5 min), divided into aliquots, and stored (cytoskeletal extract D).
Immunofluorescence. The fluorescent
imaging of EC gap formation and F-actin organization was performed on
EC monolayers grown to confluence on glass coverslips as previously
described by Schaphorst et al. (44) and Verin et al. (49). After
treatment, the cells were fixed by exchanging medium with 5%
paraformaldehyde, 50 µM phosphate, 75 mM NaCl, and 25 mM Tris, pH 7, on ice for 10 min. The cells were thoroughly rinsed with buffer
containing 150 mM NaCl and 50 mM Tris, pH 7.6, and then permeabilized
by a 3.5-min treatment with 0.2% Triton X-100 in the rinse buffer. The
cells were again rinsed three times and incubated at room temperature for 1 h with 1% BSA in the rinse buffer and then with 1 U/ml of rhodamine phalloidin (Molecular Probes, Eugene, OR) to identify F-actin. Time-dependent changes in intracellular distribution of the
actin cytoskeleton after a 5 µM DPV challenge were analyzed on a
Zeiss axioplan fluorescent microscope. To study colocalization of actin
and MLCK, the fixed permeabilized cells were exposed overnight at
4°C to a 1:50 dilution of anti-EC MLCK antibody (V-368) (12) in BSA buffer. This antibody was generated against
the peptide GEERKRP present in the unique
NH2-terminal part of EC MLCK
(49). After being rinsed to remove unbound primary antibody, the
cells were incubated for 1 h at room temperature with labeled secondary
antibody (30 mg/ml; FITC-conjugated donkey anti-rabbit IgG, Jackson
Immuno- Research, West Grove, PA) and rhodamine phalloidin. The cells
were examined with a ×60 oil objective with the Bio-Rad MLC 1024 confocal microscope and excitation with a Ar-Kr laser at 568-nm
excitation and 598-nm emission for rhodamine and 488-nm excitation and
522-nm emission for FITC at a 3-mm aperture. Data were collected for
7-17 planar sections at 0.5-µm intervals by Bio-Rad LaserSharp
acquisition software, processed by MetaMorph Imaging software
(Universal, West Chester, PA), and printed on a thermal dye diffusion
printer (Kodak, Rochester, NY). EC monolayers that were not exposed to
primary antibody did not stain with the secondary antibody.
 |
RESULTS |
Effect of DPV on EC myosin phosphorylation and MLCK
activity. To assess the linkage between DPV stimulation
and the development of a contractile EC phenotype, initial experiments
measured the capacity of DPV to increase the level of phosphorylated
MLCs. Confluent EC monolayers were stimulated for specified periods, and the stoichiometry of MLC phosphorylation was determined by densitometric scanning of the immunoblots of MLCs separated by urea gel
electrophoresis. Figure
1A
demonstrates that the combination of 10 µM
H2O2
and 10 µM vanadate to generate DPV potently increases EC MLC
phosphorylation, whereas neither
H2O2
alone (10 µM) nor vanadate alone (10 µM) altered the basal level of
MLC phosphorylation (stoichiometry of ~0.4 mol phosphate/mol MLC).
The maximal stoichiometric increase produced by DPV was ~1.8 mol
phosphate/mol MLC achieved with a 30-min stimulation with 10 µM DPV.
Temporal analysis demonstrated that the increase in MLC phosphorylation
after DPV occurs as early as 5 min (Fig.
1B).


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Fig. 1.
Effect of diperoxovanadate (DPV) on bovine endothelial cell (EC) myosin
light chain (MLC) phosphorylation. A:
representative immunoblot of phosphorylated [monophosphorylated
(Mono-P) and diphosphorylated (Di-P)] and nonphosphorylated
(Non-P) MLC species from confluent bovine pulmonary EC monolayers
challenged with vehicle or agonist (n = 3) and separated by urea gel electrophoresis as described in
METHODS. Lane
1, vehicle [control (C); 15 min];
lane 2, 100 nM thrombin [Thr; 2 min (2')]; lane 3, 10 µM
vanadate (Van); lane 4, 10 µM
H2O2;
lane 5, 10 µM
H2O2
and 10 µM vanadate [DPV; all for 15 min (15')].
