(Received for publication, June 1, 1995)
From the
Pretreatment of
The calcium sensitivity of force output from smooth muscle is
variable(1) , and the mechanism responsible for this has been
studied in permeabilized smooth muscles that retain receptor-coupled
signal transduction
mechanisms(2, 3, 4, 5, 6) .
In these preparations, either agonist activation in the presence of GTP
or direct activation of G proteins with GTP Several groups (14, 15, 16) have shown recently that the
myosin-bound phosphatase of smooth muscle is composed of three subunits
of 130, 37-38, and 20 kDa. The 37-38-kDa component is the
The quantity of
thiophosphate incorporated into muscle protein was determined from the Many protein kinases use ATP A typical force trace
for an
Figure 1:
Contractile response to
various calcium concentrations prior to and following treatment with
ATP
Fig. 2shows a summary of the
force responses and corresponding myosin light chain phosphorylation
measurements in protocols similar to that shown in Fig. 1B. As expected, maximal activation with pCa 4.5 was associated with a high level of light chain
phosphorylation, whereas pCa 6 resulted in submaximal force
and light chain phosphorylation. Treatment of the muscle with ML-9
reduced the levels of phosphorylation and force elicited by pCa 4.5 and 6. Muscles that were pretreated with 1 mM ATP
Figure 2:
Force and myosin light chain
phosphorylation under various conditions. Force (openbars) and myosin light chain phosphorylation (solidbars) at 5 min for muscles activated under the following
conditions: treatment with pCa 4.5 and pCa 6 in both
the presence and absence of the myosin light chain kinase inhibitor
ML-9 (300 µM), pCa 6 in the presence of 10
µM GTP
For comparison,
we also show the calcium-sensitizing effect of GTP
Figure 3:
Time course of myosin light chain
dephosphorylation following removal of ATP for muscles activated under
various conditions. Fractional light chain phosphorylation for muscles
that were transferred to rigor solution (pCa > 8) and
frozen at various times after having been activated for 5 min under the
following conditions: pCa 4.5 (filledcircles), pCa 6 in the presence of 10 µM GTP
There is a much slower decrease in
myosin light chain phosphorylation in the muscles pretreated with
ATP The decrease in myosin light chain phosphatase
activity in the presence of GTP Control experiments
confirmed that the increase in calcium sensitivity was in fact due to
ATP It is possible that the decrease in myosin light chain
phosphatase activity results from thiophosphorylation of a protein by a
kinase involved in regulation of the phosphatase.
[
Figure 4:
Separation and identification of
thiophosphorylated proteins following treatment with
[
Further analysis of the high molecular weight components was carried
out by SDS-PAGE on low porosity gels (Fig. 4B). The
Western blot using the mAb to the gizzard 130-kDa subunit of myosin
light chain phosphatase and its associated autoradiogram show that one
of the thiophosphorylated components (i.e. The fact that the 130-kDa
subunit of the myosin light chain phosphatase is one of the relatively
few proteins that are thiophosphorylated under conditions that result
in a large decrease in activity of the enzyme in the permeabilized
muscle strongly suggests that the thiophosphorylation of the subunit
caused the decrease in activity. If this is the case, then it is likely
that phosphorylation and dephosphorylation of the 130-kDa subunit
represents an in vivo mechanism for regulation of phosphatase
activity that is potentially important for the regulation of smooth
muscle contraction. Studies on other cellular phosphatases have
demonstrated that phosphorylation of subunits or binding proteins play
a role in regulation of activity. Phosphorylation of a site on the
targeting subunit of glycogen-associated protein phosphatase 1 causes a
decrease in activity by decreasing the affinity of the catalytic
subunit for the targeting subunit while phosphorylation of another site
increases activity(27) . In addition, phosphorylation of
inhibitor 1 and DARPP-32 by the cAMP-dependent protein kinase markedly
increases their inhibition of PP1(28) . In summary,
thiophosphorylation of the 130-kDa regulatory subunit of myosin light
chain phosphatase is associated with a 5-fold decrease in the activity
of the enzyme and a large increase in the calcium sensitivity of force
and myosin light chain phosphorylation in
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-toxin-permeabilized smooth muscle with
ATP
S (adenosine 5`-O-(thiotriphosphate)) under conditions
resulting in minimal (<1%) thiophosphorylation of the myosin light
chain increases the subsequent calcium sensitivity of force output and
myosin light chain phosphorylation. The change in calcium sensitivity
results at least in part from a 5-fold decrease in myosin light chain
phosphatase activity. One of the few proteins thiophosphorylated under
these conditions is the 130-kDa subunit of myosin light chain
phosphatase. These results suggest that thiophosphorylation of this
subunit leads to a decrease in the activity of the phosphatase, and
that phosphorylation and dephosphorylation of the subunit may play a
role in regulating myosin light chain phosphatase activity.
