From the Smooth Muscle Research Group and Canadian Institutes of Health Research Group in Regulation of Vascular Contractility, Department of Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, Calgary, Alberta T2N 4N1, Canada
Received for publication, December 22, 2000, and in revised form, February 7, 2001
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ABSTRACT |
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Smooth muscle contraction follows an increase in
cytosolic Ca2+ concentration, activation of myosin
light chain kinase, and phosphorylation of the 20-kDa light chain of
myosin at Ser19. Several agonists acting via G
protein-coupled receptors elicit a contraction without a change in
[Ca2+]i via inhibition of myosin light chain
phosphatase and increased myosin phosphorylation. We showed that
microcystin (phosphatase inhibitor)-induced contraction of skinned
smooth muscle occurred in the absence of Ca2+ and
correlated with phosphorylation of myosin light chain at Ser19 and Thr18 by a kinase distinct from
myosin light chain kinase. In this study, we identify this kinase as
integrin-linked kinase. Chicken gizzard integrin-linked kinase cDNA
was cloned, sequenced, expressed in E. coli, and shown to
phosphorylate myosin light chain in the absence of Ca2+ at
Ser19 and Thr18. Subcellular fractionation
revealed two distinct populations of integrin-linked kinase, including
a Triton X-100-insoluble component that phosphorylates myosin in a
Ca2+-independent manner. These results suggest a novel
function for integrin-linked kinase in the regulation of smooth muscle
contraction via Ca2+-independent phosphorylation of myosin,
raise the possibility that integrin-linked kinase may also play a role
in regulation of nonmuscle motility, and confirm that integrin-linked
kinase is indeed a functional protein-serine/threonine kinase.
Smooth muscle contraction is activated primarily by an increase in
cytosolic free Ca2+ concentration
([Ca2+]i) in response to membrane depolarization,
a variety of blood-borne agonists such as angiotensin II and
All of these signaling pathways terminate in inhibition of MLCP and an
increase in myosin phosphorylation, which accounts for the contractile
response. The kinase responsible for the increase in myosin
phosphorylation without a change in [Ca2+]i could
be MLCK itself that is partially activated at the prevailing
[Ca2+]i, or it could be a distinct kinase capable
of phosphorylating myosin at Ser19 in a
Ca2+-independent manner. We recently obtained evidence for
the presence in smooth muscle of a Ca2+-independent kinase
that can phosphorylate LC20 at Ser19 and
Thr18 (18). The addition of the phosphatase inhibitor
microcystin-LR to Triton X-100-demembranated (skinned) rat tail
arterial smooth muscle strips in the absence of Ca2+
elicited a contraction that reached a steady-state level of force comparable with that evoked in skinned muscle by Ca2+ or in
intact muscle by K+ depolarization. This
Ca2+-independent contraction correlated with
phosphorylation of myosin at Ser19 and Thr18 of
LC20. The kinase responsible for this phosphorylation was not MLCK for the following reasons: (i) its activity was independent of
Ca2+ and CaM; (ii) it did not bind to CaM in either the
presence or absence of Ca2+; (iii) its activity was
insensitive to AV25, a synthetic peptide inhibitor of MLCK
corresponding to the autoinhibitory (pseudosubstrate) domain of MLCK;
(iv) it phosphorylated Ser19 and Thr18 of
LC20, whereas MLCK only phosphorylates Thr18 at
high kinase concentrations and only after Ser19 has been
fully phosphorylated (6, 19); and (v) the activity could be separated
from MLCK. The kinase appeared to be associated with the myofilaments,
since Ca2+-independent kinase activity directed toward
endogenous myosin LC20 was recovered in myofilament
preparations. We were able to separate Ca2+-independent
kinase activity from MLCK by differential extraction from myofilaments
and by affinity chromatography on a column of CaM-Sepharose (18).
We report here the identification of this Ca2+-independent
kinase as integrin-linked kinase (ILK) and provide evidence for two distinct pools of this enzyme in smooth muscle, one of which is associated with the myofilaments and is responsible for the
Ca2+-independent phosphorylation of myosin. ILK could
account, at least in part, for the phosphorylation of myosin and
contraction that occurs in smooth muscle in response to agonists that
trigger a contractile response without a change in
[Ca2+]i. Furthermore, ILK could play a role in
the contractile response to agonists that induce both an increase in
[Ca2+]i and inhibition of MLCP. These results
also raise the possibility that ILK may play an important role in
regulation of nonmuscle motile processes such as cell migration,
chemotaxis, and cytokinesis. Finally, this work confirms that ILK is
indeed a functional protein-serine/threonine kinase.
Materials--
[ Purification of Ca2+-independent Myosin
LC20 Kinase--
Chicken gizzard myofilaments were
prepared as follows. All procedures were carried out at 0-4 °C.
Fresh gizzards (100 g) were trimmed, minced in a meat grinder, and
homogenized in 500 ml of buffer A (20 mM imidazole-HCl, pH
6.9, 60 mM KCl, 1 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol (DTT), 1 µg/ml
leupeptin, 0.5 mM phenylmethylsulfonyl fluoride) with the
aid of a Brinkman Polytron (setting 6). The homogenate was centrifuged
at 20,000 × g for 20 min. The supernatant was
discarded. The pellet was resuspended in 500 ml of buffer A plus 0.5%
Triton X-100, homogenized with the aid of a Brinkman Polytron (setting
5), and centrifuged as before. The pellet was resuspended in 500 ml of
buffer A plus 0.5% Triton X-100, homogenized, and centrifuged as
before. The pellet was resuspended in 500 ml of buffer A plus 0.3%
Triton X-100, homogenized, and centrifuged as before. The pellet was resuspended in 500 ml of buffer A, homogenized, and centrifuged as
before. The pellet (myofilament preparation) was resuspended in 500 ml
of buffer B (20 mM Tris-HCl, pH 7.5, 80 mM KCl,
30 mM MgCl2, 1 mM EGTA, 1 mM DTT, 1 µg/ml leupeptin, 0.5 mM
phenylmethylsulfonyl fluoride), stirred gently for 30 min, and
centrifuged as before. This extraction with 30 mM
MgCl2 removes almost all the MLCK (18). The pellet was
resuspended in 473.7 ml of buffer B, and 31.3 ml of buffer C (buffer B
plus 4 M NaCl) were added slowly with stirring. The mixture
was stirred gently for 30 min and centrifuged as before. The
supernatant, which contained the Ca2+-independent myosin
LC20 kinase, was dialyzed overnight against buffer C (20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 0.1 M NaCl, 1 mM DTT) to
lower the NaCl concentration. The dialysate was centrifuged as before
to remove particulate material, and the supernatant was subjected to
chromatography at a flow rate of 40 ml/h on a column of DEAE-Sephacel
(1.5 × 20 cm) previously equilibrated with buffer C. Fractions
containing Ca2+-independent kinase activity were identified
by assaying myosin phosphorylation in the absence of Ca2+
as previously described (18). The flow-through fractions, which contained the Ca2+-independent LC20 kinase,
were dialyzed overnight against buffer D (20 mM NaHepes, pH
7.5, 5 mM MgCl2, 1 mM EGTA, 0.1 M NaCl, 1 mM DTT). The dialysate was applied at
a flow rate of 60 ml/h to a column of SP-Sepharose Fast Flow (1.5 × 15 cm) previously equilibrated with buffer D, and the column was
washed with 100 ml of buffer D. Bound proteins, including the
Ca2+-independent LC20 kinase, were eluted with
a linear [NaCl] gradient (0.1-0.6 M) at 40 ml/h,
collecting 10-ml fractions. Fractions containing
Ca2+-independent kinase were pooled, dialyzed overnight
against buffer D, and applied to a Mono Q FPLC column, eluting bound
proteins with a linear [NaCl] gradient (0.1-0.4 M in
buffer D). Fractions of 1 ml were collected and assayed for
Ca2+-independent MLCK activity.