Similar to Thr, combination of
H2O2
and vanadate but neither agent alone dramatically increased MLC
phosphorylation. B: representative
immunoblot indicating time-dependent increase in MLC phosphorylation
after DPV challenge. Bovine EC monolayers were treated with DPV at
indicated time periods (in min).
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The level of MLC phosphorylation represents the balance between EC MLCK
and myosin-associated phosphatase activities (50). To explore the
contribution of these components to DPV-stimulated MLC phosphorylation,
we pretreated EC monolayers with KT-5926, a well-described MLCK
inhibitor (10, 12). Inhibition of EC MLCK with KT-5926 (4 µM)
resulted in marked attenuation of DPV-mediated MLC phosphorylation
(Fig. 2), consistent with DPV-induced EC
MLCK activation. We next immunoprecipitated EC MLCK from endothelium challenged with either vehicle, thrombin, or DPV and assessed in vitro
kinase activity at specific times. Table 1
demonstrates a significant enhancement of MLCK activity after treatment
with DPV, with maximal activity noted at 10 min, a time frame
consistent with prior results by Gilbert-McClain et al. (16) that
examined in vitro MLCK activity after vanadate stimulation.

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Fig. 2.
Effect of MLC kinase (MLCK) inhibitor KT-5926 on DPV-mediated MLC
phosphorylation. MLCK inhibitor KT-5926 (4 µM) was utilized to assess
whether DPV-mediated MLC phosphorylation involved activation of EC
MLCK. Values are pooled data from 5 separate experiments in which
KT-5926 was applied to EC monolayers for 15 min before DPV challenge.
Analysis of MLC profiles with scanning densitometry revealed a
significant inhibitory effect of KT-5926 on basal and DPV-induced MLC
phosphorylation. Significant difference from: * control value;
# DPV-stimulated value.
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Effect of DPV on EC MLCK translocation to the actin
cytoskeleton. Having biochemically established that DPV
activates EC MLCK and increases MLC phosphorylation, we next examined
the effect of DPV on the endothelial contractile apparatus and
actomyosin cytoskeletal architecture by immunofluorescence confocal
microscopy. Similar to thrombin and consistent with the presence of a
contractile phenotype, DPV increases F-actin-based stress-fiber
formation and time-dependent reorganization of the F-actin-containing
dense peripheral band normally present in a circumferential
distribution in resting endothelium (Fig.
3) (9, 49). In unstimulated endothelium, immunoreactive MLCK was primarily cytosolic and did not
colocalize with polymerized actin. Within 10 min after DPV challenge,
however, there was a marked increase in stress-fiber assembly and
polymerized actin and significant colocalization of EC MLCK with
F-actin. This finding is consistent with EC MLCK translocation to the
actin cytoskeleton as previously described in thrombin-challenged
endothelium (49).

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Fig. 3.
DPV-induced EC MLCK colocalization with polymerized actin. Endothelium
cultured on glass coverslips was exposed to either vehicle (control;
left) or 5 µM DPV for 10 (middle) or 30 min
(right) and then fixed with 5%
paraformaldehyde. Cells were stained for F-actin with rhodamine
phalloidin as described in METHODS and
photographed with an 8-s exposure with a ×60 objective. Fixed
permeabilized cells were reacted with primary antibody to MLCK at a
1:50 dilution in Tris wash buffer with 1% BSA overnight at 4°C.
Rinsed cells were then stained with 1:50 FITC-conjugated donkey
anti-rabbit antibody and 1:200 rhodamine phalloidin for 1 h at room
temperature. Punctate staining of MLCK (green) at base of control cells
was observed. Usual circumferential actin network (red) was observed in
control endothelium, with minimal costaining of cortical actin bands
with MLCK. DPV induced time-dependent increases in F-actin staining,
organized into parallel bundles of stress fibers, and a marked increase
in MLCK and actin costaining as indicated by shift in MLCK staining
from predominantly green in control cells to yellow (mixture of red and
green fluorescence) after DPV challenge.