S (
)increases myosin light chain phosphorylation at submaximal
calcium concentrations(7, 8, 9) . At a
constant free calcium concentration, the increase in phosphorylation is
correlated with the increase in force, and appears to result from an
inhibition of myosin light chain phosphatase activity ( (9) and (10) ; see (11) for review). The mechanism linking
receptor activation and inhibition of the myosin light chain
phosphatase is not known. Gong et al.(12) have
suggested that arachidonic acid may play a role, while Masuo et al.(13) have shown that activation of protein kinase C
decreases phosphatase activity.
-isoform of the protein phosphatase type 1 (PP1) catalytic
subunit(15) , also referred to as the
-isoform(14) . The other two subunits are thought to be
involved in targeting the phosphatase to its substrate, myosin, and
thus are putative regulatory subunits. Binding of the trimeric complex
to myosin has been demonstrated(15) , and it has also been
shown that the holoenzyme has enhanced activity to its substrate myosin
(or HMM) compared to the isolated catalytic
subunit(14, 16) . Derived sequences from the cDNAs of
the large subunit from gizzard (15) and rat (17) indicate the presence of several potential phosphorylation
sites. We report here experiments that suggest phosphorylation of the
130-kDa subunit of myosin light chain phosphatase plays a role in the
regulation of its enzymic activity.
Muscle Treatment
Longitudinal strips of rabbit
portal vein were dissected and permeabilized as described
previously(18) . Briefly, muscles were pretreated with 5 mM DIDS to reduce ecto-ATPase activity and then permeabilized with
4200 units/ml Staphylococcus aureus -toxin (Life
Technologies, Inc.) in a relaxing solution containing 10 mM sodium azide as an ecto-ATPase inhibitor. All subsequent solutions
contained sodium azide, and the compositions were described
previously(18) . Experiments were performed at 20 °C.
Mechanical Measurements
Muscle strips were mounted
under isometric conditions at their in vivo length and bathed
in stirred solutions (600-800 µl). Active force output was
compared to that during an initial activation in pCa 4.5. For
inhibition of myosin light chain kinase activity, muscles were bathed
in a relaxing solution containing 300 µM ML-9 (19) for at least 5 min before exposure to other
ML-9-containing solutions.Measurement of Phosphatase Activity
The rate
constant for myosin light chain phosphatase activity was determined
from the time course of decrease in myosin light chain phosphorylation
when the muscle was placed in a rigor solution(10) . Muscles
were activated for 5 min by transfer to an activating solution (pCa 4.5), by exposure to pCa 6 in the presence of
GTPS, or by transfer to a pCa 6 solution containing ML-9
after treatment with ATP
S. They were then transferred to a series
of rigor solutions and frozen at various times. Myosin light chain
phosphorylation was determined by two-dimensional gel
electrophoresis(20) , and the time course of dephosphorylation
of the light chain was fitted to a single exponential (Sigmaplot,
Jandel Scientific).
Identification of Thiophosphorylated
Proteins
Muscle strips were incubated in a rigor solution with
ML-9 and 100 µM ATPS containing 0.5-10 mCi/ml
[
S]ATP
S for 10 min. Muscles were frozen
following several rigor washes to remove the ATP
S, and extracted
in either 0.5 N HClO
or 10% trichloroacetic acid,
containing unlabeled ATP
S. Proteins were subjected to the
following procedures: SDS-PAGE on 7.5-20% gradient
gels(21) , SDS-PAGE on low porosity gels for higher molecular
weight proteins(22) , and two-dimensional electrophoresis over
the pH range 5-7 (2% Pharmalyte (Sigma) 5-7 and 1%
Pharmalyte 3-10). Proteins of known pI (Bio-Rad two-dimensional
SDS-PAGE standards) were used to calibrate the two-dimensional gels.
