From the Division of Pulmonary and Critical Care
Medicine, Johns Hopkins University School of Medicine, Baltimore,
Maryland 21224, the § Middle Atlantic Mass Spectrometry
Laboratory, Department of Pharmacology and Molecular Sciences,
Johns Hopkins University School of Medicine, Baltimore, MD 21205, and the ¶ Department of Biochemistry, Albert Einstein College of
Medicine, Bronx, New York 10461
Received for publication, June 16, 2000, and in revised form, December 11, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The
Ca2+/calmodulin-dependent endothelial
cell myosin light chain kinase (MLCK) triggers actomyosin contraction
essential for vascular barrier regulation and leukocyte diapedesis. Two
high molecular weight MLCK splice variants, EC MLCK-1 and EC MLCK-2 (210-214 kDa), in human endothelium are identical except for a deleted
single exon in MLCK-2 encoding a 69-amino acid stretch (amino acids
436-505) that contains potentially important consensus sites for
phosphorylation by p60Src kinase (Lazar, V., and
Garcia, J. G. (1999) Genomics 57, 256-267). We have
now found that both recombinant EC MLCK splice variants exhibit
comparable enzymatic activities but a 2-fold reduction of
Vmax, and a 2-fold increase in
K0.5 CaM when compared with the SM MLCK
isoform, whereas Km was similar in the three
isoforms. However, only EC MLCK-1 is readily phosphorylated by purified
p60Src in vitro, resulting in a 2- to 3-fold
increase in EC MLCK-1 enzymatic activity (compared with EC MLCK-2 and
SM MLCK). This increased activity of phospho-MLCK-1 was observed over a
broad range of submaximal [Ca2+] levels with comparable
EC50 [Ca2+] for both phosphorylated and
unphosphorylated EC MLCK-1. The sites of tyrosine phosphorylation
catalyzed by p60Src are Tyr464 and
Tyr471 within the 69-residue stretch deleted in the MLCK-2
splice variant. These results demonstrate for the first time that
p60Src-mediated tyrosine phosphorylation represents an
important mechanism for splice variant-specific regulation of nonmuscle
MLCK and vascular cell function.
The family of myosin light chain kinases
(MLCK)1 expressed in
vertebrates are Ca2+/calmodulin-regulated enzymes that
catalyze the transfer of phosphate from Mg2+-ATP to a
serine residue (Ser19) of regulatory myosin light chain
(MLC20) (1, 2). Members of this MLCK family share
structural similarity, with a catalytic core that binds
Mg2+-ATP and MLC20 and a regulatory segment
involved in Ca2+/calmodulin-dependent
activation. Comparisons of primary sequences deduced from cDNA
clones demonstrate that skeletal and cardiac muscle MLCK isoforms
represent gene products that differ from the gene encoding the smooth
muscle and nonmuscle MLCK isoforms localized on human chromosome 3 (3-6). Furthermore, the physiological role of the gene-specific MLCK
isoforms differs significantly in the regulation of actomyosin
contraction. In skeletal and cardiac muscles, Ca2+ binds to
the regulatory thin filament protein complex containing troponin and
tropomyosin and thus allows actin to activate sarcomeric myosin
Mg2+-ATPase. Although MLCK does not initiate muscle
contraction in these tissues, MLCK-mediated phosphorylation of
MLC20 may potentiate the rate and extent of force
development (7, 8). In contrast, Ser19 phosphorylation of
MLC20 by Ca2+/calmodulin-dependent
enzyme MLCK is essential for the initiation of nonmuscle and smooth
muscle contraction (1, 3, 9-11). In specific nonmuscle tissues, such
as the vascular endothelium, this kinase is known to be involved in
endothelial cell migration, cell retraction (12), endothelial cell
barrier regulation (13), transendothelial migration of neutrophils (14,
15), and possibly apoptosis (16). The MLCK isoform abundantly expressed
in smooth muscle (SM MLCK) generally exists as a 130- to 150-kDa
protein that has been well characterized (for review see Refs. 3 and 11). However, Western blot screening of a variety of embryonic and
adult smooth muscle and nonmuscle tissues revealed expression of a high
molecular weight MLCK variant with electrophoretic mobility in the
range of 208-214 kDa (17-19). Garcia and colleagues (20) subsequently
sequenced the high molecular weight MLCK isoform cloned from a human
endothelial cell cDNA library, revealing an open reading frame,
which encodes a protein of 1914 amino acids. Both the low molecular
mass (130-150 kDa) and high molecular mass (208-214 kDa) MLCK
isoforms share essentially identical actin binding, MLC binding,
catalytic, and Ca2+/CaM-regulatory domains. The extreme
C-terminal kinase-related protein (KRP) domain, which binds myosin, is
contained within both EC MLCK and SM MLCK but can be also expressed as
an independent protein capable of stabilizing myofilaments in
vitro (3, 21-23). The C-terminal half of the endothelial MLCK
isoform (residues 923-1914) exhibits 99.8% homology to the human low
molecular weight MLCK from hippocampus and substantial homology to
published SM MLCK sequences from rabbit (94% homology), bovine (95%
homology), and chicken (85% homology) (5, 6, 24, 25). However, the
exact biological function of the 922-amino acid N-terminal portion,
which is unique to the high molecular weight MLCK isoform, is
completely unknown. We have previously found that increased levels of
endothelial cell protein tyrosine phosphorylation evoked by thrombin or
diperoxovanadate are tightly linked to increased MLC20
phosphorylation, activation of actomyosin contraction, and a dramatic
decrease in endothelial cell barrier function (26, 27). Further studies
demonstrated that the increased kinase activity in MLCK
immunoprecipitates strongly correlated with increased EC MLCK
phosphorylation on tyrosine residues (26). Endothelial cell MLCK was
found to be stably associated with p60Src kinase after
stimulation (26), consistent with potential direct regulation of
endothelial MLCK activity by tyrosine phosphorylation. More recently,
detailed analysis of MLCK transcripts expressed in human endothelial
cells revealed several splice variants of the EC MLCK isoform with
predominant expression of the full-length isoform (MLCK-1) and a
variant, which is identical to MLCK-1 except for a deleted 69-residue
stretch (amino acid residues 437-505) encoded by a single exon
(MLCK-2) (28). Within this deleted 69-amino acid stretch is an
SH2-binding domain and consensus sites for phosphorylation by the Src
family kinases (Tyr464, Tyr485). To better
understand the role and significance of tyrosine phosphorylation and
the function of the novel N terminus in EC MLCK regulation, we have
expressed both EC MLCK-1 and EC MLCK-2 isoforms as well as smooth
muscle MLCK in the baculovirus system and have characterized the
biochemical properties of the purified recombinant proteins.
