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INTRODUCTION |
Phosphorylation of Ser-19 at the N terminus of smooth muscle
myosin regulatory light chain
(RLC)1 by
Ca2+/calmodulin-dependent myosin light chain
kinase results in an increase in actin-activated Mg2+ATPase
activity. It is now known that this activity plays a key role in a
variety of biological events, including, in part, initiation of smooth
muscle contraction (1), potentiation of skeletal and cardiac muscle
contraction (2), fibroblast contraction (3, 4), endothelial cell
retraction (5-7), platelet aggregation and contraction (8, 9), growth
cone motility in nerve cells (10), and receptor capping in lymphocytes
(11). When the intracellular Ca2+ concentration increases
upon stimulation of cells by Ca2+ influx through
Ca2+ channels in the plasma membrane or through
Ca2+ release from intracellular Ca2+ stores,
Ca2+ binds to calmodulin. Ca2+/calmodulin binds
to the calmodulin-binding sequence of the kinase, resulting in
displacement of the autoinhibitory segment and exposure of the
catalytic site for RLC phosphorylation (12, 13).
Previous studies in vitro showed that myosin light
chain kinase binds to purified F-actin as well as myosin filaments and that binding in both cases is regulated by Ca2+/calmodulin
(14-18). Biochemical studies by co-sedimentation assays in
vitro indicated that the N terminus of myosin light chain kinase was responsible for binding to purified F-actin (14-16), whereas its C
terminus bound to purified myosin (17, 18) with binding affinities in
the range of 10
5 to 10
6 M.
These binding affinities were decreased in the presence of Ca2+/calmodulin (18). Additionally,
Ca2+/calmodulin may interact with an N-terminal
F-actin-binding site of myosin light chain kinase, distinct from the
calmodulin-binding sequence at the C terminus of the catalytic core
(15). The F-actin binding site in residues 1-41 had a
Ka value of approximately 105
M
1 with an IQ-like sequence in residues
26-41 that bound calmodulin (15).
Studies have also shown that myosin light chain kinase binds to
cellular actomyosin-containing filaments. Immunocytochemistry studies
found myosin light chain kinase localized to actomyosin-containing stress fibers or myofilaments in nonmuscle and smooth muscle cells, respectively (19, 20). Recent investigations in vitro and in vivo showed that the N-terminal half of the kinase
(residues 1-655), not the C terminus, was both necessary and
sufficient for binding to isolated smooth muscle myofilaments, stress
fibers in permeabilized fibroblasts, and myofilaments in intact smooth muscle cells (21). In addition, the apparent binding affinities of
myosin light chain kinase to smooth muscle myofilaments and actin-containing thin filaments were greater than to purified smooth
muscle F-actin and skeletal muscle myofilaments (21, 22). Thus, myosin
light chain kinase binds to actomyosin-containing filaments or
actin-containing thin filaments in cells with an affinity greater than
to F-actin alone, perhaps due to the presence of a distinct anchoring
protein or a protein that facilitates binding to F-actin.
Other reports support the idea that myosin light chain kinase binds
tightly to actomyosin-containing filaments in cells or tissues. For
example, smooth muscle tissue strips made permeable with 1% Triton
X-100 and stored for several weeks at
20 °C in 50% glycerol were
still able to contract in the presence of Mg2+ATP and
Ca2+/calmodulin (23). Quantitation of myosin light chain
kinase showed no significant loss of the kinase after storage. Thus, myosin light chain kinase appears to bind tightly to myofilaments in
permeable smooth muscle cells with an apparent affinity greater than
values reported for binding to purified F-actin (21). This raises the
question of whether myosin light chain kinase must dissociate from
actin-containing thin filaments to phosphorylate myosin RLC in the
thick filaments (14-18). Stimulation of smooth muscle tissues can
result in a high extent (50-100%) of RLC phosphorylation, particularly in the presence of a cell-permeable protein phosphatase inhibitor (24-27). Yet, the concentration of RLC in smooth muscle is
estimated as 75-80 mM, a concentration 25 times greater
than the concentration of myosin light chain kinase (28, 29). Smooth muscle myosin light chain kinase associated with actin-containing filaments may thus dissociate and diffuse to myosin-containing filaments to achieve a high extent of RLC phosphorylation during contraction (14). Thus, Ca2+/calmodulin may not only
activate the kinase but also dissociate it from actin-containing
filaments via interaction with a second binding site in the
N terminus (15). To test this hypothesis, we transiently expressed
myosin light chain kinase with green fluorescent protein and
established a system to study its binding to stress fibers in permeable
A7r5 cells derived from smooth muscle.
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EXPERIMENTAL PROCEDURES |
Myosin Light Chain Kinase Binding to Smooth Muscle Myofilaments
and F-actin on Nitrocellulose-coated Coverslips--
Extracted smooth
muscle myofilaments and purified F-actin filaments (21) were diluted to
1 mg/ml in air-dried BODIPY FL phallacidin (5 units, Molecular Probes,
Inc., Eugene, OR) containing 10 mM MOPS at pH 7.0, 50 mM NaCl, 2 mM dithiothreitol, 1 mg/ml bovine
serum albumin, and 1 mM MgCl2. After adding to
nitrocellulose-coated coverslips (advice kindly provided by Dr. Joseph
Haeberle, University of Vermont) at room temperature for 30 min, the
coverslips were washed with buffer three times to remove nonadhering
filaments. Cy3-labeled full-length myosin light chain kinase (0.2 mM) (21) was added to both types of filaments. After
incubation at room temperature for 10 min, coverslips were washed with
buffer three times to remove unbound kinase. Coverslips were sealed on
glass slides for fluorescence imaging.