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Effect of Rho GTPase inhibition on MLC phosphorylation
in human and bovine endothelia. Having clearly
demonstrated DPV-mediated activation of the 214-kDa EC MLCK isoform, we
next examined the possible contribution of MLC phosphatase activity to
the DPV contractile response. Although Gilbert-McClain et al. (16) have
previously failed to identify a significant effect of millimolar
vanadate on total Ser/Thr phosphatase activity, the lack of complete
inhibition of DPV-mediated MLC phosphorylation with the MLCK inhibitor
KT-5926 (Fig. 2) suggested the possible contribution of Ser/Thr
phosphatase inhibition to the integrated DPV response. To assess this
possibility, we utilized the increasingly appreciated knowledge that
MLC phosphorylation levels are potently modified by Rho
GTPases via Rho kinase phosphorylation of the regulatory
subunit of MLC phosphatase (2). In cultured fibroblasts, both thrombin
and lysophosphatidic acid increase Rho kinase activity, stress-fiber
formation, and MLC phosphorylation. Figure
4A
demonstrates that, unlike fibroblasts, thrombin, but not
lysophosphatidic acid, increases MLC phosphorylation in human lung
microvascular ECs. We next pretreated human and bovine lung endothelia
with C3 exotoxin, a specific Rho
GTPase inhibitor derived from C. difficile. Figure 4 demonstrates that
C3 exotoxin totally abolishes
basal and both thrombin-mediated (Fig.
4B) and DPV-mediated (Fig.
4C) MLC phosphorylation. These
results indicate that thrombin- and DPV-mediated Rho activation
increases MLC phosphorylation and that in addition to MLC phosphatase
inhibition, one key target for this regulation is EC MLCK. Together,
these results indicate that DPV produces a contractile phenotype in
cultured endothelium involving Rho-regulated, MLCK-driven MLC
phosphorylation and subsequent actomyosin-mediated contraction.



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Fig. 4.
Effect of C3 exotoxin on Thr- and
DPV-stimulated MLC phosphorylation. A:
confluent bovine pulmonary arterial endothelial cells (BPAEC) and human
lung microvascular ECs (HLMVEC) were challenged with Thr for
2 min or lysophosphatidic acid (LPA) for 15 min and assessed for level
of phosphorylated MLC by urea gel electrophoresis. Unlike bovine
endothelium, human endothelium exhibits both smooth muscle and
nonmuscle isoforms, and this gives rise to 6 MLC species. Thr, but not
LPA, a known G protein activator in fibroblasts (32), produced a sharp
increase in phosphorylated MLC.
B and
C: bovine pulmonary arterial
endothelium was briefly pretreated with lipofectamine (10 µM) and
C3 exotoxin (C3 Exo or C3; 5 µg/ml) followed by replacement with lipofectamine-free medium
containing C3 Exo or vehicle [control (Cont)]. Thr (2 min;
B) or DPV (15 min;
C) was added, and MLC
phosphorylation profiles were analyzed. , Absence; +, presence.
These results indicate total inhibition of MLC phosphorylation (basal
and after agonists) by Rho GTPase inhibition. Similar results were
demonstrated in human microvascular endothelium after Thr and DPV (data
not shown).
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Effect of DPV on MLCK phosphotyrosine
accumulation. To more fully examine MLCK regulation, we
next assessed the level of EC MLCK phosphotyrosine accumulation
produced by DPV. DPV is known to dramatically increase the level of EC
tyrosine-phosphorylated proteins via profound enhancement of tyrosine
kinase activity and phosphatase inhibition (31). Figure
5 demonstrates the relatively rapid onset
of phosphotyrosine immunoreactivity of EC MLCK, with substantial
increases correlating with the extent of DPV-induced MLC
phosphorylation (Fig. 1B). This
DPV-mediated increase in both MLCK phosphotyrosine and MLCK activity,
combined with prior studies by Gilbert-McClain et al. (16) and Shi et
al. (45), suggests a strong mechanistic link between EC MLCK
phosphotyrosine status and enzymatic activity.

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Fig. 5.
Effect of DPV on EC MLCK tyrosine phosphorylation. Shown is a
representative experiment (n = 4)
where EC monolayers were treated with DPV (5 µM) at specified time
periods and MLCK immunoprecipitates were prepared under denaturing
conditions from DPV-stimulated (+) and control ( ) cells.