Furthermore, we have assessed the phosphorylation of MLCK-1, MLCK-2,
and SM MLCK by p60Src kinase in vitro, mapped the
p60Src-phosphorylation sites to Tyr464 and
Tyr471 of MLCK-1, and investigated the effects of MLCK-1
phosphorylation by p60Src kinase on the MLCK enzymatic activity
and regulation by Ca2+/calmodulin. Our studies indicate the
novel up-regulation of high molecular weight endothelial MLCK-1 isoform
activity by p60Src-induced tyrosine phosphorylation and
demonstrate isoform-specific endothelial cell MLCK regulation.
Reagents
Chemicals used in these studies were obtained from Sigma
Chemical Co. (St. Louis, MO), unless otherwise specified. Restriction and modification enzymes were purchased from Amersham Pharmacia Biotech
(Arlington Heights, IL), Roche Molecular Biochemicals (Indianapolis,
IN), Life Technologies (Gaithersburg, MD), and Promega (Madison, WI).
Radioactive nucleotides, [ Recombinant Donor Plasmids for Baculovirus Expression
Smooth-muscle MLCK--
A rabbit uterine smooth muscle MLCK
full-length cDNA in a pGEM vector (24) was a generous
gift from Dr. Patricia Gallagher (Indiana University). The plasmid
was cut at the Eco52I site, followed by blunt-ending with
Klenow enzyme. The MLCK insert was then released by digesting plasmid
with XbaI, separated from the vector by agarose gel
electrophoresis, and purified using a Prep-A-Gene (Bio-Rad, Hercules,
CA) kit. The smooth muscle MLCK cDNA was ligated with the pFastBac
Hta baculovirus donor plasmid (Bac-To-Bac baculovirus expression
system, Life Technologies), which had been digested with
StuI and XbaI enzymes.
Nonmuscle MLCK-1 and MLCK-2--
MLCK-2 cDNA was obtained
from human umbilical vein endothelial cells (HUVEC) by RT-PCR,
amplified, and subcloned as described earlier (28), using sense primer
5'-ACT GAA TTC ACC ATG GGG GAT GTG AAG CTG and antisense primer 5'-GTC
AGA ATT CTT GTT TCA CTC TTC TTC CTC TTC C, both containing an
EcoRI site. The EC MLCK-2 cDNA was inserted into
pFastBac Hta vector at the EcoRI site. To generate MLCK-1
cDNA, an ~1.85-kb 5'-end fragment of HUVEC MLCK was amplified by
RT-PCR using a SuperScript preamplification system (Life Technologies).
The first-strand cDNA was synthesized from HUVEC total RNA with
oligo-dT primer. The PCR primers were as follows, sense: 5'-ACT GCG GCC
GCA CCA TGG GGG ATG TGA AGC TG (NotI restriction site
followed by HUVEC MLCK-specific 5'-end sequence), and antisense: 5'-GTA
CTC ACT CTT CCT GCT ACT C (~1.85-kb downstream HUVEC MLCK sequence).
The same region (with a 207-bp deletion compared with EC MLCK-1) of the
nonmuscle MLCK-2/pFastBac Hta plasmid was excised and replaced with
this PCR-amplified fragment encoding the N-terminal portion of MLCK-1.
Briefly, the PCR product was digested with NotI,
blunt-ended, and cut by BlpI. The respective ~1.66-kb
fragment from the nonmuscle MLCK-2/pFastBac Hta plasmid was excised
with EheI and BlpI, and separated on agarose gel. The PCR-amplified 5'-end fragment of MLCK-1 and the 3'-end MLCK region
previously subcloned into pFastBac Hta vector were ligated to create
the recombinant donor plasmid of the whole EC MLCK-1 coding region
reported previously (20). All three constructs (SM MLCK, EC MLCK-1, and
EC MLCK-2) were verified by restriction analysis, PCR, and complete sequencing.
Recombinant Regulatory Myosin Light Chain--
A plasmid
pET3-MLC encoding rabbit vascular smooth muscle regulatory MLC was
generously provided by Dr. Patricia Gallagher (Indiana University) and
amplified by PCR using primers with unique restriction sites for
directional cloning, MLC-sense-EcoRI: 5'-TGC TTT GAA TTC ATG
TCC AGC AAG CGG GCC AAA GCC AAG-3', and MLC-antisense- XbaI:
5'-AAG GAC TCT AGA CTA GTC GTG TTT ATC CTT GGC GCC ATG-3', and
then ligated with the pFastBac Hta baculovirus donor plasmid (Bac-To-Bac baculovirus expression system), which had been digested with EcoRI and XbaI enzymes.
Baculovirus Expression of MLC and MLCK Isoforms
Human endothelial cell MLCK-1 and MLCK-2, rabbit smooth muscle
MLCK and MLC recombinant baculovirus stocks were prepared using the
Bac-To-Bac baculovirus expression system (Life Technologies) according
to manufacturer's instructions. The system uses site-specific transposition of foreign genes into a baculovirus shuttle vector, bacmid, propagated in Escherichia coli. Sf9 and Hi5
insect cell suspension cultures at 2 × 106 cells/ml
were infected with the respective viral stock at a multiplicity of
infection range of 0.1-10, and after 1-h incubation, diluted 5-fold
with fresh media and grown for 2-4 days at 28 °C with continuous shaking. The optimal conditions varied for the three different MLCK
isoforms expressed (not shown). For large scale expression and
purification, Sf9 cells were infected with baculovirus
(multiplicity of infection = 1), and the cells producing SM MLC,
rabbit SM MLCK, or human endothelial MLCK-1 and -2 were harvested. The
recombinant MLCK and MLC proteins contained a histidine tag at their
N-terminal region introduced during subcloning MLCK cDNAs into the
pFastBac Hta baculovirus donor vector. Expression of the MLCK isoforms was confirmed by SDS-polyacrylamide gel electrophoresis (29) and
Western blot (30) using MLCK- specific D119 antiserum (31) or
commercial anti-His tag antibodies.
Purification of Recombinant Proteins
For the isolation of the recombinant MLCK isoforms or MLC, the
infected Sf9 cells were harvested by centrifugation at 3000 × g for 5 min and frozen at Verification and Characterization of MLCK Constructs
Human endothelial MLCK-1 and MLCK-2 splice variants as well as
the rabbit smooth muscle MLCK isoform cDNAs were subcloned into
baculovirus vector pFastBac Hta as described above. The alternatively spliced regions in MLCK-1 and MLCK-2 mRNAs were verified by PCR amplification (not shown) using primers located upstream and downstream of nucleotides 1428-1634 as well as by complete sequencing of the
MLCK-1 and MLCK-2 cDNA inserts. Translation of completely sequenced
cDNA inserts encoding EC MLCK splice variants revealed four amino
acid residue differences between the unique N-terminal sequence of the
EC MLCK-1 and MLCK-2 (Phe629, Cys681,
Gly714, and Leu806) and the previously reported
EC MLCK cDNA sequence (GenBankTM accession number U48959).