In-gel Kinase Assay--
Protein samples were mixed with an
equal volume of 2 × SDS gel sample buffer (50 mM
Tris-HCl, pH 6.8, 1% SDS, 30% glycerol, and 0.01% bromphenol blue)
and incubated at 20 °C for 2 h prior to electrophoresis in SDS
gels with a 10-20% acrylamide gradient (23) with LC20
throughout the 0.75-mm thick gel (5 µg/ml running gel solution). Gels
were washed at room temperature with 25 mM Tris-HCl, pH
7.5, 60 mM KCl, 10 mM MgCl2, 10 mM DTT, 10 mM EGTA, 2.5% Triton X-100 for
2 h to remove the SDS and then washed for a further 2 h in 20 mM Tris-HCl, pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 mM DTT, 10 mM EGTA, 0.1% Tween 80 (kinase assay buffer). The buffer
was replaced with 25 ml of fresh kinase assay buffer, and LC20 phosphorylation was initiated by the addition of 60 µM ATP containing 100 µCi of
[ Cloning of Chicken Gizzard ILK cDNA--
A partial ILK
cDNA was cloned from chicken gizzard mRNA by reverse
transcriptase-polymerase chain reaction using primers
(5'-TGTGGCTGGACAACACAGAG-3' and 5'-GAGAGGTCAGCAAAGGGCAC-3')
corresponding to nucleotides 53-72 and 1168-1187, respectively,
within the coding region of the human cDNA (24). Full-length ILK
cDNA was cloned by 5'- and 3'-rapid amplification of cDNA ends
(25). The full-length cloned cDNA was sequenced by the Sanger
dideoxynucleotide chain termination method (26).
Mass Spectrometric Analysis--
Matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)
was performed on the trypsin-digested 59-kDa kinase band from the Mono
Q column (see Fig. 2B), and one of the peptides (1594 Da)
was directly sequenced by MALDI-TOF MS in the post-source decay mode by
Borealis Biosciences Inc. (Toronto, Ontario, Canada).
Expression and Purification of Glutathione S-Transferase
(GST)-ILK--
Full-length chicken ILK cDNA was inserted into the
EcoRI/BamHI site of the vector pGEX-2T (Amersham
Pharmacia Biotech). The recombinant plasmid pGEX-2T-ILK and the empty
vector as a negative control were used to transform competent
BL21(DE3)pLysS cells. Expression was induced with 0.1 mM
isopropyl The Ca2+-independent Myosin Light Chain Kinase Is
Integrin-linked Kinase--
We showed previously that myofilaments
isolated from chicken gizzard smooth muscle contain endogenous
Ca2+-independent kinase activity toward myosin
LC20, phosphorylation occurring at Ser19 and
Thr18 (18). These myofilaments also retain the full
complement of cellular MLCK (18). To estimate the relative activities
of these two myofilament-associated kinases, we examined the effect of synthetic peptide inhibitors of MLCK, corresponding to the
autoinhibitory domain of MLCK, on the phosphorylation of endogenous
myosin in the presence of Ca2+ and CaM. These peptides are
the most highly selective inhibitors of MLCK (27-29). SM-1 corresponds
to the autoinhibitory sequence of MLCK (residues 783-804 of chicken
gizzard MLCK), and AV25 is a similar peptide corresponding to residues
783-807 of chicken gizzard MLCK with three substitutions
(Trp800 was replaced by Leu to effectively eliminate
interaction of the peptide with CaM, and Ser787 and
Thr803 were replaced by Ala to eliminate phosphorylatable
residues). The two peptides proved to be equipotent inhibitors of MLCK,
so data obtained from their use were combined. As shown in Fig.
1, most of the MLCK activity in
myofilaments can be accounted for by
Ca2+/CaM-dependent MLCK, but a significant
proportion was found to be insensitive to inhibition by the
autoinhibitory domain of MLCK (Fig. 1A). For comparison,
purified MLCK was completely inhibited by the MLCK inhibitory peptides,
with half-maximal inhibition at 1 µM peptide (Fig.
1B). [Ala9]autocamtide 2, a peptide inhibitor
of Ca2+/CaM-dependent protein kinase II (CaM
kinase II), an enzyme known to phosphorylate myosin in vitro
(30), had no effect on Ca2+-independent LC20
phosphorylation in myofilaments but potently inhibited the
phosphorylation of caldesmon in myofilaments by exogenous CaM kinase
II, as expected (18). CaM kinase II, or a constitutively active
proteolytic fragment of CaM kinase II, is therefore not involved in
myosin phosphorylation in myofilaments. On the other hand, the
Ca2+-independent phosphorylation of myosin in myofilaments
was inhibited by staurosporine, a relatively nonselective kinase
inhibitor, with half-maximal inhibition at ~0.2 µM
(18). Staurosporine also inhibited microcystin-induced,
Ca2+-independent contraction of skinned smooth muscle (18).
The Ca2+-independent phosphorylation of myosin in
myofilaments was insensitive to the PKC-selective inhibitor
chelerythrine and the ROK inhibitor HA-1077 (18).