Construction of MLCK-GFP Plasmids--
MLCK-GFP expression
vectors were constructed from modified pGREEN LANTERNTM-1
(Life Technologies, Inc.), a cytomegalovirus promoter-driven expression
vector containing GFP coding sequences. This GFP coding sequence
contains a Ser65
Thr mutation and "humanized" codon
usage for increasing its brightness and better expression in mammalian
cells (30). Plasmid pGREEN LANTERNTM-1 was modified to
delete two nucleotides (GC) between SpeI and NotI
cloning sites. This modification shifts an in frame TAG stop codon
(within the SpeI restriction enzyme site) at the 5'-end of
GFP coding sequences to out of frame. A two-nucleotide-deleted SpeI-BclI GFP DNA fragment (1 kilobase pair) was
synthesized by polymerase chain reaction using pGREEN
LANTERNTM-1 as a template and a primer pair:
5'-GCTGACTAGTGCGGCCGCCGCCAC-3' and 5'-GGCTGATTATGATCATGAAC-3'. This
polymerase chain reaction DNA fragment and vector pGREEN
LANTERNTM-1 were digested with SpeI and
BamHI sites. The 0.54- and 4.5-kilobase-paired DNA fragments
from the digested polymerase chain reaction product and the vector,
respectively, were ligated and transformed. The modified plasmid (pGFP)
was confirmed by DNA sequencing and further used to construct MLCK-GFP
fusion protein expression vectors. Full-length and N-terminal (amino
acids 2-142) deleted myosin light chain kinase DNA fragments were
synthesized without a stop codon at their 3'-ends by polymerase chain
reaction using Pat1/J1-J3 (containing full-length rabbit smooth muscle
myosin light chain kinase coding sequences) as a template and primer
pairs 5'-TCCCCCCGGGATGGATTTCCGCGCCAAC-3' and
5'-GACTAGTTGACTCCTCTTCCTCCTCTTCCCC-3', and
5'-TCCCCCCGGGATGGAGAGCTCGAAACCTGTGGGC-3' and
5'-GACTAGTTGACTCCTCTTCCTCCTCTTCCCC-3', respectively. Synthesized myosin light chain kinase DNA fragments (3.6 and 3.0 kilobase pairs)
were ligated into pGFP by XmaI and SpeI sites.
The resultant plasmids (pFL-GFP and pDN-GFP) were confirmed by
restriction enzyme analyses and DNA sequencing.
Cell Culture and DNA Transfection--
A7r5 rat thoracic aorta
smooth muscle cells (obtained from ATCC, CRL-1444) were maintained in
Dulbecco's modified Eagle's medium containing 4 mM
L-glutamine, 1.5 g/liter sodium bicarbonate, 4.5 g/liter
glucose, 1 mM sodium pyruvate, 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37 °C with 5%
CO2. For DNA transfection, A7r5 cells were seeded onto
40-mm round coverslips (40 Circles-1D; Fisher) in 60-mm Petri dishes at
30-50% confluence 1 day before transfection. DNA was transfected by a
liposome-mediated method according to the manufacturer's instructions.
In brief, 12 ml of FuGENETM-6 (Boehringer Mannheim) was
added into 288 ml of serum-free medium. After a 5-min incubation at
room temperature, the FuGENETM-6/medium mixture was added
slowly to 3 mg of DNA (15 ml of 0.2 mg/ml DNA). After a 15-min
incubation at room temperature, the FuGENETM-6/DNA mixture
was added to cells. Transfected cells were incubated for 2 days before experiments.
Determination of Myosin Light Chain Kinase Activity and Binding
in Vitro--
Ca2+/calmodulin-dependent
activity of myosin light chain kinase was determined by measuring rates
of 32P incorporation into myosin RLC (31). Transfected or
untransfected cells were lysed on ice for 10 min in 50 mM
MOPS at pH 7.0, 50 mM MgCl2, 0.5 mM
EGTA, 1% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, and protease inhibitors (100 mg/ml phenylmethylsulfonyl fluoride, 20 mg/ml leupeptin, 30 mg/ml aprotenin, 60 mg/ml tosyllysylchlomethyl ketone, and 60 mg/ml tosylphenylalanyl chloromethyl ketone). Cell lysates were clarified by centrifugation at 14,000 × g
for 2 min and diluted for kinase activity assays so that
32P incorporation was linear in respect to time. The
minimal kinase activities from untransfected cells were measured as
background values. The amounts of expressed MLCK-GFP proteins were
quantitated by immunoblotting using either monoclonal antibodies raised
to the full-length myosin light chain kinase or polyclonal antibodies raised against the catalytic core of rabbit smooth muscle myosin light
chain kinase. Various amounts of rabbit smooth muscle myosin light
chain kinase purified from Sf9 cells (21) were used for a
standard curve.
For binding analyses in vitro, A7r5 cell lysates containing
expressed myosin light chain kinase were further dialyzed against 50 mM MOPS at pH 7.0, 0.5 mM EGTA, 10% glycerol,
1 mM dithiothreitol, and protease inhibitors to remove
MgCl2. Binding of myosin light chain kinases to smooth
muscle myofilaments in vitro was measured by a
co-sedimentation procedure (21). The amounts of myosin light chain
kinase in the supernatant and pellet fractions were compared by
measurements of kinase activity.