These sequence differences, which may represent polymorphisms within
the human MLCK gene, did not involve either the
consensus sequence for potential tyrosine phosphorylation catalyzed by
p60Src (Tyr464, Tyr471,
Tyr485), the putative SH2-binding sites (Tyr59,
Tyr464), the SH3 domains
(Pro314-Arg318,
Arg373-Pro379) previously proposed for EC MLCK
(26, 28), and did not affect MLCK enzymatic properties (shown below).
Rigorous analysis of the cDNA sequences encoding the C terminus of
EC MLCK common to both high and low molecular weight MLCK isoforms,
revealed three variances (Phe925/Leu,
Ala1179/Val, and Lys1233/Glu) in the
baculovirus-expressed recombinant MLCK-1 and MLCK-2 isoforms that do
not correspond to GenBankTM accession number 48959 or to homologous
regions of the reported human, rabbit, and bovine SM MLCK variants,
respectively (6, 24, 25). Leu925 resides within the
putative actin-binding domain spanning residues 910-1036 of MLCK-1,
whereas Val1179 and Glu1233 do not lie within
functional domains described for smooth muscle MLCK (32).
Myosin Light Chain Phosphorylation Assays
Baculovirus-expressed SM MLC was used as a substrate after
His-tag excision by rTEV protease. In addition, several types of recombinant Xenopus regulatory MLC, including wild type MLC
(Ser19Thr18) and
Ser19Ala18 and
Ala19Thr18 mutants expressed in E. coli (33) were used as substrates in studies of EC MLCK substrate
specificity. The purified MLCKs were diluted in 50 mM MOPS,
pH 7.4, 10 mM Mg2+ acetate, 0.05%
2-mercaptoethanol containing 1 mg/ml bovine serum albumin to a
1.25 × 10 Phosphorylation of MLCK by p60Src in Vitro
Purified MLCKs were dialyzed and brought to 0.1 mg/ml
concentration using reaction buffer containing 25 mM
Tris-HCl, pH 7.5, 20 mM KCl, 5 mM
Mg2+ acetate, 0.5 mM leupeptin. Phosphorylation
of MLCK diluted in reaction buffer was started by adding 0.2 mM ATP, 10 µCi/ml [ Tryptic Cleavage and MALDI TOF Analysis of MLCK-1
Phosphopeptides
A 200-µl aliquot of MLCK-1 (0.3 mg/ml) phosphorylated by
p60src in vitro was partially digested by incubation
with trypsin (1:100 w/w) for 5 min at room temperature in buffer
containing 50 mM Tris-HCl, pH 7.5, 5 mM EGTA, 5 mM MgCl2. The reaction was terminated by adding
phenylmethylsulfonyl fluoride at 2 mM final
concentration. The reaction mixture was lysed in SDS-sample buffer,
peptides were separated on SDS-polyacrylamide gel electrophoresis, and phosphotyrosine peptides were identified by autoradiography and Western
blot with anti-phosphotyrosine antibody. A portion of the
polyacrylamide gel containing a major 55-kDa MLCK-1 tryptic peptide,
which incorporated 32P and cross-reacted with
anti-phosphotyrosine antibody, was excised and further processed. After
3 × 30 min washes in distilled water, the gel piece was trimmed
and the phosphopeptide incorporated into gel was subjected to
destaining and complete trypsinolysis. Gel destaining required the
addition of 100 µl of 1:1 (v/v) acetonitrile:25 mM
ammonium bicarbonate for ~30 min. The gel was then dried down completely using a Speed Vac concentrator (Savant), and further trypsinolysis was performed. The gel was incubated overnight at 37 °C with 1 µl of trypsin solution (0.1 µg/µl in 1% acetic
acid) and 25 µl of 25 mM ammonium bicarbonate, pH 7.8. After trypsinolysis, the MLCK peptides were eluted from the gel with a
1:1 (v/v) acetonitrile:water, 5% trifluoroacetic acid solution
and concentrated to several microliters by Speed Vac. This peptide
mixture was then analyzed by mass spectrometry. Mass spectra were
acquired on a Kratos Axima CFR (Manchester, United Kingdom)
time-of-flight mass spectrometer. Briefly, an aliquot of the peptide
digest (0.3 µl) was placed on the sample plate followed by the
addition of 0.3 µl of saturated ammonium sulfate and 0.3 µl of
matrix solution (saturated solution of Kinetic Characteristics of Recombinant Endothelial Cell MLCK Splice
Variants--
The domain organization of the endothelial cell MLCK
isoforms is schematically presented in Fig.
1A and demonstrates that the
two endothelial isoforms: MLCK-1 and MLCK-2 are identical with the
exception of the deletion of a 69-residue stretch encoded by a single
exon in MLCK-2 (28). The baculovirus-expressed and -purified MLCK
isoforms (MLCK-1, MLCK-2, and SM MLCK) were analyzed by gel
electrophoresis and revealed protein bands with expected sizes 214, 206, and 150 kDa, respectively (Fig. 1B), which reacted on
the Western blot with MLCK-specific D119 antiserum (Fig.
1C). After purification and rigorous sequencing analysis,
the three recombinant MLCK isoforms were examined for their intrinsic
enzymatic properties. Both EC MLCK-1 and EC MLCK-2 exhibited comparable Vmax values (11.9 ± 3.2 and 10.9 ± 1.8 µmol/min/mg, respectively), which were slightly reduced compared
with the rabbit SM MLCK isoform (17.0 ± 2.5 µmol/min/mg) (Table
I). The Km values
reflecting substrate affinity were not significantly different among
the three recombinant MLCK preparations, whereas the
K0.5 calmodulin was higher for the EC MLCK
isoforms (0.49 and 0.42 nM) compared with the rabbit SM
MLCK isoform (0.21 nM) (Table I). Overall, the
Km, Vmax,
K0.5 calmodulin values for the
baculovirus-expressed endothelial MLCK-1 and MLCK-2 splice variants are
in good agreement with published values for MLCK activity derived from
other tissues (24, 35-37). Furthermore, all three recombinant MLCK
isoforms possess identical substrate specificity and preferentially
phosphorylate Ser19 and Thr18 of regulatory
MLC20 (Table I), because the substitution of Ala for either
Ser19 alone or both Ser19 and Thr18
in the MLC mutants resulted in dramatic reduction in MLC20
phosphorylation. These results are in complete agreement with the
previously reported preferred sequential phosphorylation of
Ser19 followed by phosphorylation at the MLC second site
(Thr18), which is phosphorylated more slowly than
Ser19 and requires relatively high concentrations of myosin
light chain kinase (2).