Resulting samples were subjected to a 4-15% gradient SDS-PAGE,
electrotransferred to nitrocellulose membranes, and reacted with
anti-phosphotyrosine antibodies followed by enhanced chemiluminescence.
Immunoprecipitation was performed with anti-MLCK D119 antibodies. No.
on left, molecular-mass marker. DPV
treatment increased total MLCK phosphotyrosine protein content in MLCK
immunoprecipitates.
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Detection of stably associated proteins in MLCK
immunoprecipitates. To further define the potential
regulation of EC contraction evoked by DPV, we next immunoprecipitated
EC MLCK under nondenaturing conditions and analyzed the proteins
retrieved in stable association with EC MLCK. Figure
6A depicts
the marked increase in phosphotyrosine immunoreactive proteins (range
29-214 kDa) present in MLCK immunoprecipitates after DPV
treatment. Immunoblotting of the stripped membranes with specific
antisera revealed p60src and the
p214 EC MLCK, a finding consistent with Fig. 2 as well as with a
previous report by Gilbert-McClain et al. (16) that EC MLCK is a
phosphotyrosine protein. To identify the strongly positive
phosphotyrosine p85 signal, EC MLCK immunoprecipitates were blotted
with antibodies directed against two candidate proteins with a
molecular mass known to be ~85 kDa:
1) the p85 regulatory subunit of
phosphatidylinositol 3-kinase, which has been shown to be tyrosine
phosphorylated after activation of several receptor tyrosine kinases
(35), and 2) cortactin, a
cytoskeleton-associated protein with two isoforms of 80 and 85 kDa,
which has been recently identified as a
p60src substrate in
thrombin-activated platelets and in v-Src-transformed cells (20, 33,
41, 52). Western blotting studies to detect the p85 regulatory subunit
of phosphatidylinositol 3-kinase were entirely negative (data not
shown); however, cortactin antisera readily detected the highly
immunoreactive 85-kDa phosphoprotein in MLCK immunoprecipitates. Figure
6B demonstrates that, under basal
conditions, cortactin immunoreactivity in nondenatured EC MLCK
immunoprecipitates is present as both the p80 and p85 isoforms. After
DPV stimulation, there is a significant increase in the level of the
p85 cortactin isoform present within MLCK immunoprecipitates in close
association with enhanced tyrosine phosphorylation of cortactin. Both
c-Src and cortactin immunoprecipitates under basal conditions yielded
both p80 and p85 cortactin isoforms, with an increase in the p85
cortactin isoform after DPV stimulation (Fig. 6B). Figure
7 demonstrates the rapid translocation of
p80 and p85 cortactin from the cytosol to the EC cytoskeleton after
DPV, with dramatic time-dependent increases in cortactin
phosphotyrosine accumulation beginning at 5 min. Together, these data
are consistent with the stable association of both
p60src and p80/85 cortactin with
EC MLCK and suggests a potential mechanism for regulation of EC MLCK by
tyrosine phosphorylation catalyzed by
p60src.

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Fig. 6.
DPV-mediated association of EC MLCK with
p60src and cortactin.
A: EC MLCK immunoprecipitates (I/P)
from bovine EC extracts were prepared under nondenaturing conditions as
described in METHODS and subjected to
a 4-15% gradient SDS-PAGE. This was followed by Western
immunoblotting with anti-phosphotyrosine (PTyr) antibodies,
demonstrating a large number of phosphoproteins under these
nondenaturing conditions. Membranes were then stripped and reprobed
with specific smooth muscle MLCK D119 antibodies,
anti-p60src, or anti-cortactin
antisera. Arrows, positions of corresponding contractile proteins and
rabbit IgG. These results indicate presence of an Src kinase
(p60c-src) and several Src
kinase substrates, including cortactin, in stable association with EC
MLCK. B: immunoprecipitates prepared
by utilizing MLCK antisera, c-Src antisera, or cortactin antisera under
nondenaturing conditions were reblotted with anti-p80/85 cortactin
antisera. An increase in immunoreactive cortactin after DPV (15 min) is
observed in each experiment, primarily in p85 species. Nos. on
left, molecular mass.