Measurement of the Amount of Expressed MLCK-GFP in A7r5
Cells--
A7r5 cells transfected with cDNA for MLCK-GFP were
sorted by flow cytometry (FACStar Plus; Becton Dickinson, San Jose,
CA). After cells were detached by 0.05%/0.53 mM
trypsin-EDTA (Life Technologies, Inc.), fetal bovine serum was added to
inhibit trypsin activity. Cells were centrifuged at 200 × g for 10 min and washed with phosphate-buffered saline twice
before sorting. Cells were sorted based on fluorescence intensity
(excitation at 488 nm). About 15% transfected A7r5 cells showed green
fluorescence. Positive (fluorescent) and negative (nonfluorescent)
cells were collected separately. Numbers of the collected cells after
sorting were recorded. Cells were lysed, and the amount of expressed
MLCK-GFP was measured by immunoblotting as described previously herein. The average concentration of expressed MLCK-GFP in a single cell was
calculated as follows: amount of MLCK-GFP/(number of cells × volume of a single cell). The volume of a single cell was assumed to be
2 picoliters.
Ca2+/EGTA Buffers and Cell
Permeabilization--
Ca2+-free buffer contained 20 mM PIPES, 4 mM EGTA, 5 mM
MgSO4, 90 mM K+-gluconate, 5.3 mM Na2ATP, 0.1% bovine serum albumin, 0.1 mM ionomycin (Calbiochem), 1.5 mM thapsigargin
(Sigma), 0.1 mM phenylmethylsulfonyl fluoride (Sigma), and
10 mg/ml leupeptin (Sigma) at pH 6.8 (23). Concentrated
Ca2+ solutions buffered with Ca2+-free buffer
(containing 4 mM EGTA) were used to achieve free [Ca2+] of 1 mM and 10 mM. Buffers
containing 1 and 10 mM [Ca2+] were measured
and calibrated with the Ca2+ indicators fluo-3 and calcium
green-5N (Molecular Probes), respectively. A7r5 cells were treated with
0.02% saponin in the Ca2+-free, 1 mM
[Ca2+], 100 nM calmodulin or the 10 mM [Ca2+], 1 mM calmodulin, 10 mM wortmannin buffer at 37 °C for 10 min. After
treatment, the permeable cells were washed with the buffer without
saponin for three times and/or treated with the indicated buffers.
Fluorescence Imaging--
Fluorescence imaging was performed as
described previously (32). Twelve-bit fluorescence images were acquired
by a cooled CCD camera (Quantix Photometrics, Tucson, AZ) and
Oncor-Image software (Oncor, Gaithersburg, MD). Narrow bandpass
interference filters (Omega, Brattleboro, VT) were used to select
BODIPY fluorescein or GFP (excitation at 490 nm and emission at 520 nm)
and rhodamine or Cy3 (excitation at 550 nm and emission at 575 nm)
fluorescence. Cells were kept in an open thermal controlled chamber
(Custom Scientific, Dallas, TX) at 37 °C during fluorescence
imaging. Additional imaging experiments were performed with purified
F-actin and detergent-washed gizzard myofilaments with Cy3-labeled
myosin light chain kinase (21).
Fluorescence Recovery after Photobleaching (FRAP)--
FRAP was
performed on the saponin-permeable A7r5 cells as described previously
(23) with the following modification. An argon ion laser
(Spectra-Physics 2017) operated at 1 watt on the 488 nm line was
directed into the microscope (Zeiss Axiovert 35) with the beam radius
4.43 mm at the specimen plane. The proportion of the cell photobleached
was approximately 3%. Photobleaching time was 2-30 ms, resulting in a
20-70% decrease in the fluorescence intensity. At least three
half-lives of data were recorded for each recovery. The recovery time
for some experiments was monitored for 15 min. Cells were maintained at
37 °C in a sealed chamber. A control experiment using A7r5 cells
microinjected with 167-kDa fluorescein-labeled dextran (Sigma; 4 mg/ml
in 10 mM MOPS, 30 mM magnesium acetate, 100 mM NaCl at pH 7.1) was also included in FRAP measurements
under similar conditions, except the cells were permeabilized with 30 mg/ml
-escin at 37 °C for 10 min (23).
Measurement of RLC Phosphorylation by Urea-Glycerol
PAGE--
A7r5 cells were treated with 0.02% saponin in
Ca2+-free buffer at 37 °C for 10 min and washed with
Ca2+-free buffer three times. After washing, permeable
cells were treated with various buffers at room temperature for 3 or 30 min: Ca2+-free buffer, Ca2+ buffer alone,
Ca2+ buffer plus 10 mM wortmannin (Sigma), and
Ca2+ buffer plus 4 mM concentraetion of a
peptide (KKRAARATSNVFS-amide, synthesized by Genosys Biotechnologies,
Inc.) containing the sequence around the phosphorylatable serine in the
smooth muscle RLC (33), respectively. The Ca2+ buffer
solution included 10 mM Ca2+ and 100 nM calmodulin with or without 3 mM okadaic
acid, a protein phosphatase type 1 and 2A inhibitor (Calbiochem). In
addition to other chemicals described above, after treatments the
solutions were aspirated completely. Ice-cold 10% trichloroacetic acid
and 10 mM dithiothreitol were added, and cells were frozen
by putting culture dishes into liquid nitrogen immediately. Cell
protein was collected and incubated on ice for 20 min, and insoluble
proteins were collected by centrifugation at 4,700 × g
for 1 min. Pellets were washed with ethyl ether (Fisher) three times
and resuspended in 8 M urea sample buffer as described
previously (34). The samples were analyzed by urea-glycerol PAGE and
immunoblotting using a monoclonal antibody against smooth muscle RLC
(34). The relative amounts of nonphosphorylated, monophosphorylated, and diphosphorylated RLCs were measured by quantitative immunoblots.
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RESULTS |
Myosin Light Chain Kinase Binding to Smooth Muscle Myofilaments and
F-actin on Nitrocellulose-coated Coverslips--
Extracted smooth
muscle myofilaments and purified smooth muscle F-actin mixed with
BODIPY FL phallacidin were bound to nitrocellulose-coated coverslips.