In Vitro Phosphorylation of MLCK Isoforms by
p60Src--
Augmentation of tyrosine protein
phosphorylation in endothelial cells in vivo correlates with
increased phosphotyrosine content in MLCK immunoprecipitates, increased
MLCK activity, and increased MLC phosphorylation (26, 38). More
recently, EC MLCK has been shown to be stably associated with
p60Src and a well recognized p60Src substrate, the
actin-binding protein cortactin (26). To further characterize the role
of tyrosine phosphorylation in EC MLCK regulation, the three
recombinant MLCK isoforms were used as substrates for in
vitro phosphorylation catalyzed by p60Src. EC MLCK-1
exhibited substantial time-dependent
p60src-catalyzed incorporation of radioactive phosphate (Fig.
2), whereas p60src-mediated
32P incorporation did not occur in either MLCK-2, the EC
MLCK splice variant, lacking the 69-amino acid stretch containing the
p60src consensus site, nor in SM MLCK, which completely lacks
the novel N terminus (Fig. 2). The 32P incorporation into
MLCK-1 was essentially abolished by the specific p60src kinase
inhibitor PP-2 (250 nM). However, all three MLCK isoforms exhibited low level of 32P incorporation even in the
absence of p60src (0.63 ± 0.21 mol of PO4/mol
of protein) consistent with the MLCK autophosphorylation previously
described for smooth muscle MLCK (39, 40). This was confirmed by heat
treatment of the MLCK preparations (70 °C for 5 min), which
completely abolished incorporation of 32P into MLCK in the
absence of p60src (data not shown). The preferential
p60src-catalyzed phosphorylation of MLCK-1 compared with other
MLCK isoforms strongly suggested that the specific site of tyrosine phosphorylation of endothelial MLCK by p60src resides within
the 69-amino acid residue stretch in the N-terminal part of the MLCK
molecule encoded by a single exon (28), which is not expressed in
MLCK-2. However, the stoichiometry of phosphate incorporation into
MLCK-1 catalyzed by p60src (2.32 ± 0.32 mol of
PO4/mol of protein) strongly suggested the potential
presence of a secondary tyrosine residue within MLCK-1, which may also
be phosphorylated by p60src.
Identification of p60src Phosphorylation Sites within
MLCK-1--
To search for tyrosine phosphorylation site(s) within
MLCK-1, we applied mass spectroscopy analysis of tryptic fragments
obtained from MLCK-1 phosphorylated by p60src. As an initial
step, a limited trypsinolysis of radiolabeled phospho-MLCK-1 was
performed, and tyrosine phosphorylation site(s) mapped to the tryptic
fragment with an approximate molecular weight of 55 kDa based on
autoradiography data and Western blot analysis with
anti-phosphotyrosine antibody (inset, Fig.
3A). Mass spectrometric analysis of MLCK-1 peptides obtained after complete tryptic digestion of a 55-kDa MLCK-1 fragment revealed a group of peptides corresponding to the N-terminal portion of MLCK-1 (Fig. 3A). Furthermore,
we identified a single characteristic peak (Fig. 3A) not
seen in the digests of MLCK-1 preincubated with ATP without
p60src (data not shown). This peak has m/z ratio
2486 corresponding to the theoretical mass of the tryptic peptide
457-476 of MLCK-1 (457QEGSIEVYEDAGSHYLCLLK476)
with two incorporated phosphate groups (underlined). MALDI
TOF analysis allowed isotopic resolution of the m/z 2486 peak (isotopic variants with m/z ratios 2484.9, 2485.9, and
2486.9, respectively) further supporting mapping of the peak to the
diphospho-(457-476)-MLCK-1 fragment. To further prove phosphorylation
of sites Y464 and Y471 by p60src, we
used synthetic peptides
460SIEVYEDAGSHYLCLL475
and 478RTRDSGTYSCTASNA492
corresponding to amino acid residues 460-475 and 478-492 of
full-length MLCK-1 protein, respectively, for in vitro
p60src phosphorylation assay (Fig. 3B). Only the
peptide containing Y464 and Y471 was readily
phosphorylated in the presence of p60src, whereas another
candidate peptide containing the potential p60src consensus
phosphorylation site, Y485, as well as irrelevant peptide
PEKVPPPKPATPDFRSVL (residues 968-985 of MLCK-1), which lacks tyrosine
residues, were not phosphorylated. The relatively low stoichiometry of
p60src-mediated phosphate incorporation into peptide 460-475
(~0.015 mol of PO4/mol of peptide, 10-min reaction)
suggests that other MLCK-1 epitope(s) in proximity to amino acid
residues 460-475 may be required for the optimal p60src
activity. As recently demonstrated, the interaction of the
p60src SH3 domain with the ligand sequence of the substrate is
important for activation of the catalytic domain and
autophosphorylation (41), and the addition of an SH3 domain ligand to a
substrate peptide increases its phosphorylation 10-fold via lowering of the Km value of the substrate and kinase activation
(42). Thus, putative SH3- and SH2-binding domains present in MLCK-1 N-terminal portion (Fig. 1A) may be important for
p60src-catalyzed MLCK-1 phosphorylation, and further studies
are underway to address this question. Finally, mass spectrometry
analysis of the fragments obtained after cyanogen bromide cleavage of
the MLCK-1 suggested that the two phosphate groups are contained within the Lys1721-Met1761 EC MLCK fragment (data not
shown) consistent with the presence of the EC MLCK-1
autophosphorylation sites Thr1748 and Ser1760,
which are homologous to the previously described SM MLCK
autophosphorylation sites Thr803 and Ser815
(39).
Differential Activation of MLCK-1 and MLCK-2 by
p60src-mediated Phosphorylation--
To explore whether
phosphorylation by p60src alters endothelial MLCK-1 enzymatic
properties, MLCK-1 samples were preincubated with either ATP and
p60src ("phospho-MLCK") or ATP alone ("dephospho-EC
MLCK"), followed by assessment of in vitro kinase
activity. Phosphorylation of EC MLCK-1 by p60src increased EC
MLCK-1 kinase activity 2-fold (Fig. 4),
whereas the enzymatic activities of EC MLCK-2 and SM MLCK were not
affected by p60src and were comparable to that measured for EC
MLCK-1 in the absence of p60src. These results are again
consistent with the inability of p60src to phosphorylate EC
MLCK-2 (as shown in Fig. 2) and indicate a significant enhancement of
MLCK-1 kinase activity by p60src-mediated phosphorylation.
Enzymatic activity of all three isoforms was
Ca2+/CaM-dependent, because chelation of free
Ca2+ with 2 mM EGTA (Fig. 4) or removal of
calmodulin from the kinase reaction mixture (data not shown) completely
abolished MLC phosphorylation catalyzed by either phospho- or
dephospho-MLCK-1 preparations by MLCK-2 and SM MLCK. An inhibitor of
smooth muscle MLCK activity, ML-7 (5 × 10 In contrast to smooth muscle, only the high molecular weight MLCK
isoform (208-214 kDa) is expressed in endothelium (18-20, 22).