|
|

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of DPV on cortactin tyrosine phosphorylation. ECs grown to
confluence were challenged with 5 µM DPV for up to 30 min (30')
and then partitioned by serial detergent fractionation as described in
METHODS. Proteins present in
digitonin-extracted fraction (cytosol) and SDS-extracted fraction
(cytoskeleton) for each condition were separated on 8% acrylamide gels
by SDS-PAGE and electrophoretically transferred to nitrocelluose
followed by Western immunoblotting. Each lane represents 10 µg of
total protein. These data indicate rapid translocation of
immunoreactive cortactin (CA) from cytosol to cytoskeleton in concert
with alterations in phosphotyrosine immunoreactivity. Similarly, an
increase in cytoskeleton-associated actin is noted beginning 5 min
(5') after DPV and persists throughout period tested. Nos. on
left, molecular mass.
|
|
 |
DISCUSSION |
This study was undertaken to explore the poorly understood relationship
between tyrosine kinase or phosphatase activities and nonmuscle
contraction. The results reported are highly consistent with a major
role of tyrosine phosphorylation in the regulation of the EC
contractile apparatus, a process that appears to be regulated by Rho
GTPases and potentially driven by
p60src-regulated activity of a
novel high-molecular-mass EC MLCK isoform. Prior structure or function
studies (10, 14) indicated that activity of the
Ca2+/CaM-dependent EC MLCK isoform
is not solely regulated by
Ca2+/CaM availability. Additional
regulatory mechanisms have been postulated, including the subcellular
localization of the EC MLCK isoform and posttranslational modifications
such as Ser/Thr phosphorylation (12, 49). Although studies of the
nonmuscle MLCK isoform are limited, the vast majority of
studies dealing with phosphorylation of the smooth muscle
MLCK isoform have demonstrated an inhibitory effect of this
modification (46). In cultured endothelium, increases in cAMP-dependent
protein kinase A activity or Ser/Thr phosphatase inhibition results in
EC MLCK hyperphosphorylation in association with decreased MLCK
activity in MLCK immunoprecipitates (12, 49). Analysis of the EC MLCK
amino acid sequence reveals a number of consensus sites for Ser/Thr
phosphorylation catalyzed by a variety of enzymes including protein
kinase C, cAMP-dependent protein kinase A, cGMP-dependent protein
kinase, casein kinase II, and
Ca2+/CaM-dependent protein kinase
II (11, 49). Although the Ser/Thr phosphatase associated with EC MLCK
is, by implication, an essential regulator of MLCK activity, the exact
nature of this key enzyme remains incompletely understood. Importantly,
both prior reports by Gilbert-McClain et al. (16) and Shi et al. (45)
and current data indicate that phosphorylation of EC MLCK on tyrosine
residues, in contrast to the inhibitory effects of Ser/Thr
phosphorylation, may participate in EC MLCK regulation in a stimulatory capacity.
DPV is a particularly well-suited tool with which to study the
signaling cascades that regulate the EC contractile apparatus through
tyrosine phosphorylation. Via its potent stimulatory effect on tyrosine
kinases and robust inhibition of tyrosine phosphatases, DPV induces
association of multiple tyrosine-containing proteins with Src homology
(SH2 and SH3) domains (24) such as phospholipase C-
, Src family
tyrosine kinases, and potentially important adaptor proteins such as
p59 Shc (22, 34, 38, 42). Careful analysis of the 1,914-amino acid
sequence of EC MLCK reveals two SH2 binding sites
(Tyr59 and
Tyr464), two potential SH3
domains (amino acid residues 314-318 and 373-379), and
consensus sites for tyrosine kinase phosphorylation catalyzed by the
Src family of kinases (Tyr485,
Tyr1449, and
Tyr1575). Each SH2 and SH3
binding site and the Tyr485 Src
kinase consensus site are present in the novel
NH2 terminus of the EC MLCK
isoform, a sequence not shared by the smooth muscle MLCK isoform. In
addition to these sites of tyrosine phosphorylation, potential sites
for EC MLCK phosphorylation by tyrosine kinase-regulated Ser/Thr
kinases, such as mitogen-activated protein or extracellular signal-regulated (ERK) kinase, have also been identified (49). Interestingly, DPV has been shown to increase ERK1 and ERK2 activity in
several cell systems including the endothelium, presenting a potential
mechanism that may regulate the activity of the smooth muscle MLCK
isoform (25, 29, 31, 54). Taken together, these findings are consistent
with the novel regulation of the nonmuscle EC MLCK isoform by tyrosine phosphorylation.