Cy3-labeled myosin light chain kinase was added to the filaments to
test its binding ability. Fig. 1 shows
phallacidin binding to actin in both smooth muscle myofilaments and
smooth muscle F-actin. The Cy3-labeled myosin light chain kinase
strongly bound only to smooth muscle myofilaments (Fig. 1). These
results are similar to previous results obtained with a cosedimentation assay where myosin light chain kinase bound to gizzard smooth muscle
myofilaments with a greater affinity compared with purified smooth
muscle F-actin (21).

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Fig. 1.
Myosin light chain kinase binding to smooth
muscle myofilaments and F-actin on nitrocellulose-coated
coverslips. Extracted smooth muscle myofilaments and purified
F-actin filaments (1 mg/ml) were mixed with BODIPY FL phallacidin (5 units; Molecular Probes) in a buffer containing 10 mM MOPS
at pH 7.0, 50 mM NaCl, 2 mM dithiothreitol, 1 mg/ml bovine serum albumin, and 1 mM MgCl2 to
identify actin-containing filaments. The respective filamentous
solutions were adhered to nitrocellulose-coated coverslips, and
Cy3-labeled full-length myosin light chain kinase was added. After
incubation for 10 min, coverslips were washed three times to remove the
unbound kinase. Filaments were double imaged under a fluorescence
microscope to detect BODIPY fluorescein and Cy3 fluorescence (for
details, see "Experimental Procedures"). Typical fluorescence
micrographs are presented for three experiments.
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Expression and Biochemical Properties of MLCK-GFP in Intact A7r5
Smooth Muscle Cells--
To study the physiological effects and
binding properties of myosin light chain kinase in cells, MLCK-GFP
fusion protein expression vectors were constructed. The cDNA
encoding GFP was fused at the C terminus of full-length (FL) or
N-terminal deleted (DN) myosin light chain kinase (Fig.
2A). The N-terminal 2-142
amino acids were deleted in DN MLCK-GFP. This region is within the
N-terminal half of the kinase, which is responsible for high affinity
myofilament binding in vitro and in vivo (21) and
contains the actin binding segment (14-16). GFP, FL MLCK-GFP, and DN
MLCK-GFP were transiently expressed in A7r5 smooth muscle cells (Fig.
2B). As expected, fluorescence from the 27-kDa GFP was
diffuse and was found in both the nucleus and cytoplasm in transfected
cells (Fig. 2B, left part). The 153-kDa FL
MLCK-GFP was localized primarily to filaments with some diffuse
cytoplasmic fluorescence (Fig. 2B, middle part).
The amount of diffuse cytoplasmic fluorescence was greater in cells
expressing larger amounts of the kinase. The 137-kDa DN MLCK-GFP showed
only diffuse fluorescence in the cytoplasm without apparent binding to
filaments (Fig. 2B, right part). Both FL MLCK-GFP
and DN MLCK-GFP were excluded from nuclei, most likely due to their
masses. MLCK-GFP with deletion of the kinase C terminus (amino acids
1004-1147) was also expressed and found primarily on filaments similar
to FL MLCK-GFP (data not shown). These results show that the site for
high affinity binding resides in residues 2-142 and that the GFP tag
at the C terminus of full-length myosin light chain kinase did not
affect kinase binding to stress fibers in cells. Similar results were
also obtained in transfected NIH 3T3 and CV1 fibroblast cells (data not
shown).

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Fig. 2.
Expression of myosin light chain kinases with
green fluorescent protein. A, scheme of domain
organization of myosin light chain kinases with green fluorescent
protein at the C terminus. Plasmids carrying the coding sequences of
GFP and MLCK-GFP fusion proteins were constructed as described under
"Experimental Procedures." The regulatory segment contains the
respective autoinhibitory and calmodulin-binding sequences.
Ig-1, Ig-2, and Ig-3, immunoglobulin
C2-like motifs; Fn, fibronectin type III-like motif.
B, transient expression of GFP, FL MLCK-GFP, and DN MLCK-GFP
in A7r5 cells. DNA was transiently transfected into A7r5 cells and
fluorescence images obtained as described under "Experimental
Procedures."
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The specific activity of FL MLCK-GFP from transfected cell lysates was
compared with full-length myosin light chain kinase purified from
Sf9 cells (Fig. 3A).
The kinase activity of FL MLCK-GFP was
Ca2+/calmodulin-dependent, and the specific
activity was 1280 pmol/min/pmol, comparable with that of purified
full-length myosin light chain kinase without GFP (1980 pmol/min/pmol).
The ability of FL MLCK-GFP to bind to smooth muscle myofilaments
in vitro was also measured by the co-sedimentation assay.
Cell lysates containing 5 nM FL MLCK-GFP were used to
compare the binding ability with purified full-length myosin light
chain kinase to detergent-washed myofilaments from gizzard smooth
muscle (Fig. 3B). Myosin light chain kinase with or without
GFP bound smooth muscle myofilaments similarly under these conditions.
Thus, the introduction of green fluorescent protein at the C terminus
of myosin light chain kinase had no significant effect on kinase
catalytic or binding properties. These results indicate that the FL
MLCK-GFP may be used to characterize the cellular binding properties of
myosin light chain kinase in cells.

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Fig. 3.