Molecular cloning of MLCK from human endothelial cells (20) revealed a
high molecular weight MLCK variant containing a unique 922-residue
N-terminal domain not expressed in the low molecular weight MLCK
isoform, which is abundantly expressed in smooth muscle. Comparison of
the cDNA encoding human high and low molecular weight MLCKs, when
combined with results of chromosome mapping of human MLCK to
single locus with chromosomal localization to 3qcen-q21 (6), suggests
that mammalian MLCK genomic organization is highly similar
to the "gene within a gene" organization of the avian smooth
muscle/nonmuscle MLCK gene expressing two size class MLCK
variants and one nonkinase protein (KRP), which are encoded by exons
1-31, 15-31, and 29A-31, respectively (17, 43). The complexity of the
human MLCK genomic organization was recently further
emphasized by the detection of five splice variants of high molecular
weight MLCK in nonmuscle and smooth muscle tissues using RT-PCR
approaches (28). These data, which elucidated the considerable
expression of EC MLCK-2, were strongly consistent with the potential
functional diversity of the expressed smooth muscle and nonmuscle MLCK
proteins. Among endothelial cell MLCK splice variants, the MLCK-1 and
MLCK-2 appear to be preferentially expressed (28), although all five
have been identified in tissues. Using purified recombinant MLCK-1 and
MLCK-2 expressed in a baculovirus system, we have now characterized for
the first time the kinetic parameters of the high molecular weight MLCK
isoforms from human endothelial cells. Comparisons of the
Vmax and Km of these high
molecular weight isoforms to recombinant rabbit uterine smooth muscle
MLCK reveal very similar enzymatic properties of the three MLCK
isoforms. However, a 2-fold increase in
K0.5 calmodulin observed in endothelial MLCK
splice variants, may suggest a lower sensitivity of the intracellular
EC MLCK for regulation by Ca2+/calmodulin as compared with
SM MLCK.
A number of protein kinases, including cAMP-dependent
protein kinase A, protein kinase C,
Ca2+/CaM-dependent protein kinase II, and
p21-activated kinase have been demonstrated to phosphorylate the smooth
muscle MLCK isoform in vitro and in vivo
(44-47). Serine/threonine phosphorylation within MLCK
calmodulin-binding domain results in a 10-fold increase in
KCaM reflecting a 3.5-fold decrease in the
association rate and a 6-fold increase in the dissociation rate between
MLCK and Ca2+/CaM (3, 45, 46, 48, 49) and thus reduced MLCK
enzymatic activity. In turn, phosphorylation by p21-activated kinase
decreases MLCK-1 catalytic activity by ~50% via decrease in maximum
velocity (Vmax) without affecting
KCaM (47). In addition, Thr803,
Ser815, and Ser823 of the smooth muscle MLCK
isoform undergo autophosphorylation in vitro also resulting
in decreased MLCK affinity to Ca2+/calmodulin (39).
In contrast to serine/threonine phosphorylation of SM MLCK and EC MLCK,
which attenuates MLCK activity (3, 20, 31, 46, 48), information is
limited regarding phosphorylation sites within the MLCK molecule, which
serve to enhance its enzymatic activity. Phosphorylation of smooth
muscle MLCK by mitogen-activated protein kinase in vitro has
been reported to stimulate smooth muscle MLCK activity (50), although
we have not yet found mitogen-activated protein kinase to affect EC
MLCK activity in this manner. However, recent studies have defined the
involvement of tyrosine phosphorylation in EC MLCK regulation (26, 27,
51). Augmentation of protein tyrosine phosphorylation increased
MLC20 phosphorylation and cell contraction in endothelial
cells, which strongly correlated with an increase in MLCK
phosphotyrosine content, enhanced MLCK enzymatic activity, and the
stable association of EC MLCK with activated p60src (26). Our
present results appear to be consistent with the hypothesized novel
role of the unique N terminus in EC MLCK regulation via tyrosine
phosphorylation. We now demonstrate for the first time the in
vitro phosphorylation of the full-length EC MLCK-1 by
p60Src kinase on Tyr464 and Tyr471,
post-translational modifications not observed in the EC MLCK-2 splice
variant lacking the 69-residue stretch (amino acids 436-505) in the N
terminus, which is encoded by a single exon deleted in the EC MLCK-2
isoform (28). Our future studies using site-directed mutagenesis
approach are aimed at the determination of the sequence of
phosphorylation events and the role of each tyrosine phosphorylation site in the regulation of MLCK-1.
In summary, we have characterized the kinetic properties of endothelial
MLCK splice variants and demonstrated a novel mechanism of MLCK-1
regulation by p60src phosphorylation. The phosphorylation sites
(Tyr464 and Tyr471) are located within unique
N-terminal domain (436) of endothelial MLCK-1 isoform not
expressed in smooth muscle MLCK or in the alternatively spliced
endothelial isoform MLCK-2. Consistent with this finding, only MLCK-1
activity is regulated by p60src-catalyzed phosphorylation.
These data demonstrate the importance of the novel N-terminal domain in
the specific regulation of the MLCK isoform present in nonmuscle cells.
As we have previously demonstrated, the tyrosine phosphorylation of EC
MLCK increases its association with both p60src kinase, as well
as with the actin-binding protein and the p60src substrate
cortactin (26), we speculate that MLCK-1 tyrosine phosphorylation may
be involved in contractile complex scaffolding and contribute to
Ca2+ sensitization of the endothelial contractile
apparatus. Based on our data, we speculate that
p60src-catalyzed tyrosine phosphorylation contributes to the
local and selective activation of endothelial cell MLCK-1 under
submaximal Ca2+ concentrations providing a mechanism that
may tightly orchestrate critical cytoskeletal rearrangements and
ultimately the cellular contraction, which is critical for endothelial
cell-dependent biological processes, such as vascular
barrier regulation, transendothelial leukocyte diapedesis, and angiogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and
[
-33P]ATP, were obtained from PerkinElmer Life
Sciences (Boston, MA). MLCK inhibitor (ML-7) and the p60Src
inhibitor (PP2) were obtained from Calbiochem-Novabiochem Corp. (La
Jolla, CA). Purified p60C-src kinase was obtained from
Upstate Biotechnology (Lake Placid, NY). Sf9 and Hi5 insect
cells (Invitrogen, Carlsbad, CA) were grown in serum-free Sf-900 II SFM
media obtained from Life Technologies.
80 °C. Frozen insect cells
were lysed (1:5 w/v ratio) in ice-cold lysis buffer (50 mM
Tris-HCl, pH 8.5, 5 mM 2-mercaptoethanol, 100 mM KCl, 1 mM phenylmethylsulfonyl fluoride, and
1% Nonidet P-40) at 4 °C for 2 min. The lysate was centrifuged at
10,000 × g for 10 min, and the supernatant was loaded
onto Ni-NTA resin (Qiagen, Santa Clarita, CA). After a wash step with
buffer A (20 mM Tris-HCl, pH 8.5, 500 mM KCl, 5 mM 2-mercaptoethanol, 10% glycerol), the expressed MLCK
isoforms were eluted with 100 mM imidazole, 20 mM Tris-HCl, pH 8.5, 100 mM KCl, 5 mM 2-mercaptoethanol, 10% glycerol. The protein
concentration was determined by Bio-Rad protein assay. The yield of
MLCK isoforms ranged from 1.5 to 4 mg of MLCK from a 10-g Sf9
cell pellet. The purified enzymes were aliquoted and stored at
80 °C.