The MLCK activity that was resistant to the MLCK inhibitor peptides was
partially purified from myofilaments by first removing MLCK by
extraction with 30 mM Mg2+ (18). The
Ca2+-independent kinase was then extracted with 0.25 M NaCl and further purified by sequential chromatography on
columns of DEAE-Sephacel, SP-Sepharose, and Mono Q. The elution profile
of Ca2+-independent MLCK activity from the final column is
shown in Fig. 2A. An in-gel
kinase assay in the absence of Ca2+, with isolated
LC20 as substrate, showed that this activity resides in a
59-kDa protein (Fig. 2B). This band, which was barely
visible in a Coomassie Blue-stained gel of the corresponding Mono Q
fractions (Fig. 2C), was cut out of the gel, digested with
trypsin, and analyzed by MALDI-TOF MS (Table
I) (31). Comparison of the measured
masses of the tryptic peptides with predicted masses from the
protein sequence data bases revealed a single match to ILK (24).
Peptides 1-11 perfectly matched predicted tryptic peptides from human
ILK, and together the 11 peptides accounted for 31% of the complete
sequence. To confirm its identity as ILK, one of the tryptic peptides
(peptide 6) was selected and sequenced directly by MALDI-TOF MS in the
postsource decay mode (31). Its sequence was confirmed as
GMAFLHTLEPLIPR, corresponding to residues 304-317 of human ILK. From
the mass spectrometric analysis, there was no evidence of a second
protein in the 59-kDa band used for trypsin digestion.
Identification of the Ca2+-independent MLCK as ILK was
confirmed by Western blotting with an antibody to human ILK. Fig.
3 shows a correlation of
Ca2+-independent MLCK activity (eluted from the Mono Q
column) in the in-gel kinase assay (Fig. 3A) and
immunoreactivity with anti-ILK (Fig. 3B), both at 59 kDa.
ILK Is Not Contaminated with a MLCK
Fragment--
Ca2+/CaM-dependent MLCK can be
proteolytically degraded to a constitutively active fragment that
retains the catalytic domain but lacks the autoinhibitory and
CaM-binding domains (32). To rule out the possibility that ILK purified
through the Mono Q column is contaminated with a constitutively active
fragment of Ca2+/CaM-dependent MLCK, pooled ILK
fractions from the Mono Q column were subjected to Western blotting
with an antibody that recognizes both intact MLCK (130 kDa) and the
61-kDa catalytically active fragment of
Ca2+/CaM-dependent MLCK (18). Fig.
4 shows that the ILK preparation contains
no trace of Ca2+/CaM-dependent MLCK fragments
(lane 3), whereas both intact
Ca2+/CaM-dependent MLCK (lane
1) and the 61-kDa constitutively active tryptic fragment
(lane 2) are clearly recognized by the antibody. Furthermore, Ca2+-independent phosphorylation of myosin by
ILK purified through the Mono Q column was unaffected by AV25,
supporting the conclusion that the ILK preparation is not contaminated
with proteolyzed MLCK (Fig. 5).
ILK Phosphorylates Myosin at Ser19 and
Thr18 of LC20--
Mono Q-purified ILK
catalyzed mono- and diphosphorylation of isolated LC20
(Fig. 6A, lanes
2 and 3) and intact myosin (Fig. 6A,
lane 5). Isolated LC20 was
phosphorylated to a stoichiometry of 1.64 mol of Pi/mol of
LC20 (36.2% monophosphorylated LC20 and 63.8%
diphosphorylated LC20) and intact myosin to 1.14 mol of Pi/mol of myosin (45.4% monophosphorylated
LC20 and 5.7% diphosphorylated LC20). The
sites of phosphorylation were identified as Ser19 and
Thr18 using phosphorylation site-specific antibodies (Fig.
6B).
ILK Exists in Two Distinct Subcellular Fractions in Smooth
Muscle--
While a portion of the ILK in chicken gizzard smooth
muscle was recovered in the cytosolic fraction following homogenization of the tissue in the absence of detergent (Fig.
7, lane 2), a substantial fraction remained in the Triton-insoluble fraction composed
of myofilaments and cytoskeleton (Fig. 7, lane
6), consistent with its retention in Triton-skinned smooth
muscle, where it phosphorylates myosin in situ in the
absence of Ca2+ and elicits a Ca2+-independent
contraction (18). From in-gel kinase assays, ~73% of total ILK
activity was recovered in the Triton-insoluble fraction.
Chicken ILK Is Very Similar to Human ILK--
ILK cDNA was
cloned from chicken gizzard mRNA by reverse
transcription-polymerase chain reaction (33) and sequenced (26). The
nucleotide and deduced amino acid sequences of chicken ILK are shown in
Fig. 8 and are available from
GenBankTM under accession number AF296130. A comparison of
the chicken and human ILK amino acid sequences is shown in Fig.
9. The deduced amino acid sequence of
chicken ILK is 90.7% identical and 95.4% similar to the human ILK
sequence (GenBankTM accession number U40282), with 21 conservative and 21 nonconservative substitutions in the 452-residue
protein. Compared with many other protein-serine/threonine kinases, ILK
contains a number of amino acid substitutions in highly conserved
subdomains within the catalytic domain (34). The
GXGXXG sequence in subdomain I of most protein kinases is NENHSG in human ILK and NENQSG in the chicken enzyme. Conserved residues in subdomain VIB (HDRL) are PRHA in human ILK and
PRHH in chicken ILK, and conserved residues in subdomain VII (DFG)
are MAD in both ILKs. On the basis of these amino acid substitutions, it has been suggested that ILK may not be a functional protein kinase
(35, 36). The fact that we isolated the Ca2+-independent
MLCK activity and then identified it as ILK confirms that ILK is indeed
an active protein-serine/threonine kinase.
Reexamination of the mass spectrometric analysis of chicken ILK based
on the deduced chicken sequence identified nine more peptide matches
covering an additional 18% of the sequence (Table II).
Expression of Chicken ILK--
Full-length chicken ILK was
expressed in E. coli as a GST fusion protein and purified
from a bacterial cell lysate by glutathione-Sepharose affinity
chromatography (Fig. 10A).
Purified GST-ILK was shown to phosphorylate LC20 in intact
smooth muscle myosin in the absence of Ca2+ (Fig.
10B).
The mechanism underlying the phenomenon of Ca2+
sensitization of smooth muscle contraction has received a great deal of
attention recently (9). Ca2+ sensitization refers to the
process whereby agonist-induced contraction of smooth muscle occurs
without a change in [Ca2+]i and appears to
involve signal transduction pathways that terminate in inhibition of
MLCP, an increase in myosin LC20 phosphorylation, and
resultant cross-bridge cycling. Inhibition of MLCP per se
cannot induce an increase in LC20 phosphorylation, but
rather alters the balance of kinase/phosphatase activity in favor of
the kinase. The question then arises as to which kinase is responsible
for LC20 phosphorylation following inhibition of MLCP. An
obvious candidate is Ca2+/CaM-dependent MLCK.