Autoradiography was performed either with x-ray film on gels using a
liquid scintillant enhancer (Autofluor, National Diagnostics Corp.) or
with a storage phosphor screen (PhosphorImager, Molecular Dynamics).
Monoclonal antibodies (mAb) to the 130-kDa subunit of myosin light
chain phosphatase were prepared by conventional methods (23) using the gizzard holoenzyme as antigen. For Western
blots, the antimouse immunoglobulin-peroxidase conjugate was recognized
either by enhanced chemiluminescence (ECL, Amersham) or
colorimetrically with 4-chloro-1-napthol.
S present in the acid-insoluble precipitate after
pretreatment with 100 µM ATP
S (
S, 0.5
mCi/ml,). The precipitate was solubilized (Solvable, DuPont NEN), total
S dpm measured, and thiophosphate content calculated from
the specific activity of [
S]ATP
S in the
labeling solution. Muscle volume was measured as described previously (18) by including 1 mM mannitol (
C, 0.01
mCi/ml) in the solution prior to freezing the muscle.
S to thiophosphorylate a
protein substrate, which is then resistant to phosphatase
activity(24) . In permeabilized smooth muscle, ATP
S
treatment results in a calcium-dependent thiophosphorylation of the
myosin light chain, which is then a very poor substrate for phosphatase
activity (25) . When the light chain is thiophosphorylated,
force output is independent of calcium concentration(25) , and
the actin-activated myosin ATPase is irreversibly
activated(26) . These observations were important in
determining the central role that myosin light chain phosphorylation
plays in the regulation of smooth muscle contraction. While performing
experiments to investigate the factors that control calcium sensitivity
of force output, we found that under certain conditions, ATP
S
pretreatment can increase the calcium sensitivity of contraction in
-toxin-permeabilized smooth muscle when there is minimal
thiophosphorylation of the myosin light chain.
-toxin-permeabilized muscle prior to and following
treatment with ATP
S is shown in Fig. 1A.
Pretreatment with 1 mM ATP
S at pCa > 8 in the
presence of the myosin light chain kinase inhibitor ML-9 resulted in no
increase in force when the muscles were transferred to an
ATP-containing relaxing solution. Upon transfer to a pCa 6
solution, however, there was a rapid rise in force to its maximum (106
± 5% of initial pCa 4.5 contraction) in contrast to a
much lower force output observed at the same calcium concentration
prior to ATP
S treatment (n = 6). If the muscle was
placed in pCa 7 after ATP
S pretreatment, there was a slow (t
13 min) increase in force to 71
± 7% of maximum, although there was no force output in this
calcium concentration before treatment (n = 4). Fig. 1B shows a similar protocol except that ML-9 was
included in the solutions after ATP
S treatment. Upon transfer to a pCa 6 solution with ML-9, there was a rapid rise in force
which was much greater than that observed at the same calcium
concentration in the presence of ML-9 prior to ATP
S treatment (n = 19). In this case the enhanced contractile
response was usually phasic.
S. Typical isometric force traces for muscles activated under
various conditions prior to and following treatment with 1 mM ATP
S in the presence of 300 µM ML-9 for 10 min
at low calcium (pCa > 8). Muscles were initially contracted
in pCa 4.5. Openbars indicate exposure to
relaxing solution (pCa > 8). PanelA,
response to pCa 6 before and after ATP
S treatment. PanelB, response to pCa 6 in the presence
of ML-9 before and after ATP
S treatment. PanelC, response to pCa 7 following ATP
S
treatment, with subsequent addition of GTP
S (100
µM).
S for 10 min under low calcium conditions (pCa
> 8) in the presence of ML-9 produced no force when transferred to a
relaxing solution containing 1 mM ATP. There was no detectable
thiophosphorylation of the myosin light chain in two-dimensional gels,
and phosphorylation was low (12 ± 4%, n = 5).