11 M final assay
concentration. The MLCK activity was determined by measuring
32P incorporation into the regulatory MLC used as
substrate. The MLCK kinase assays were performed in 50 mM
MOPS, pH 7.4, 10 mM Mg2+-acetate, 0.025%
2-mercaptoethanol, in the presence 0.3 mM
CaCl2, 10
6 M calmodulin,
10
7 M [
-32P]ATP at 0.5 Ci/mmol specific activity, and 1.25-15 × 10
6
M myosin light chain at 22 °C as previously described
(31). The concentration of free [Ca2+] in kinase
reactions was calculated as described by Imai and Okeda (34) using the
equation: p[Ca2+] = 2 pH
7.28 + log([EGTA]added/[CaCl2]added
1). Km and Vmax values were
determined from Lineweaver-Burk double-reciprocal plots using
SigmaPlot software (SPSS Inc., Chicago, IL).
-32P]ATP and 75 units/ml recombinant p60src kinase (Upstate Biotechnology,
Inc., Lake Placid, NY; final concentrations). Synthetic MLCK-1
peptides were used for p60src phosphorylation assays at 0.1 mg/ml final concentration. In certain experiments, a putative specific
p60src inhibitor, PP-2 (Calbiochem-Novabiochem Corp., La Jolla,
CA), was added to reaction tubes at 500 nM final
concentration. The phosphorylation reaction was performed at 22 °C,
and 10-µl aliquots of reaction mixture were applied onto cellulose
phosphate filters P81 (Whatman, UK) at specified periods of time. The
filters were washed to remove unincorporated label, and specific
incorporation of 32P into MLCK was determined by
scintillation counting. The p60Src phosphorylation of the MLCK
synthetic peptides as substrates was performed during 30 min at
22 °C under the same conditions. For scintillation counting,
synthetic MLCK peptides were spotted onto nitrocellulose, and unbound
radioactive label was washed out with solution containing 20% methanol
and 2% disodium pyrophosphate. After completion of the phosphorylation
reaction, phospho-MLCKs were aliquoted and stored at
80 °C until
use in kinase assays.
-cyano-4-hydroxycinnamic acid
in 1:1 (v/v) ethanol:water). The mixture was air-dried (~10 min), and
the sample plate was inserted into the mass spectrometer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (49K):
[in a new window]
Fig. 1.
Baculovirus expression and purification of
recombinant MLCK isoforms. A, schematic representation
of the domain organization of recombinant endothelial MLCK-1, MLCK-2,
and smooth muscle MLCK expressed in baculovirus system. Depicted are
the actin-binding domain, catalytic core, (catalyst), the regulatory
segment containing the inhibitory and calmodulin-binding domains, and
the kinase-related protein (KRP) domain. The unique N-terminal portion
of the endothelial MLCK isoforms contains putative SH3- and SH2-binding
domains. A 69-residue fragment (hatched region) containing a
putative SH2-binding domain and the potential p60Src
phosphorylation sites (Tyr464, Tyr471, and
Tyr485) is not expressed by either MLCK-2 or SM MLCK. The
histidine tag (HIS) has been introduced at the C termini of
recombinant MLCKs for purification purposes. B, purification
of recombinant MLCK expressed in the baculovirus expression system
using metal ion chromatography. The Coomassie Blue-stained gel
demonstrates the results of MLCK purification using MLCK-1 as an
example. Lane 1, molecular weight standards; lane
2, control cell lysate; lane 3, total lysate of
MLCK-1-producing cells; lane 4, flowthrough fraction after
Ni-NTA column chromatography; lane 5, purified MLCK-1 eluted
from Ni-NTA resin with elution buffer containing 100 mM
imidazole (see "Materials and Methods"). Lanes 6 and
7 represent the purified MLCK-2 and smooth muscle MLCK,
respectively, after identical purification procedures. C,
Western blot verification of recombinant MLCK isoforms using anti-MLCK
antiserum D119. Lane 1 represents control Sf9 lysate;
lanes 2-4 depict anti-MLCK immunoreactivity of purified
recombinant MLCK-1, MLCK-2, and smooth muscle MLCK, respectively, after
elution from the Ni-NTA column.
Kinetic properties and substrate specificity of recombinant MLCK-1,
MLCK-2, and SM MLCK isoforms
6 M smooth muscle
MLC20 final concentration and at saturating Ca2+
concentration. Kinase activity of recombinant MLCKs toward wild type
(wt) and mutant MLC20 is expressed as 10
8 mol of
phosphate incorporated per mg of MLCK per min. The concentrations of
wild type and mutant MLC20 were 5 × 10
6
M. The kinetic data represent the mean ± S.D. of at
least five separate experiments. The difference in
K0.5 CaM and Vmax between the EC
MLCK and SM MLCK isoforms is statistically significant
(p < 0.01).
View larger version (25K):
[in a new window]
Fig. 2.
Phosphorylation of MLCK-1, MLCK-2, and SM
MLCK by p60Src in vitro. Purified
recombinant MLCK-1 (left panel) and MLCK-2 (middle
panel) and SM MLCK (right panel) expressed in the
baculovirus system were phosphorylated using purified p60Src
kinase as outlined under "Materials and Methods." The
insets depict the representative autoradiograms of
time-dependent 32P incorporation into each MLCK
isoform in the presence of [32P]ATP and p60Src.
The graphs demonstrate the time dependence of
32P incorporation into MLCK-1, MLCK-2, and SM MLCK
incubated in the presence of p60Src alone (solid
lines) or in combination with 500 nM PP-2, a specific
inhibitor of p60Src (broken lines) or in the absence
of p60Src (dotted lines). The results obtained in
four independent experiments are expressed as the stoichiometry of
32P incorporation into MLCK.
View larger version (24K):
[in a new window]
Fig. 3.
Identification of the tyrosine
phosphorylation sites within the N-terminal 55-kDa MLCK-1 tryptic
fragment. The p60src-catalyzed phosphorylation of the
55-kDa tryptic MLCK-1 fragment was detected by autoradiography and
immunoreactivity with anti-phosphotyrosine antibody (upper
inset). After excision from the gel, the phosphoprotein was
subjected to complete trypsinolysis as described under "Materials and
Methods," and the peptide digest was analyzed using mass
spectrometry. A, high mass spectrum resolution of the peak
with an average m/z ratio 2486, which mathematically
corresponds to tryptic fragment 457-476 of MLCK-1 with two
incorporated phosphate groups. Shown are peaks with m/z
ratios 2484.9, 2485.9, and 2486.9 corresponding to isotopic variants of
the diphosphorylated MLCK-1 457-476 peptide. B, in
vitro p60Src-catalyzed phosphorylation of synthetic
peptides consisting of amino acids 968-985, 460-475, and 478-492 of
MLCK-1. Each peptide was tested in kinase assay as outlined under
"Materials and Methods." Each point represents the mean ± S.E. of three experiments. *, results are significant p < 0.005.