In this case, in the absence of agonist, the activity of MLCP would
presumably mask the basal activity of MLCK that exists at the
prevailing [Ca2+]i, and agonist-induced
inhibition of MLCP would unmask this basal MLCK activity, leading to an
increase in LC20 phosphorylation and contraction. However,
resting [Ca2+]i in smooth muscle cells, which is
in the range of 130-140 nM (37, 38), would be insufficient
to induce significant activation of MLCK (39). This raises the
possibility that a distinct, Ca2+-independent kinase may be
responsible for the observed increase in LC20
phosphorylation and contraction at constant
[Ca2+]i, i.e. Ca2+
sensitization. In support of this possibility, we demonstrated recently
that the phosphatase inhibitor microcystin can trigger LC20
phosphorylation and contraction of skinned smooth muscle in the absence
of Ca2+, phosphorylation occurring at Ser19 and
Thr18, Ser19 being the principal site of
phosphorylation by Ca2+/CaM-dependent MLCK
(18). This Ca2+-independent phosphorylation was catalyzed
by a kinase distinct from MLCK, since it was found to be
Ca2+/CaM-independent, resistant to the selective MLCK
peptide inhibitors AV25 and SM-1, and could be separated from MLCK. In
this study, we show that, under the conditions used in the experiments
of Fig. 1, a significant proportion of the myosin phosphorylating activity was resistant to the MLCK-selective peptide inhibitors AV25
and SM-1.
Using other selective kinase inhibitors, we have also shown that this
kinase is not CaM kinase II, PKC, or ROK (18). It is also distinct from
a kinase that was identified in preparations of MLCP that
phosphorylated both the myosin targeting subunit of MLCP and myosin
LC20, since that kinase was shown to be sensitive to
chelerythrine (40). It is, however, sensitive to the nonselective kinase inhibitor staurosporine; the myofilament-associated,
Ca2+-independent kinase activity was half-maximally
inhibited at ~0.2 µM and the partially purified kinase
at ~0.3 µM staurosporine. As shown in Figs. 4 and 5, we
confirmed that the kinase preparation is not contaminated with a
fragment of MLCK that has lost the requirement for Ca2+ and
CaM due to partial proteolysis. Consistent with its ability to
phosphorylate myosin in skinned smooth muscle strips, this kinase is
retained in myofilament preparations, suggesting its association with a
component of the myofilaments.
The principal objective of this work was to isolate and identify this
Ca2+-independent myosin LC20 kinase, starting
from myofilaments prepared from chicken gizzard, a rich source of
smooth muscle tissue. The bulk of MLCK was first extracted from the
myofilaments by treatment with 30 mM Mg2+,
following which the Ca2+-independent kinase was extracted
with high [NaCl]. The extracted kinase was then further purified by
sequential chromatography on columns of DEAE-Sephacel, SP-Sepharose,
and Mono Q. The partially purified kinase phosphorylated isolated
LC20 and intact myosin at Ser19 and
Thr18 of LC20 in a Ca2+-independent
manner (Figs. 5 and 6). The kinase activity was associated with a
59-kDa protein, as shown by the ability of fractions from the Mono Q
column to phosphorylate myosin in the absence of Ca2+ in
solution and to phosphorylate isolated LC20 in a
Ca2+-independent manner in the in-gel kinase assay (Fig.
2). Tryptic digestion, peptide mass fingerprinting by mass
spectrometry, and de novo sequencing of a selected tryptic
peptide, followed by data base searching, identified the kinase as ILK
(Tables I and II). Its identity was confirmed by Western blotting with
antibodies to ILK (Fig. 3).
ILK was originally discovered, through yeast two-hybrid screening, as
an enzyme that binds to the cytoplasmic domain of Gohla et al. (43) recently demonstrated that receptors for
contractile agonists such as endothelin-1, angiotensin II, and vasopressin are coupled to both Gq/11 and
G12/13, the latter being upstream of RhoA and ROK. Such
agonists, therefore, appear to induce contraction via both activation
of Ca2+/CaM-dependent MLCK (downstream of
Gq/11) and inhibition of MLCP (downstream of
G12/13). The possibility arises that ILK may also play a
role in these contractile responses, contributing to the overall
increase in LC20 phosphorylation (Fig.
11).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
1-adrenergic agonists, or stretch (1, 2).
Ca2+ originates from the extracellular space and/or
intracellular stores, principally the sarcoplasmic reticulum (3).
Ca2+ binds to calmodulin
(CaM)1 (4), which activates
myosin light chain kinase (MLCK) (5), resulting in phosphorylation of
myosin II, specifically at Ser19, and sometimes to a small
extent at Thr18 (6-8), of the pair of 20-kDa light chain
subunits (LC20). This simple phosphorylation reaction
triggers cross-bridge cycling and the development of force or
shortening of the muscle (1, 2). Several agonists that act via
seven-transmembrane domain-containing, G protein-coupled receptors
elicit a contractile response without a change in
[Ca2+]i (9). This generally results from
inhibition of myosin light chain phosphatase (MLCP), a
myosin-associated type 1 protein-serine/threonine phosphatase that
dephosphorylates LC20 phosphorylated by MLCK (10). MLCP
inhibition occurs via signaling pathways that involve (i) protein
kinase C (PKC) and its substrate protein CPI-17 (17-kDa phosphatase
inhibitor and substrate of protein kinase C), which becomes a very
potent inhibitor of type 1 phosphatase after phosphorylation by PKC
(11, 12); (ii) the small GTPase RhoA, which activates Rho-associated
kinase (ROK), which in turn phosphorylates the myosin-targeting
subunit of MLCP, inhibiting phosphatase activity (13, 14); or
(iii) arachidonic acid, which inhibits MLCP activity either directly
(15) or via activation of an atypical PKC isoenzyme (16) or ROK (17). Of these signal transduction pathways, the RhoA-ROK pathway, appears to
be the most important quantitatively (9).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-32P]ATP (>5000 Ci/mmol) was
purchased from Amersham Pharmacia Biotech, Triton X-100 was from Roche
Molecular Biochemicals, Tween 80 was from Fisher, anti-ILK was from
Upstate Biotechnology Inc., and molecular mass marker proteins were
from New England Biolabs. MLCK inhibitor peptides AV25
(AKKLAKDRMKKYMARRKLQKAGHAV) and SM-1 (AKKLSKDRMKKYMARRKWQKTG) were
synthesized in the Peptide Synthesis Core Facility at the University of
Calgary. The purity of the peptides (>95%) was confirmed by
analytical high performance liquid chromatography and amino acid
analysis. Chicken gizzard myosin II (20), LC20 (21), MLCK
(22), and the 61-kDa constitutively active fragment of MLCK (18) were
purified as described. Anti-MLCK was generously provided by Dr. David
Hartshorne (University of Arizona). Phosphorylation site-specific
antibodies to LC20 were generously provided by Drs. Y. Sasaki and M. Seto (Asahi Chemical Industry Co., Ltd., Shizuoka,
Japan); antibody pLC1 recognizes exclusively LC20
phosphorylated at Ser19, and antibody pLC2 recognizes
exclusively LC20 phosphorylated at both Ser19
and Thr18 (18). All other reagents were analytical grade or
better and were purchased from VWR Canlab or Sigma.