Specific activity and binding of FL MLCK-GFP
in A7r5 cells. Purified full-length myosin light chain kinase from
Sf9 cells (21) and cell lysates from A7r5 cells expressing FL
MLCK-GFP were used for measurements of kinase activity and binding to
detergent-washed smooth muscle myofilaments. A, specific
activity of FL MLCK-GFP. Kinase activities were measured by rates of
32P incorporation into myosin RLC in the presence of 4 mM EGTA ( Ca2+/CaM, open bar) or
0.3 mM CaCl2 and 1 mM calmodulin (+ Ca2+/CaM, hatched bar) (31). The amount of
expressed MLCK-GFP protein was quantitated by immunoblotting as
described under "Experimental Procedures." B, binding of
5 nM myosin light chain kinases to smooth muscle
myofilaments in vitro. Binding of purified full-length
smooth muscle myosin light chain kinase (filled circle) and
5 nM FL MLCK-GFP in cell lysates (open circle)
to smooth muscle myofilaments in vitro was measured by a
co-sedimentation procedure (21). The concentration of actin in smooth
muscle myofilaments was measured by densitometric scanning after
SDS-PAGE. The amounts of myosin light chain kinase in the supernatant
and pellet fractions were compared by measurements of kinase activity.
The data represent the means ± S.E. for three experiments.
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The concentration of expressed FL MLCK-GFP in a single A7r5 cell was
estimated by flow cytometry and immunoblotting (data not shown). The
estimated FL MLCK-GFP concentration was 1.1 ± 0.3 mM,
which is in a range similar for the endogenous myosin light chain
kinase in smooth muscle cells in tissues (3-4 mM), although it is at least 10 times greater than the amounts found in
nonmuscle cells (35).
Myosin Light Chain Kinase Binding in Permeable A7r5 Cells--
To
examine Ca2+ regulation of myosin light chain kinase
binding to cellular actomyosin filaments, transfected A7r5 cells were made selectively permeable by saponin. A7r5 cells were transfected with
DN MLCK-GFP or FL MLCK-GFP and intact cells were imaged (Fig. 4, top panels). After
treatment with saponin for 10 min, the diffuse cytoplasmic fluorescence
due to DN MLCK-GFP was completely removed, indicating release of the
kinase from cells (Fig. 4, middle left panel). This was not
due to loss of actin-containing stress fibers as shown by staining with
rhodamine-labeled phalloidin (Fig. 4, bottom left panel). In
the FL MLCK-GFP-transfected A7r5 cells, the diffuse fluorescence was
released from the cells by saponin treatment, while the
filament-associated FL MLCK-GFP remained (Fig. 4, middle right
panel).

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Fig. 4.
Myosin light chain kinase binding in
permeable A7r5 cells. DN MLCK-GFP (left panels) and FL
MLCK-GFP (right panels) in transfected A7r5 cells were
imaged before permeabilization (Intact, top
panels). After imaging the intact cells, cells were treated with
0.02% saponin in Ca2+-free buffer (20 mM PIPES
at pH 6.8, 4 mM EGTA, 5 mM MgSO4,
90 mM K+-gluconate, 5.3 mM
Na2ATP, 0.1% bovine serum albumin, 0.1 mM
ionomycin, 1.5 mM thapsigargin, 0.1 mM
phenylmethylsulfonyl fluoride, and 10 mg/ml leupeptin) at 37 °C for
10 min. After permeabilization, cells were washed three times with the
Ca2+-free buffer without saponin, and the same cells were
imaged (Saponin, middle panels).
Rhodamine-labeled phalloidin was added to the Ca2+-free
buffer for 2 min and then washed with the Ca2+-free buffer
three times following by imaging (phalloidin, bottom
panels).
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Effect of Ca2+/Calmodulin on Myosin Light Chain Kinase
Binding to Filaments in Permeable Cells--
To test whether
Ca2+/calmodulin affects myosin light chain kinase binding
in cells, fluorescence intensities of FL MLCK-GFP bound to filaments in
saponin-permeable cells were compared in the presence of either 4 mM EGTA or 10 mM Ca2+ plus 1 mM calmodulin and 10 mM wortmannin (Fig.
5A). Although calmodulin is
not completely removed from cellular filaments by EGTA (23), calmodulin
was added to the buffer containing Ca2+ to assure a
sufficient amount for binding to the kinase. Wortmannin was added to
the Ca2+-containing buffer to prevent the robust
contraction of cell filaments for fluorescence measurements. Cells were
either treated with EGTA buffer first and then washed with the
Ca2+/calmodulin/wortmannin buffer or treated with
Ca2+/calmodulin/wortmannin buffer first and then washed
with the EGTA buffer. The fluorescence intensities of FL MLCK-GFP
remained unchanged in both circumstances (Fig. 5A). Similar
results were obtained by comparison with EGTA buffer and 1 mM Ca2+/100 nM calmodulin buffer
(data not shown). Thus, myosin light chain kinase binding to
actin-containing stress fibers is not released by
Ca2+/calmodulin.

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Fig. 5.
Effect of Ca2+/calmodulin on
myosin light chain kinase binding to filaments in permeable A7r5
cells. A, measurements of fluorescence intensities of
filament-bound FL MLCK-GFP in saponin-permeable A7r5 cells in the
presence of 4 mM EGTA Ca2+-free buffer
(open bar) or 10 mM Ca2+/1
mM calmodulin/10 mM wortmannin buffer
(closed bar). Cells were either treated with the EGTA buffer
first and then washed with the Ca2+/calmodulin/wortmannin
buffer (left) or treated with the
Ca2+/calmodulin/wortmannin buffer first and then washed
with the EGTA buffer (right). Fluorescence intensities were
measured as described under "Experimental Procedures." Fluorescence
intensities in the same areas of the cells were monitored for 5 s
at 500-ms intervals. The fluorescence intensities after buffer changes
were normalized to the fluorescence intensities before buffer changes.