6
M) also abolished the enzymatic activity of EC MLCK-1
(phospho- and dephospho-), EC MLCK-2, and SM MLCK (Fig. 4). Finally,
phosphorylation of MLCK-1 by p60src did not alter
Ca2+/CaM-dependent regulation, because the
values for half-maximal activation of phospho- and dephospho-MLCK-1
determined over a range of free Ca2+ concentrations
(10
8 to 10
5 M) were comparable
(pCa 6.56 versus pCa 6.50, respectively) despite an increase of ~2-fold in enzymatic activity
toward MLC20 in the phospho-MLCK-1 preparation (Fig.
5). These data suggest that, although
similar Ca2+ concentrations are required for MLCK-1
activity, tyrosine phosphorylation promotes increased MLC
phosphorylation at lower Ca2+ concentrations within the
cells.
View larger version (22K):
[in a new window]
Fig. 4.
Effect of phosphorylation of endothelial MLCK
by p60src on MLCK activity in vitro.
A, kinase activity of MLCK-1 after 60-min incubation with
either p60src (solid bars) or vehicle
(crosshatched bars). The MLC kinase assay utilized
recombinant rabbit smooth muscle MLCs as substrate as described under
"Materials and Methods." B and C are similar
to A with the exception that MLCK-2 or SM MLCK were used in
each reaction mixture. MLCK-2 and SM MLCK incubated with (solid
bars) or without (open bars) p60src under
identical conditions were also tested for kinase activity. Removal of
Ca2+ from the kinase reaction mixture with EGTA or the
addition of specific MLCK inhibitor ML-7 (5 × 10 6
M) abolishes MLCK-1, MLCK-2, and SM MLCK activity.
MLCK-catalyzed 32P incorporation into myosin light chains
in the presence of Ca2+ and calmodulin in the reaction
mixture as described under "Materials and Methods" was taken as
100% MLCK activation. Each point represents the mean ± S.E. of
at least three experiments. *, results are significant
p < 0.005.
View larger version (21K):
[in a new window]
Fig. 5.
Effect of p60src-mediated MLCK-1
phosphorylation on Ca2+-dependent MLCK-1
activation. The enzymatic activities of p60src-treated
MLCK-1 (solid line) or MLCK-1 incubated with vehicle and
referred as dephospho-MLCK-1 (broken line) were measured in
the presence of 5 × 10 7 M calmodulin
over a range of Ca2+ concentrations (10
8 to
10
5 M) as described under "Materials and
Methods." The maximal activity of phospho-MLCK-1 observed in the
presence of 10
6 M [Ca2+] was
taken as 100%. Maximal activation of dephospho-MLCK-1 (incubated at
10
6 M [Ca2+]) represents 55%
of phospho-MLCK-1 activity. Each point represents the
mean ± S.E. of at least three experiments. *, results are
significant p < 0.005. EC50 values for
Ca2+ dependence of dephospho- and phospho-MLCK-1 activation
are outlined by vertical lines.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Amina Woods (National Institute on Drug Abuse, Baltimore, MD) for her helpful assistance with the mass spectrometry analysis of the MLCK-1 autophosphorylation sites. We gratefully acknowledge the invaluable secretarial support of Ellen Reather.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants HL50533 and HL58064.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.
Recipient of an American Heart Association Grant-In-Aid.
** To whom correspondence should be addressed: Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, JHAAC, 4B.77A, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. Tel.: 410-550-5960; Fax: 410-550-6985; E-mail: drgarcia@welch.jhu.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M005270200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MLCK, myosin light chain kinase; MLC20, regulatory myosin light chain; SM, smooth muscle; EC, endothelial cell; HUVEC, human umbilical vein endothelial cells; RT-PCR, reverse-transcriptase-polymerase chain reaction; kb, kilobase(s); bp, base pair(s); Ni-NTA, nickel-nitrilotriacetic acid; MOPS, 4-morpholinepropanesulfonic acid; MALDI TOF, matrix-assisted laser desorption time of flight; KRP, kinase-related protein; CaM, calmodulin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Kamm, K. E., and Stull, J. T. (1985) Annu. Rev. Pharmacol. Toxicol. 25, 593-620[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Ikebe, M.,
and Hartshorne, D. J.
(1985)
J. Biol. Chem.
260,
10027-10031 |
3. | Gallagher, P. J., Herring, B. P., and Stull, J. T. (1997) J. Muscle Res. Cell. Motil. 18, 1-16[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Roush, C. L.,
Kennelly, P. J.,
Glaccum, M. B.,
Helfman, D. M.,
Scott, J. D.,
and Krebs, E. G.
(1988)
J. Biol. Chem.
263,
10510-10516 |
5. | Olson, N. J., Pearson, R. B., Needleman, D. S., Hurwitz, M. Y., Kemp, B. E., and Means, A. R. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2284-2288[Abstract] |
6. | Potier, M. C., Chelot, E., Pekarsky, Y., Gardiner, K., Rossier, J., and Turnell, W. G. (1995) Genomics 29, 562-570[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Sweeney, H. L.,
Bowman, B. F.,
and Stull, J. T.
(1993)
Am. J. Physiol.
264,
C1085-C1095 |
8. | Sweeney, H. L., and Stull, J. T. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 414-418[Abstract] |
9. | Kamm, K. E., and Stull, J. T. (1986) Science 232, 80-82[Medline] [Order article via Infotrieve] |
10. | Tan, J. L., Ravid, S., and Spudich, J. A. (1992) Annu. Rev. Biochem. 61, 721-759[CrossRef][Medline] [Order article via Infotrieve] |
11. | Allen, B. G., and Walsh, M. P. (1994) Trends Biochem. Sci 19, 362-368[CrossRef][Medline] [Order article via Infotrieve] |
12. | Wysolmerski, R. B., and Lagunoff, D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 16-20[Abstract] |
13. | Garcia, J. G., Davis, H. W., and Patterson, C. E. (1995) J. Cell. Physiol. 163, 510-522[Medline] [Order article via Infotrieve] |
14. |
Garcia, J. G. N.,
Verin, A. D.,
Herenyiova, M.,
and English, D.
(1998)
J. Appl. Physiol.
84,
1817-1821 |
15. |
Saito, H.,
Minamiya, Y.,
Kitamura, M.,
Saito, S.,
Enomoto, K.,
Terada, K.,
and Ogawa, J.