-32P]ATP. After incubation at 20 °C for 3 h,
the gel was washed extensively with 5% trichloroacetic acid, 1%
sodium pyrophosphate until the radioactivity in the wash solution was
negligible. The gel was stained, destained, dried, and exposed to x-ray
film (23).
-D-thiogalactopyranoside for 3 h. GST-ILK
was purified from the bacterial lysate by glutathione-Sepharose 4B
affinity chromatography, and the GST was cleaved off by thrombin treatment following the manufacturer's instructions (Amersham Pharmacia Biotech).
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
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REFERENCES
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Fig. 1.
Smooth muscle myofilaments contain two
distinct myosin light chain kinase activities. A,
phosphorylation of the endogenous LC20 in chicken gizzard
myofilaments was quantified in the presence of the indicated
concentrations of the Ca2+/CaM-dependent MLCK
inhibitory peptides AV25 or SM-1 (the effects of the two related
peptides were identical, and therefore data have been combined).
Conditions were as follows: 25 mM Tris-HCl, pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 mM DTT, 0.1 mM CaCl2, 10 µM okadaic acid, 10 µg/ml CaM, 0.63 mg/ml myofilament
protein (0.111 mg/ml myosin), 0-100 µM peptide, and 1 mM [ -32P]ATP. Reactions (60 µl) were
started with ATP, incubated at 30 °C for 75 s, and stopped by
the addition of an equal volume of 2× SDS gel sample buffer and
boiled. Samples were electrophoresed in 10-20% polyacrylamide
gradient gels and autoradiographed. LC20 phosphorylation
was quantified by scanning densitometry, and results are expressed as
mol of Pi incorporated/mol of LC20
(urea/glycerol gel electrophoresis (18) indicated that the level of
LC20 phosphorylation in the absence of peptide was 1.0 mol
of Pi/mol of LC20). Numbers beside
the symbols indicate n values. B, the
activity of purified Ca2+/CaM-dependent MLCK
was assayed at the indicated concentrations of AV25 or SM-1 (again
effects were identical, and therefore data have been combined).
Conditions were as follows: 25 mM Tris-HCl, pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 mM DTT, 0.1 mM CaCl2, 0.1% Tween
80, 10 µM microcystin-LR, 10 µg/ml CaM, 10 µg/ml
MLCK, 0.5 mg/ml myosin, 0-100 µM peptide, and 0.2 mM [
-32P]ATP (300 cpm/pmol). Reactions (30 µl) were started with ATP, incubated at 30 °C for 75 s, and
stopped by spotting 20 µl on P81 paper. LC20
phosphorylation was quantified as described (18). The inset
shows a replot of the data on a log scale to show the peptide
concentration dependence of the inhibition more clearly.
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Fig. 2.
Elution profile of
Ca2+-independent MLCK from Mono Q. A,
fractions eluted from the Mono Q column were assayed for MLCK activity
in the absence of Ca2+ with intact myosin as substrate.
Phosphorylation of LC20 was analyzed by SDS-PAGE and
autoradiography. The autoradiogram is shown. B, fractions
eluted from the Mono Q column were subjected to an in-gel kinase assay
to identify the Ca2+-independent MLCK. Isolated
LC20 was incorporated throughout the gel by the addition to
the gel buffers prior to polymerization. Mono Q fractions were then
subjected to SDS-PAGE, following which the proteins in the gel were
renatured and [ -32P]ATP was added to the bathing
solution. Incorporation of 32P into LC20 was
identified by autoradiography. The arrow indicates the
Ca2+-independent MLCK of 59 kDa. Numbers to the
right (in kDa) indicate the positions of molecular mass
marker proteins. C, Coomassie Brilliant Blue-stained SDS gel
of Mono Q fractions. The faintly staining band at 59 kDa in fractions
11-15 (indicated by the arrow) correlates with
Ca2+-independent MLCK activity. This band was recognized by
anti-ILK (see Fig. 3). The stronger staining band of the same molecular
weight in fractions 16-21 is a distinct protein (it was not recognized
by anti-ILK).
MALDI-TOF MS analysis of Ca2+-independent myosin light chain
kinase
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Fig. 3.
Correlation between in-gel kinase activity
and ILK immunoreactivity. Mono Q fractions corresponding to the
elution of Ca2+-independent MLCK activity were subjected to
the in-gel kinase assay (A), and the same fractions were
immunoblotted with anti-ILK (B). Lanes
1-10 correspond to successive fractions in the peak of
Ca2+-independent MLCK activity (identified by solution
assay with intact myosin as substrate; cf. Fig.
2A).
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Fig. 4.
ILK purified through the Mono Q column is not
contaminated with MLCK. Intact MLCK (1.3 µg; lane
1), a constitutively active fragment of MLCK (61 kDa)
generated by trypsin digestion (0.61 µg; lane
2), and pooled ILK-containing fractions from the Mono Q
column (6 µg of protein; lane 3)
were subjected to Western blotting with anti-MLCK.
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Fig. 5.
Phosphorylation of myosin by Mono Q-purified
Ca2+-independent myosin LC20 kinase is
unaffected by AV25. Smooth muscle myosin was incubated under
phosphorylating conditions in the absence (lanes
1 and 2) and presence (lanes
3 and 4) of pooled ILK-containing fractions from
the Mono Q column (178.5 µl/ml) and in the absence and presence of 50 µM AV25, as described previously (18). At the end of the
reaction, samples (40 µl) were subjected to SDS-PAGE and
autoradiography. The autoradiogram is shown. Numbers to the
right indicate molecular mass markers.