Although our data provide a strong linkage between MLCK activity and
the phosphotyrosine status of the kinase, we cannot exclude the
possibility that additional DPV-mediated effects, aside from direct
tyrosine phosphorylation of MLCK, may be contributing to kinase
activation. For example, the increase in MLCK activities elicited by
DPV may be related to DPV-mediated increases in EC cytosolic
Ca2+, an essential cofactor in
enhanced EC MLCK activation (10). However, even submillimolar
concentrations of
H2O2
alone do not increase EC MLC phosphorylation (Fig.
1A), and previously published findings by Gilbert-McClain et al. (16) and Natarajan et al. (31)
indicated that DPV and 100 µM
H2O2
only weakly increase cytosolic
Ca2+ concentration, whereas
millimolar concentrations of vanadate fail to alter
Ca2+ in confluent endothelium.
Furthermore, recent studies (10, 14) have unequivocally demonstrated
that rises in cytosolic Ca2+,
including those elicited by Ca2+
ionophores such as A-23187 and ionomycin, although necessary, are
insufficient to activate the EC MLCK isoform. Ionomycin not only fails
to activate MLCK but produces rapid MLC dephosphorylation via
activation of the type 2B Ser/Thr phosphatase known as calcineurin (47,
50). Because extensive homology exists between the 130- to 160-kDa
smooth MLCK isoform and the 214-kDa EC MLCK isoform in the CaM-binding
region (residues 1749-1759), differences in CaM affinity are
unlikely to explain the lack of enzymatic activation by
Ca2+ alone (12, 48). It is much
more likely that additional regulatory elements contained in the unique
NH2-terminal region (residues 1-922), not shared by smooth muscle MLCK isoforms, participate in
EC MLCK regulation. Our data now indicate the strong possibility that
MLCK tyrosine phosphorylation mediated by
p60src is an important event in
the regulation of MLCK enzymatic activity and activation of the EC
contractile apparatus.
One of the primary functions of the endothelium is to change its shape
in response to agonists, a response that requires an extensive and
rapid reorganization of the EC cytoskeleton. The Rho GTPase members of
the Ras superfamily are clearly involved in the regulation of the
nonmuscle cytoskeleton and stress-fiber formation (2, 32, 40). Although
Gilbert-McClain et al. (16) have previously failed to identify a
significant effect of millimolar vanadate on total Ser/Thr phosphatase
activity, the lack of total inhibition of DPV-mediated MLC
phosphorylation with KT-5926 suggested the possible contribution of
Ser/Thr phosphatase inhibition to the full integrated DPV-induced MLC
phosphorylation response similar to what Verin et al. (47, 50) have
observed after thrombin stimulation. MLC phosphorylation in smooth
muscle and nonmuscle tissues is potently modified by Rho GTPases via Rho kinase-mediated phosphorylation of the regulatory subunit of MLC
phosphatase (26). Our experiments utilizing
C3 exotoxin, a specific Rho GTPase
inhibitor, indicate that thrombin and DPV mediate significant Rho
activation in human lung microvascular and bovine pulmonary endothelia,
with C3 exotoxin totally
abolishing basal thrombin- and DPV-mediated MLC phosphorylation. The
mechanism of DPV-stimulated Rho activation is unknown; however, these
results are entirely plausible because Rho activation has been shown to involve tyrosine kinase activities (32, 40). Nevertheless, the highly
significant inhibition of DPV-mediated MLC phosphorylation by KT-5926,
an MLCK inhibitor with relative specificity, in conjunction with the
complete abolishment of MLC phosphorylation by
C3 exotoxin, argue for an
important role of the EC MLCK isoform in the DPV-mediated MLC response.