(1998)
J. Immunol.
161,
1533-1540 |
16. |
Mills, J. C.,
Stone, N. L.,
Erhardt, J.,
and Pittman, R. N.
(1998)
J. Cell Biol.
140,
627-636 |
17. | Birukov, K. G., Schavocky, J. P., Shirinsky, V. P., Chibalina, M. V., Van Eldik, L. J., and Watterson, D. M. (1998) J. Cell. Biochem. 70, 402-413[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Gallagher, P. J.,
Garcia, J. G.,
and Herring, B. P.
(1995)
J. Biol. Chem.
270,
29090-29095 |
19. | Fisher, S. A., and Ikebe, M. (1995) Biochem. Biophys. Res. Commun. 217, 696-703[CrossRef][Medline] [Order article via Infotrieve] |
20. | Garcia, J. G., Lazar, V., Gilbert-McClain, L. I., Gallagher, P. J., and Verin, A. D. (1997) Am. J. Respir. Cell Mol. Biol. 16, 489-494[Abstract] |
21. |
Shirinsky, V. P.,
Vorotnikov, A. V.,
Birukov, K. G.,
Nanaev, A. K.,
Collinge, M.,
Lukas, T. J.,
Sellers, J. R.,
and Watterson, D. M.
(1993)
J. Biol. Chem.
268,
16578-16583 |
22. | Watterson, D. M., Collinge, M., Lukas, T. J., Van, E.ldik, L. J., Birukov, K. G., Stepanova, O. V., and Shirinsky, V. P. (1995) FEBS Lett. 373, 217-220[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Verin, A. D.,
Lazar, V.,
Torry, R. J.,
Labarrere, C. A.,
Patterson, C. E.,
and Garcia, J. G.
(1998)
Am. J. Respir. Cell Mol. Biol.
19,
758-766 |
24. |
Gallagher, P. J.,
Herring, B. P.,
Griffin, S. A.,
and Stull, J. T.
(1991)
J. Biol. Chem.
266,
23936-23944 |
25. | Kobayashi, H., Inoue, A., Mikawa, T., Kuwayama, H., Hotta, Y., Masaki, T., and Ebashi, S. (1992) J Biochem. (Tokyo) 112, 786-791[Abstract] |
26. | Garcia, J. G., Verin, A. D., Schaphorst, K., Siddiqui, R., Patterson, C. E., Csortos, C., and Natarajan, V. (1999) Am. J. Physiol. 276, L989-L998[Medline] [Order article via Infotrieve] |
27. | Shi, S., Verin, A. D., Schaphorst, K. L., Gilbert-McClain, L. I., Patterson, C. E., Irwin, R. P., Natarajan, V., and Garcia, J. G. (1998) Endothelium 6, 153-171[Medline] [Order article via Infotrieve] |
28. | Lazar, V., and Garcia, J. G. (1999) Genomics 57, 256-267[CrossRef][Medline] [Order article via Infotrieve] |
29. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
30. | Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract] |
31. |
Verin, A. D.,
Gilbert-McClain, L. I.,
Patterson, C. E.,
and Garcia, J. G.
(1998)
Am. J. Respir. Cell Mol. Biol.
19,
767-776 |
32. | Shoemaker, M. O., Lau, W., Shattuck, R. L., Kwiatkowski, A. P., Matrisian, P. E., Guerra-Santos, L., Wilson, E., Lukas, T. J., Van, E.ldik, L. J., and Watterson, D. M. (1990) J. Cell Biol. 111, 1107-1125[Abstract] |
33. | Bresnick, A. R., Wolff-Long, V. L., Baumann, O., and Pollard, T. D. (1995) Biochemistry 34, 12576-12583[Medline] [Order article via Infotrieve] |
34. | Imai, S., and Takeda, K. (1967) Nature 213, 1044-1045 |
35. |
Adelstein, R. S.,
and Klee, C. B.
(1981)
J. Biol. Chem.
256,
7501-7509 |
36. | Kemp, B. E., Pearson, R. B., and House, C. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7471-74715[Abstract] |
37. |
Ikebe, M.,
Reardon, S.,
Schwonek, J. P.,
Sanders, C. R., 2nd,
and Ikebe, R.
(1994)
J. Biol. Chem.
269,
28165-28172 |
38. | Gilbert-McClain, L. I., Verin, A. D., Shi, S., Irwin, R. P., and Garcia, J. G. (1998) J. Cell. Biochem. 70, 141-155[CrossRef][Medline] [Order article via Infotrieve] |
39. | Tokui, T., Ando, S., and Ikebe, M. (1995) Biochemistry 34, 5173-5179[Medline] [Order article via Infotrieve] |
40. | Abe, M., Hasegawa, K., and Hosoya, H. (1996) Cell Struct. Funct. 21, 183-188[Medline] [Order article via Infotrieve] |
41. | Moarefi, I., LaFevre-Bernt, M., Sicheri, F., Huse, M., Lee, C. H., Kuriyan, J., and Miller, W. T. (1997) Nature 385, 650-653[CrossRef][Medline] [Order article via Infotrieve] |
42. | Scott, M. P., and Miller, W. T. (2000) Biochemistry 39, 14531-14537[CrossRef][Medline] [Order article via Infotrieve] |
43. | Collinge, M., Matrisian, P. E., Zimmer, W. E., Shattuck, R. L., Lukas, T. J., Van Eldik, L. J., and Watterson, D. M. (1992) Mol. Cell. Biol. 12, 2359-2371[Abstract] |
44. |
Nishikawa, M.,
de Lanerolle, P.,
Lincoln, T. M.,
and Adelstein, R. S.
(1984)
J. Biol. Chem.
259,
8429-8436 |
45. |
Nishikawa, M.,
Shirakawa, S.,
and Adelstein, R. S.
(1985)
J. Biol. Chem.
260,
8978-8983 |
46. | Hashimoto, Y., and Soderling, T. R. (1990) Arch. Biochem. Biophys 278, 41-45[Medline] [Order article via Infotrieve] |
47. |
Sanders, L. C.,
Matsumura, F.,
Bokoch, G. M.,
and de Lanerolle, P.
(1999)
Science
283,
2083-2085 |
48. |
Kasturi, R.,
Vasulka, C.,
and Johnson, J. D.
(1993)
J. Biol. Chem.
268,
7958-7964 |
49. | Miller, J. R., Silver, P. J., and Stull, J. T. (1983) Mol. Pharmacol. 24, 235-242[Abstract] |
50. |
Klemke, R. L.,
Cai, S.,
Giannini, A. L.,
Gallagher, P. J.,
de Lanerolle, P.,
and Cheresh, D. A.
(1997)
J. Cell Biol.
137,
481-492 |
51. |
Garcia, J. G.,
Schaphorst, K. L.,
Shi, S.,
Verin, A. D.,
Hart, C. M.,
Callahan, K. S.,
and Patterson, C. E.
(1997)
Am. J. Physiol.
273,
L172-L184 |