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Fig. 6.
Ca2+-independent MLCK catalyzes
mono- and diphosphorylation of isolated LC20 and intact
myosin at Ser19 and Thr18. A,
LC20 (lanes 1-3) or myosin
(lanes 4 and 5) was incubated under
phosphorylating conditions in the absence (lanes
1 and 4) or presence of Mono Q-purified
Ca2+-independent MLCK (lanes 2,
3, and 5). Two different kinase preparations were
used in lanes 2 and 3.
Unphosphorylated and mono- and diphosphorylated forms of
LC20 (designated P0-, P1-, and
P2-LC20, respectively) were separated by
urea/glycerol gel electrophoresis (18). The two fastest migrating bands
in lanes 4 and 5 are CaM and the
17-kDa light chain of myosin. B, intact myosin was incubated
under phosphorylating conditions in the absence (lanes
1 and 3) or presence of Mono Q-purified
Ca2+-independent MLCK (lanes 2 and
4) prior to SDS-PAGE and Western blotting with antibody pLC1
that recognizes only LC20 phosphorylated at
Ser19 (lanes 1 and 2) or
antibody pLC2 that recognizes only LC20 phosphorylated at
both Ser19 and Thr18 (lanes
3 and 4).
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Fig. 7.
Subcellular fractionation indicates two
distinct populations of ILK in smooth muscle. Fractions obtained
during the purification of ILK as described under "Experimental
Procedures" were analyzed by Western blotting with anti-ILK.
Lane 1, chicken gizzard total homogenate;
lane 2, the homogenate supernatant (cytosolic
fraction prepared in the absence of detergent); lanes
3-5, three successive washes of the pellet with Triton
X-100 (0.5, 0.5, and 0.3%, respectively) following centrifugation of
the homogenate; lane 6, the final
detergent-washed pellet or myofilament preparation; lane
7, pooled fractions from the DEAE-Sephacel column;
lane 8, pooled fractions from the SP-Sepharose
column; lane 9, pooled fractions from the Mono Q
columns. Samples of 30 µl were applied to each lane.
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Fig. 8.
The nucleotide sequence and deduced amino
acid sequence of chicken ILK. The cloned ILK cDNA was 1934 nucleotides in length, and the encoded protein consists of 452 amino
acids. The asterisk denotes a stop codon. These sequences
are available from GenBankTM under accession number
AF296130.
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Fig. 9.
Comparison of the amino acid sequences of
chicken and human ILK. The upper sequence
corresponds to chicken ILK, and the lower sequence
corresponds to human ILK.
Further MALDI-TOF MS analysis of Ca2+-independent myosin light
chain kinase
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Fig. 10.
Expression and characterization of chicken
ILK. A, Coomassie Blue-stained gel of purified GST-ILK
expressed in E. coli (lane 3) and of
purified ILK (lane 2) and GST (lane
4) following digestion of GST-ILK with thrombin and
purification. Molecular mass marker proteins are shown in
lane 1 with their masses in kDa indicated to the
left. B, phosphorylation of myosin by bacterially
expressed ILK. Intact myosin (0.5 mg/ml) was incubated at 30 °C for
60 min in 25 mM Tris-HCl, pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 mM DTT, 0.1% Tween
80, 10 mM EGTA, 10 µM microcystin, and 0.2 mM ATP (containing 100 µCi/ml [ -32P]ATP)
in the absence (lane 1) or presence
(lane 2) of purified GST-ILK. LC20
phosphorylation was analyzed by SDS-PAGE and autoradiography. The
autoradiogram shows the ILK-dependent phosphorylation of
LC20 in intact myosin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-integrins and
plays a role in integrin-mediated signal transduction (24, 41). Since
integrins are plasma membrane proteins, the identification of the
Ca2+-independent LC20 kinase as ILK raised the
question of how a plasma membrane-associated kinase would have access
to a substrate (myosin) located in the myofilaments. Using a
subcellular fractionation approach, we identified two distinct
populations of ILK, one with the expected properties of
integrin-associated ILK and the other that was retained in myofilament
preparations (Fig. 7). Presumably, the latter population is responsible
for the observed Ca2+-independent phosphorylation of myosin
in myofilaments and skinned smooth muscle preparations. It is
noteworthy that ILK contains four ankyrin repeat motifs in the
N-terminal domain and that the myosin-targeting subunit of
MLCP, which anchors the phosphatase to the myosin filaments, contains
seven N-terminal ankyrin repeats, and this region of the
molecule is involved in the interaction with myosin (42). It is
feasible, therefore, that ILK may associate with the myosin filaments
of smooth muscle via its ankyrin repeats. In support of this
possibility, purified myosin contains a low level of
Ca2+-independent LC20 kinase activity that is
resistant to the MLCK inhibitor AV25 (Fig. 5, lanes
1 and 2). It remains possible, however, that ILK
may be associated with a protein(s) other than myosin in the myofilaments.
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Fig. 11.
Proposed mechanism of regulation of smooth
muscle contraction via inhibition of MLCP via the RhoA/ROK pathway and
phosphorylation of myosin by ILK. Contractile agonists such as
endothelin-1, angiotensin II, and vasopressin act via receptors that
are coupled to both Gq/11 and G12/13
heterotrimeric G proteins (43). Gq/11 activates
phospholipase C (PLC
), which hydrolyzes
phosphatidylinositol 4,5-bisphosphate (PIP2) to
generate inositol 1,4,5-trisphosphate (IP3) and
1,2-diacylglycerol. Inositol 1,4,5-trisphosphate diffuses to the
sarcoplasmic reticulum (SR), where it interacts with the
inositol 1,4,5-trisphosphate receptor, a Ca2+ release
channel in the sarcoplasmic reticulum membrane, thereby triggering
Ca2+ release from the sarcoplasmic reticulum. The increase
in [Ca2+]i activates CaM-dependent
MLCK and cross-bridge cycling. G12/13, on the other hand,
probably activates the guanine nucleotide exchange factor p115 RhoGEF
(GEF) (48). Activated guanine nucleotide exchange factor
triggers the dissociation of a guanine nucleotide dissociation
inhibitor from the small GTPase RhoA, replacement of GDP by GTP on
RhoA, and translocation of RhoA to the plasma membrane, where it
inserts into the membrane via its geranylgeranyl moiety (indicated as a
green extension) (9). Activated RhoA-GTP
activates ROK, which phosphorylates the myosin binding regulatory or
targeting subunit of MLCP, resulting in inhibition of the phosphatase
activity (13, 14). This alters the MLCP/ILK activity ratio in favor of
the kinase, resulting in an increase in myosin phosphorylation and
contraction. If this occurs without an increase in
[Ca2+]i, which would occur if the agonist is
coupled to G12/13 but not Gq/11, as in the case
of thromboxane A2, for example (9), then ILK could account entirely for
the increase in myosin phosphorylation. This mechanism would then
account for Ca2+ sensitization of contraction. On the other
hand, if the agonist is coupled to both G12/13 and
Gq/11, as in the case of endothelin-1, for example (43),
then both ILK and Ca2+/CaM-dependent MLCK would
account for the increase in myosin phosphorylation. Not depicted in
this figure are other mechanisms of inhibition of MLCP
involving PKC/CPI-17 or arachidonic acid, which are referred to in the
Introduction.