The fluorescence intensities were calculated as means ± S.E. from
three experiments. B, fluorescence recovery after
photobleaching of filament-bound FL MLCK-GFP (solid symbols) and
167-kDa fluorescein-labeled dextran (open
symbols) in the presence of Ca2+-free buffer
(circle, solid line) or 10 mM
Ca2+, 1 mM calmodulin, 10 mM
wortmannin buffer (square, dashed line). Eight to
fifteen samples from three individual experiments were tested. A
typical FRAP experiment is presented.
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To test whether myosin light chain kinase may diffuse laterally along
filaments, the mobility of FL MLCK-GFP was examined in the
saponin-permeable A7r5 cells by FRAP (Fig. 5B,
closed symbols). After photobleaching, there was
no recovery of fluorescence for up to 15 min, indicating that FL
MLCK-GFP bound to filaments with no evidence of mobility. There was no
fluorescence recovery in either EGTA buffer or 10 mM
Ca2+, 1 mM calmodulin, 10 mM
wortmannin after photobleaching. Similar results were obtained with the
addition of 1 mM Ca2+, 100 nM
calmodulin buffer for 80 s (data not shown). Additionally, similar
results for measurements of fluorescence intensities and FRAP were
obtained by perfusion of Cy3-labeled myosin light chain kinase in
Triton X-100-permeable stress fibers in Swiss 3T3 cells (data not
shown). In contrast, the fluorescence of 167-kDa fluorescein-labeled dextran in
-escin-permeable cells was recovered 100% in 1 min after
photobleaching in either EGTA or Ca2+/calmodulin (Fig.
5B, open symbols). These data
collectively indicate that myosin light chain kinase binds to
actin-containing filaments with high affinity and does not dissociate
or diffuse along the filaments in the presence of
Ca2+/calmodulin.
Cell Contraction and RLC Phosphorylation--
To determine if
filament-bound myosin light chain kinase could phosphorylate myosin RLC
in thick filaments in cells, cell contractility was first measured in
the presence of Ca2+. Fig.
6A (top to
bottom parts) shows intact cells transfected with
FL MLCK-GFP, followed by saponin-permeabilization in
Ca2+-free buffer containing Mg2+ATP. The
subsequent addition of Ca2+ in the presence of
Mg2+ATP results in contraction of filaments containing
bound-myosin light chain kinase. This
Ca2+-dependent contraction was inhibited in the
presence of 10 mM wortmannin, a myosin light chain kinase
inhibitor (Fig. 6B), or 4 mM RLC peptide, a
competitive substrate (Fig. 6C).

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Fig. 6.
Ca2+-dependent
contraction in saponin-permeable A7r5 cells. FL
MLCK-GFP-transfected A7r5 cells were imaged before permeabilization
(Intact Cell, top panels). Cells were
then treated with 0.02% saponin in the Ca2+-free buffer
(20 mM PIPES at pH 6.8, 4 mM EGTA, 5 mM MgSO4, 90 mM
K+-gluconate, 5.3 mM Na2ATP, 0.1%
bovine serum albumin, 0.1 mM ionomycin, 1.5 mM
thapsigargin, 0.1 mM phenylmethylsulfonyl fluoride, and 10 mg/ml leupeptin) at 37 °C for 10 min. After permeabilization, cells
were washed with the Ca2+-free buffer without saponin three
times, and the same cells were imaged (Ca2+
free, middle panels). Cells were further
incubated with 10 mM Ca2+ buffer (A,
bottom part) or 10 mM Ca2+ buffer
containing either 10 mM wortmannin (B,
bottom part) or 4 mM RLC peptide (C,
bottom part).
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To determine whether RLC phosphorylation mediates this
Ca2+-dependent contraction by the
filament-bound kinase, RLC phosphorylation was measured in the
saponin-permeabilized cells (Fig. 7).
Nonphosphorylated, monophosphorylated, and diphosphorylated RLCs were
separated by urea-glycerol PAGE and measured by immunoblotting (Fig.
7A). As expected, RLC phosphorylation in saponin-permeable
cells in Ca2+-free buffer alone for 3 min was low (Fig. 7,
A and B). When a protein phosphatase inhibitor,
okadaic acid, was added to the Ca2+-free buffer, RLC
phosphorylation increased from 0.14 to 0.43 mol of phosphate/mol of
RLC. When wortmannin was added with okadaic acid to the
Ca2+-free buffer for 3 min, RLC phosphorylation was similar
(0.41 mol of phosphate/mol of RLC) to the extent of phosphorylation obtained with okadaic acid alone (Fig. 7, A and
B). Thus, there was apparently some myosin light chain
kinase-independent phosphorylation of RLC. However, inclusion of 10 mM Ca2+ and 100 nM calmodulin with
okadaic acid resulted in a greater extent of RLC phosphorylation, 0.87 mol of phosphate/mol of RLC (Fig. 7, A and B).
This Ca2+/calmodulin-dependent RLC
phosphorylation was abolished by wortmannin or RLC peptide (0.40 and
0.25 mol of phosphate/mol of RLC, respectively) (Fig. 7, A
and B). When the RLC peptide substrate concentration was
50-fold in excess of RLC in a typical kinase assay, the myosin light
chain kinase activity toward RLC was inhibited greater than 90% (data
not shown).

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Fig. 7.
RLC phosphorylation by filament-bound myosin
light chain kinase in permeable A7r5 cells. FL
MLCK-GFP-transfected A7r5 cells were permeabilized with 0.02% saponin
in Ca2+-free buffer at 37 °C for 10 min and washed with
Ca2+-free buffer three times. After washing, cells were
treated at room temperature for 3 or 30 min with Ca2+-free
buffer or 10 mM Ca2+ buffer containing the
following components: 3 mM okadaic acid, 10 mM
wortmannin, and 4 mM RLC peptide. A,
representative immunoblots of RLC phosphorylation after urea-glycerol
PAGE. Migrations of the nonphosphorylated (non-P),
monophosphorylated (mono-P), and diphosphorylated
(di-P) forms of RLC are indicated. Lanes
1-9 correspond to the conditions in B from
left to right. B, quantitation of
phosphorylated RLC. The data represent the means ± S.E. for three
or four experiments.