Together, these results indicate that DPV-mediated Rho activation
increases MLC phosphorylation and that key targets for this regulation
appear to include both MLC phosphatase as well as the EC MLCK isoform.
The novel mechanisms by which Rho may alter MLCK activities are under
current investigation.
Our data examining proteins that are stably associated with EC MLCK
indicate that p80/85 cortactin, a potent F-actin-binding protein
associated with the nonmuscle cytoskeleton, may participate in a
signaling cascade that enables the cytoskeleton to reorganize and the
endothelium to remodel rapidly. Cortactin promotes sedimentation of
F-actin at centrifugation forces under which F-actin is otherwise not
able to be precipitated. However, cortactin is an in vitro substrate
for p60src (52, 53) and cortactin
phosphorylation by Src abolishes F-actin bundling properties (20).
Tyrosine phosphorylation of cortactin is immediately enhanced before
thrombin-induced platelet aggregation and subsequent cortactin
translocation to the cytoskeleton (41). Our data indicate that DPV
induces cortactin phosphotyrosine phosphorylation (with a shift to the
p85 isoform) and cortactin translocation to the endothelial
cytoskeleton in a stable association with EC MLCK. The observations
that 1) DPV significantly increases
protein tyrosine phosphorylation of cytoskeletal elements, including
both p80/85 cortactin and EC MLCK;
2) cortactin is present in c-Src immunoprecipitates; and 3) a stable
association exists between p60src
cortactin and MLCK together implicate
p60src as the tyrosine kinase
most likely responsible for phosphorylation of EC MLCK and cortactin.
Recent reports (30, 33, 43) in cells lacking the
c-src gene or after overexpression of
c-Csk, a negative regulator of
p60src, provide further
compelling evidence that cortactin is an intrinsic substrate for
p60c-src. The deduced amino acid
sequence of cortactin contains an SH3 motif at the COOH terminus and a
NH2-terminal domain composed of
five (p80) or six (p85) internal tandem repeats that represent sites of
actin binding (20). The presence of an SH3 domain within the cortactin
structure could support its function as a scaffolding protein with a
number of Src family kinases, adaptor proteins, and specific
cytoskeletal proteins as well as with signaling molecules (23). Both
structural and subcellular localization data seem to support the
concept that cortactin potentially participates in the transduction of
signals from the cell surface to the cytoskeleton. In addition, the
large number of known cytoskeletal targets for p60src, together with our
observation of DPV-induced tyrosine phosphorylation of several proteins
in the endothelial cytoskeleton, appears to be consistent with this notion.
In summary, we have used the cell-permeable oxidant and potent tyrosine
kinase activator or phosphatase inhibitor DPV to study whether tyrosine
phosphorylation is intimately involved in activation of the endothelial
actomyosin cytoskeleton. Our results indicate that DPV activates EC
MLCK in a Rho GTPase-dependent manner, stimulates EC MLCK
phosphotyrosine accumulation, and promotes the stable association of
p80/85 cortactin and p60src with
EC MLCK and the actin cytoskeleton. Both cortactin and
p60src can now be included as
potentially key components involved in the establishment of a
functional MLCK enzymatic complex known to include MLC and myosin heavy
chain, CaM, and actin (49). We speculate that tyrosine phosphorylation
of cortactin and MLCK by p60src
within a growing MLCK complex of proteins may be key steps in a cascade
of events leading to cytoskeleton changes, disassembly of lung EC
adherens junctions, and, ultimately, leukocyte infiltration and edema
formation during lung inflammation.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Lakshmi Natarajan, Anila Ricks-Cord, and
Steve Durbin for superb technical assistance; Dr. Denis English for
helpful discussions; and Ellen Reather for excellent manuscript preparation.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-50533, HL-58064, HL-57260, and HL-47671 and awards from the
American Heart Association and the American Lung Association.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. G. N. Garcia,
5501 Hopkins Bayview Circle, Baltimore, MD 21224 (E-mail:
drgarcia{at}welchlink.welch.jhu.edu).
Received 18 September 1998; accepted in final form 8 February
1999.
 |
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