Two recent reports have suggested that ILK may not be a functional protein-serine/threonine kinase, based on a number of amino acid substitutions in highly conserved regions within the catalytic domain and the inability to detect kinase activity in immunoprecipitates of ILK (35, 36). The rationale based on ILK sequence differences is unconvincing, since other active protein kinases have been identified with amino acid substitutions in highly conserved regions. For example, the kinases Mik1 and Vps15p lack all three glycine residues in the GXGXXG motif (44, 45). Furthermore, we expressed ILK in E. coli and detected Ca2+-independent kinase activity toward intact myosin, as shown in Fig. 10. However, our ability to detect this activity required denaturation followed by careful renaturation of the expressed kinase. Furthermore, the activity of the bacterially expressed ILK was low relative to that of the smooth muscle enzyme, suggesting that the kinase expressed in bacteria does not fold uniformly into the active conformation and may require post-translational modification. Based on these observations and the fact that we first detected the Ca2+-independent kinase activity in smooth muscle myofilaments and then identified the kinase as ILK, we conclude that ILK is indeed a bona fide protein-serine/threonine kinase.
ILK is widespread in terms of its tissue distribution, being expressed in most mammalian cells, with highest expression levels in cardiac and skeletal muscle, and its sequence is highly conserved across species. Regulation of nonmuscle motility involves myosin phosphorylation (9), similar to the regulation of smooth muscle contraction. The possibility arises, therefore, that ILK may also play a role in regulation of nonmuscle motile processes such as cell migration, chemotaxis, cytokinesis, and fibroblast contraction mediating orientation of collagen fibers in connective tissue. Indeed, Kolodney et al. (46) recently demonstrated that contraction of chick embryo fibroblasts in response to fetal bovine serum stimulation involves the Ca2+-independent phosphorylation of myosin II.
Finally, it is likely that myofilament-associated ILK is subject to regulation by as yet unidentified mechanisms that will be the subject of future investigations. ILK is known to be activated by phosphatidylinositol 3,4,5-trisphosphate (47), consistent with the presence of a phosphoinositide-binding domain resembling a pleckstrin homology domain in the center of the molecule (41). However, we observed no effect of phosphatidylinositol 3,4,5-trisphosphate on the phosphorylation of myosin by partially purified smooth muscle ILK (results not shown).
The results of this study provide the first indication of a novel role
for ILK in Ca2+ sensitization of smooth muscle contraction,
whereby signaling pathways that lead to inhibition of MLCP unmask the
activity of myofilament-associated ILK, which phosphorylates myosin and
activates contraction in a Ca2+-independent manner (Fig.
11). ILK is also likely to contribute to the increase in myosin
phosphorylation that occurs in response to activation of receptors,
e.g. the endothelin-1 receptor, which are coupled to both
Gq/11 and G12/13 heterotrimeric GTP-binding proteins. Given the widespread tissue distribution of ILK (24), it is
also conceivable that ILK may be involved in regulation of nonmuscle motility.
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ACKNOWLEDGEMENT |
---|
We are very grateful to Dr. S. Dedhar for critical reading of the manuscript and valuable discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Canadian Institutes of Health Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF296130.
An Alberta Heritage Foundation for Medical Research Medical
Scientist and recipient of a Canada Research Chair in Biochemistry. To
whom correspondence should be addressed: Dept. of Biochemistry and
Molecular Biology, University of Calgary Faculty of Medicine, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-3021;
Fax: 403-270-2211; E-mail: walsh@ucalgary.ca.
Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M011634200
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ABBREVIATIONS |
---|
The abbreviations used are: CaM, calmodulin; CaM kinase II, Ca2+- and calmodulin-dependent protein kinase II; DTT, dithiothreitol; ILK, integrin-linked kinase; LC20, 20-kDa light chain subunit of myosin II; MALDI-TOF MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; PKC, protein kinase C; ROK, Rho-associated kinase; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
---|
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---|
1. | Somlyo, A. P., and Somlyo, A. V. (1994) Nature 372, 231-236[CrossRef][Medline] [Order article via Infotrieve] |
2. | Allen, B. E., and Walsh, M. P. (1994) Trends Biochem. Sci. 19, 362-368[CrossRef][Medline] [Order article via Infotrieve] |
3. | Missiaen, L., De Smedt, H., Droogmans, G., Himpens, B., and Casteels, R. (1992) Pharmacol. Ther. 56, 191-231[CrossRef][Medline] [Order article via Infotrieve] |
4. | Chin, D., and Means, A. R. (2000) Trends Cell Biol. 10, 322-328[CrossRef][Medline] [Order article via Infotrieve] |
5. | 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] |
6. |
Ikebe, M.,
and Hartshorne, D. J.
(1985)
J. Biol. Chem.
260,
10027-10031 |
7. |
Colburn, J. C.,
Michnoff, C. H.,
Hsu, L-C.,
Slaughter, C. A.,
Kamm, K. E.,
and Stull, J. T.
(1988)
J. Biol. Chem.
263,
19166-19173 |
8. | Seto, M., Sasaki, Y., and Sasaki, Y. (1990) Pflügers Arch. 415, 484-489[Medline] [Order article via Infotrieve] |
9. |
Somlyo, A. P.,
and Somlyo, A. V.
(2000)
J. Physiol.
522,
177-185 |
10. | Hartshorne, D. J., Ito, M., and Erdödi, F. (1998) J. Muscle Res. Cell Motil. 19, 325-341[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Li, L.,
Eto, M.,
Lee, M. R.,
Morita, F.,
Yazawa, M.,
and Kitazawa, T.
(1998)
J. Physiol.
508,
871-881 |
12. |
Kitazawa, T.,
Eto, M.,
Woodsome, T. P.,
and Brautigan, D. L.