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In the absence of Ca2+, RLC phosphorylation was 0.17 mol of
phosphate/mol of RLC at 30 min after saponin treatment, results similar
to those obtained at 3 min (Fig. 7, A and B). In
the presence of Ca2+/calmodulin but in the absence of
okadaic acid, the extent of RLC phosphorylation increased to 0.60 mol
of phosphate/mol of RLC at 30 min. This value increased to 1.60 mol of
phosphate/mol of RLC when okadaic acid was added (Fig. 7, A
and B). These results suggest that filament-bound myosin
light chain kinase phosphorylates myosin RLC to initiate cell contraction.
 |
DISCUSSION |
Previous investigations showed that myosin light chain kinase
bound to F-actin and F-actin-containing filaments in cells (14-20). Although it was assumed the binding in cells was to F-actin, a recent
report indicated a significant difference in apparent affinities for
the two types of filaments (21). Using a cosedimentation assay, Lin
et al. (21) showed that the kinase binding affinity was
greater for detergent-washed smooth muscle myofilaments compared with
purified F-actin or detergent-washed myofilaments from skeletal muscle.
This observation was extended herein with fluorescence imaging of
Cy3-labeled kinase binding to purified F-actin or gizzard myofilaments.
Myosin light chain kinase bound to gizzard myofilaments with no
significant binding to F-actin. These results are consistent with high
affinity binding of the kinase to myofilaments, perhaps due to an
anchoring or accessory protein. They also raise a question as to
whether this binding is regulated by Ca2+/calmodulin like
the low affinity binding of kinase to purified F-actin.
A fusion protein containing myosin light chain kinase and GFP
(MLCK-GFP) was expressed to monitor kinase binding to actin-containing filaments in cells. It has similar catalytic and binding properties as
purified smooth muscle myosin light chain kinase and therefore may be
used to characterize binding of myosin light chain kinase to
actin-containing stress fibers in cells. FL MLCK-GFP, although primarily localized to the filaments in intact cells, showed some diffuse cytoplasmic fluorescence that depended on the extent of expression. This cytoplasmic fluorescence may be due to saturation of
kinase binding sites on stress fibers in A7r5 cells, which is supported
by the observations that it diffused out when cells were treated with
saponin. Smooth muscle cells in tissues have a greater capacity for
binding myosin light chain kinase, because previous reports showed that
the kinase bound completely to skinned fibers (23) or detergent-washed
myofilaments (36). The FL MLCK-GFP on actin-containing filaments
remains bound after saponin permeabilization. These results obtained
from MLCK-GFP-transfected A7r5 cells are similar to those in Triton
X-100-permeable fibroblasts and intact smooth muscle cells perfused or
microinjected with Cy3-labeled myosin light chain kinases (herein, and
see Ref. 21).
Binding of myosin light chain kinase to actin-containing filaments in
permeable cells is not affected by Ca2+/calmodulin based on
the comparison of fluorescence intensities in the presence or absence
of Ca2+/calmodulin. In addition, FRAP measurements in
permeable cells indicate that myosin light chain kinase does not
diffuse laterally along the filaments. Previous studies in
vitro showed the binding affinity of myosin light chain kinase for
purified F-actin or purified myosin was decreased in the presence of
Ca2+/calmodulin (15, 18). Additionally,
Ca2+/calmodulin also decreased the binding affinity of the
N-terminal fragment (1-114 residues) of the kinase for purified
F-actin and another potential Ca2+/calmodulin binding site
was localized within the N-terminal actin binding sequence (residues
26-41) (15). However, the binding affinities (105 to
106 M
1) of myosin light chain
kinase and its actin binding sequence for F-actin alone are low (14,
15, 18) relative to the almost irreversible binding of the kinase in
skinned fibers (23) and detergent-washed myofilaments (21, 36) from
smooth muscles. Deletion of the N-terminal 142 residues of MLCK-GFP
prevents binding to actin-containing filaments in cells consistent with
previous results (21) when the N-terminal half of myosin light chain kinase was deleted. These results indicate that a recently identified Ca2+/calmodulin-insensitive binding site in myosin light
chain kinase (residues 319-721 preceding the catalytic core; Ref. 15)
is not responsible for binding to filaments in cells. These results also show that the anchoring motif is in the N terminus of myosin light
chain kinase. Importantly, this high affinity binding of myosin light
chain kinase to actin-containing filaments in saponin-permeable A7r5
cells and in Triton X-100-permeable Swiss 3T3 fibroblasts was not
affected by Ca2+/calmodulin. Although
Ca2+/calmodulin is required for activation of catalysis, it
does not significantly dissociate myosin light chain kinase from
cellular filaments.
Filament-bound myosin light chain kinase in saponin-permeabilized cells
is sufficient for RLC phosphorylation and cell contraction in the
presence of Ca2+/calmodulin. Wortmannin and a competitive
peptide substrate inhibited Ca2+-dependent RLC
phosphorylation and smooth muscle cell contraction in saponin-permeable
cells. Previous studies have shown that wortmannin is a potent and
selective inhibitor for myosin light chain kinase and
phosphatidylinositol 3-kinase, which is involved in formation of
inositol 1,4,5-trisphosphate and Ca2+ signaling (37, 38).