(2000)
J. Biol. Chem.
275,
9897-9900 |
13. | Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori, B., Feng, J., Nakano, T., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 273, 245-248[Abstract] |
14. |
Swärd, K.,
Dreja, K.,
Susnjar, M.,
Hellstrand, P.,
Hartshorne, D. J.,
and Walsh, M. P.
(2000)
J. Physiol.
522,
33-49 |
15. |
Gong, M. C.,
Fuglsang, A.,
Alessi, D.,
Kobayashi, S.,
Cohen, P.,
Somlyo, A. V.,
and Somlyo, A. P.
(1992)
J. Biol. Chem.
267,
21492-21498 |
16. | Gailly, P., Gong, M. C., Somlyo, A. V., and Somlyo, A. P. (1997) J. Physiol. 500, 95-109[Abstract] |
17. |
Feng, J.,
Ito, M.,
Kureishi, Y.,
Ichikawa, K.,
Amano, M.,
Isaka, N.,
Okawa, K.,
Iwamatsu, A.,
Kaibuchi, K.,
Hartshorne, D. J.,
and Nakano, T.
(1999)
J. Biol. Chem.
274,
3744-3752 |
18. |
Weber, L. P.,
Van Lierop, J. E.,
and Walsh, M. P.
(1999)
J. Physiol.
516,
805-824 |
19. |
Ikebe, M.,
Hartshorne, D. J.,
and Elzinga, M.
(1986)
J. Biol. Chem.
261,
36-39 |
20. | Persechini, A., and Hartshorne, D. J. (1981) Science 213, 1381-1385 |
21. | Hathaway, D. R., and Haeberle, J. R. (1983) Anal. Biochem. 135, 37-43[Medline] [Order article via Infotrieve] |
22. | Ngai, P. K., Carruthers, C. A., and Walsh, M. P. (1984) Biochem. J. 218, 863-870[Medline] [Order article via Infotrieve] |
23. |
Winder, S. J.,
and Walsh, M. P.
(1990)
J. Biol. Chem.
265,
10148-10155 |
24. | Hannigan, G. E., Leung-Hagesteijn, C., Fitz-Gibbon, L., Coppolino, M. G., Radeva, G., Filmus, J., Bell, J. C., and Dedhar, S. (1996) Nature 379, 91-96[CrossRef][Medline] [Order article via Infotrieve] |
25. | Frohman, M. A. (1993) Methods Enzymol. 218, 340-356[Medline] [Order article via Infotrieve] |
26. | Sanger, F. (1981) Science 214, 1205-1210[Medline] [Order article via Infotrieve] |
27. | Ikebe, M. (1990) Biochem. Biophys. Res. Commun. 168, 714-720[Medline] [Order article via Infotrieve] |
28. | Foster, C. J., Johnston, S. A., Sunday, B., and Gaeta, F. C. A. (1990) Arch. Biochem. Biophys. 280, 397-404[Medline] [Order article via Infotrieve] |
29. | Knighton, D. R., Pearson, R. B., Sowadski, J. M., Means, A. R., Ten Eyck, L. F., Taylor, S. S., and Kemp, B. E. (1992) Science 258, 130-135[Medline] [Order article via Infotrieve] |
30. | Edelman, A. M., Lin, W-H., Osterhout, D. J., Bennett, M. K., Kennedy, M. B., and Krebs, E. G. (1990) Mol. Cell. Biochem. 97, 87-98[Medline] [Order article via Infotrieve] |
31. | Stults, J. T. (1995) Curr. Opin. Struct. Biol. 5, 691-698[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Ikebe, M.,
Maruta, S.,
and Reardon, S.
(1989)
J. Biol. Chem.
264,
6967-6971 |
33. | Schwabe, W., Lee, J. E., Nathan, M., Xu, R. H., Sitaraman, K., Smith, M., Potter, R. J., Rosenthal, K., Rashtchian, A., and Gerard, G. F. (1998) Focus 20, 30-33 |
34. | Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241, 42-52[Medline] [Order article via Infotrieve] |
35. | Balendran, A., Casamayor, A., Deak, M., Paterson, A., Gaffney, P., Currie, R., Downes, C. P., and Alessi, D. R. (1999) Curr. Biol. 9, 393-404[CrossRef][Medline] [Order article via Infotrieve] |
36. | Lynch, D. K., Ellis, C. A., Edwards, P. A. W., and Hiles, I. D. (1999) Oncogene 18, 8024-8032[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Williams, D. A.,
and Fay, F. S.
(1986)
Am. J. Physiol.
250,
C779-C791 |
38. | Williams, D. A., Becker, P. L., and Fay, F. S. (1987) Science 235, 1644-1648[Medline] [Order article via Infotrieve] |
39. |
Gallagher, P. J.,
Herring, B. P.,
Trafny, A.,
Sowadski, J.,
and Stull, J. T.
(1993)
J. Biol. Chem.
268,
26578-26582 |
40. |
Ichikawa, K.,
Ito, M.,
and Hartshorne, D. J.
(1996)
J. Biol. Chem.
271,
4733-4740 |
41. | Dedhar, S., Williams, B., and Hannigan, G. E. (1999) Trends Cell Biol. 9, 319-323[CrossRef][Medline] [Order article via Infotrieve] |
42. | Ichikawa, K., Hirano, K., Ito, M., Tanaka, J., Nakano, T., and Hartshorne, D. J. (1996) Biochemistry 35, 6313-6320[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Gohla, A.,
Schultz, G.,
and Offermanns, S.
(2000)
Circ. Res.
87,
221-227 |
44. | Lundgren, K., Walworth, N., Booher, R., Dembski, M., Kirschner, M., and Beach, D. (1991) Cell 64, 1111-1122[Medline] [Order article via Infotrieve] |
45. | Herman, P. K., Stack, J. H., DeModena, J. A., and Emr, S. D. (1991) Cell 64, 425-437[Medline] [Order article via Infotrieve] |
46. |
Kolodney, M. S.,
Thimgan, M. S.,
Honda, H. M.,
Tsai, G.,
and Yee, H. F., Jr.
(1999)
J. Physiol.
515,
87-92 |
47. |
Delcommenne, M.,
Tan, C.,
Gray, V.,
Rue, L.,
Woodgett, J.,
and Dedhar, S.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11211-11216 |
48. |
Kozasa, T.,
Jiang, X.,
Hart, M. J.,
Sternweis, P. M.,
Singer, W. D.,
Gilman, A. G.,
Bollag, G.,
and Sternweis, P. C.
(1998)
Science
280,
2109-2111 |