However, in experiments reported here, inhibition of
phosphatidylinositol 3-kinase is not an important consideration, since
this kinase does not phosphorylate myosin RLC, and a permeable cell
system was used so that filament-bound myosin light chain kinase could
be activated directly by the addition of Ca2+ and
calmodulin. Wortmannin has little or no effect on the activities of
other kinases including protein kinase C, cAMP-dependent
protein kinase, cGMP-dependent protein kinase,
calmodulin-dependent protein kinase II, tyrosine kinases,
and phosphatidylinositol 4-kinase (37, 38). In saponin-permeabilized
cells, both wortmannin and RLC peptide inhibited
Ca2+-dependent RLC phosphorylation and cell
contraction, which is consistent with the idea that these coupled
events (3-7) are due to filament-bound myosin light chain kinase.
In our studies, three forms (non-, mono-, and diphosphorylated) of
RLC were observed in saponin-permeable cells in the presence of
Ca2+/calmodulin and/or okadaic acid. Phosphorylation at
Ser-19 of smooth muscle myosin RLC by myosin light chain kinase has
been identified both in vitro and in smooth muscle tissues
as the major monophosphorylation site important for actin-activated
MgATPase activity and myosin motility (25, 39). However, Thr-18 is also
phosphorylated but at a slower rate by myosin light chain kinase
in vitro, leading to diphosphorylated RLC (40, 41). In
contracting smooth muscle tissues, monophosphorylated and
diphosphorylated forms of myosin RLC were also observed (34). However,
because phosphorylation at Thr-18 has no effect on the velocity of
myosin movement in motility assays in vitro (42), the
physiological importance of diphosphorylated RLC needs clarification by
further investigations.
An interesting result was observed in saponin-permeable cells in
Ca2+-free buffer. RLC phosphorylation was increased in
Ca2+-free buffer containing okadaic acid, a known
phosphatase type 1 and 2A inhibitor (26). In addition, wortmannin did
not inhibit this RLC phosphorylation. These results suggest that some
low kinase activity toward RLC is independent of the
Ca2+/calmodulin-dependent myosin light chain
kinase pathway. Ca2+-independent RLC phosphorylation in the
presence of a protein phosphatase inhibitor such as okadaic acid has
also been reported for smooth muscle tissues (26). Recent studies also
showed in vitro and in vivo that protein kinase C
and Rho-associated kinase (Rho-kinase) may phosphorylate RLC (43-45).
Although protein kinase C phosphorylated Thr-9, Ser-1, and/or Ser-2 of
myosin RLC in vitro, it did not increase actin-activated
Mg2+ATPase activity or myosin motility (42, 44). Smooth
muscle contraction still occurred at low Ca2+
concentrations, suggesting that a Ca2+-independent isoform
of protein kinase C
could conceivably phosphorylate RLC (46). On the
other hand, Rho-kinase activated by GTP-bound RhoA may increase the
extent of RLC phosphorylation and induce smooth muscle contraction in
the absence of Ca2+ (45). Increasing RLC phosphorylation by
Rho-kinase appears to be primarily indirect through the inactivation of
myosin phosphatase by phosphorylation of the myosin-binding subunit of
the phosphatase (47). However, very little Rho-kinase was present in
Triton X-100-permeable fibers (45), and both inactive and active
Rho-kinases did not appear to be localized to actomyosin-containing
fibers based on immunofluorescence staining (48). Additional
investigations are needed to identify specifically this
Ca2+-independent kinase and its potential physiological relevance.
In summary, our results suggest that myosin light chain kinase bound to
actin-containing filaments with a high affinity at its N terminus is
sufficient for RLC phosphorylation and cell contraction when activated
by Ca2+/calmodulin. This raises an interesting question as
to how the kinase phosphorylates myosin RLC in thick filaments if it is
bound to actin-containing thin filaments. Structural studies by
sedimentation velocity show that axial ratios of full-length myosin
light chain kinase are 18.0 and 16.8 in the absence and presence of
Ca2+/calmodulin, respectively (49). Based on sedimentation
velocity data, the calculated length of the recombinant smooth muscle
myosin light chain kinase is 540 Å.2 Thus, the shape of a
full-length myosin light chain kinase molecule is elongated either in
the presence or absence of Ca2+/calmodulin. Myosin light
chain kinase may reach and phosphorylate RLC on myosin thick filaments
while it remains attached to actin-containing thin filaments. In smooth
muscle, the surface-to-surface distance from thick and thin filaments
is approximately 150 Å (50). It has been shown by small angle x-ray
and neutron scattering that myosin light chain kinase containing the
catalytic core and regulatory segment forms an elongated ellipsoid with
a maximum linear dimension of 95 and 78 Å in the absence and presence
of Ca2+/calmodulin, respectively (51). The catalytic cleft
is in the middle of these ellipsoids. Smooth muscle myosin light chain
kinase contains structural motifs between the N terminus and catalytic core, including the tandem repeat region and two immunoglobulin-like (Ig) and one fibronectin-like (Fn) motifs (Fig. 2A). The
three-dimensional structures of individual Ig and Fn motifs are known
(52, 53), and calculations show that three of these motifs would extend the catalytic core at least 130 Å. The three-dimensional structures of
the tandem repeat region, the intervening sequences between the
immunoglobulin-like and fibronectin-like motifs, and the N terminus of
myosin light chain kinase are not known, but clearly the intervening
distance between the N-terminal binding segment and the
substrate-binding cleft in the catalytic core seems more than
sufficient to span the 150 Å between thin and thick filaments. Although a plausible hypothesis, additional investigations are needed
to understand how myosin light chain kinase bound to actin-containing filaments in cells phosphorylates myosin light chain in thick filaments.