Subsequent incubation in pCa 6 in the presence of ML-9
resulted in
70% maximum force and myosin light chain
phosphorylation. Note the high force and phosphorylation under these
conditions compared to pCa 6 with or without ML-9 and pCa 4.5 with ML-9 prior to treatment with ATP
S. It is
obvious that ATP
S pretreatment dramatically increases the calcium
sensitivity of myosin light chain phosphorylation.
S, and both low calcium (pCa > 8)
and pCa 6 in the presence of ML-9 following treatment with 1
mM ATP
S. Data are shown as means ± S.E. for n = 4-7.
S (10
µM) at pCa 6. As expected, both force and myosin
light chain phosphorylation are increased compared to pCa 6
alone. This G protein-mediated increase in calcium sensitivity is
thought to result primarily from inhibition of myosin light chain
phosphatase activity, and we considered the possibility that a similar
inhibition of the phosphatase might be the basis for the increase in
calcium sensitivity observed with ATP
S pretreatment. Therefore, we
estimated the rate of myosin light chain phosphatase activity under
different conditions by measuring the time course of dephosphorylation
of the myosin light chain following removal of ATP (10) . Under
these conditions, kinase activity ceases and the subsequent decrease in
phosphorylation over time reflects the activity of the phosphatase. The
data are shown in Fig. 3.
S (opencircles), and pCa 6 in
the presence of 300 µM ML-9 following pretreatment with 1
mM ATP
S (filledtriangles). Data are
shown as means ± S.E. for n = 4-7. The curves are single exponential fits to the data (see
text).
S compared to those activated in pCa 4.5 or in pCa 6 in the presence of 10 µM GTP
S. In
muscles activated in pCa 6 with ML-9 following the ATP
S
pretreatment, the phosphatase rate constant (0.0028 ± 0.0003
s
) was much lower than those activated in pCa 4.5 (0.016 ± 0.002 s
), and even
lower than those activated in pCa 6 in the presence of 10
µM GTP
S (0.011 ± 0.002 s
).
The increased calcium sensitivity of force and myosin light chain
phosphorylation seen with ATP
S pretreatment seems, at least in
part, to be due to a more than 5-fold decrease in myosin light chain
phosphatase activity.
S observed in this study is similar
to the 50% decrease reported by others for both GTP
S (10, 13) and protein kinase C activators(13) ,
but there is a much larger decrease in phosphatase rate caused by
ATP
S pretreatment. Fig. 1C shows an experimental
protocol designed to test whether GTP
S-mediated calcium
sensitization can occur after the phosphatase activity has been
decreased by ATP
S pretreatment. When the muscle is in pCa
7 after ATP
S pretreatment, the force output is less than maximum,
and there is little, if any, increase when 100 µM GTP
S is added (n = 6). Apparently, the
mechanism responsible for GTP
S-mediated calcium sensitization
cannot decrease phosphatase activity when it is already at a very low
rate as a result of ATP
S pretreatment.
S rather than other aspects of the treatment protocol. In the
absence of ATP
S, there was no change in the calcium sensitivity (n = 4), while treatment with 0.1 and 0.01 mM ATP
S gave 0.99 ± 0.04 (n = 3) and 0.55
± 0.12 (n = 4), respectively, of the response
with 1 mM ATP
S pretreatment. When 0.1 mM GTP
S pretreatment was substituted for ATP
S, no enhanced
contractile response was observed (n = 4), while
subsequent treatment with ATP
S elicited the calcium-sensitizing
effect.
S]ATP
S was used to radiolabel the proteins
that are thiophosphorylated under conditions when the phosphatase
activity is decreased. The total incorporation of thiophosphate into
muscle protein was equivalent to 3.0 ± 0.5 µmol/liter of
muscle volume (n = 6). Fig. 4A shows a
Coomassie Blue-stained gel of the total homogenate separated by
SDS-PAGE and its PhosphorImager autoradiogram; also shown is the scan
of the autoradiogram. The bands containing most of the radioactivity
were of relatively high molecular mass (>80 kDa), and in this range
five or six major components were seen. There was very little
radioactivity associated with low molecular weight proteins. Based on
the complexity of the total homogenate, it is surprising that a more
extensive pattern of thiophosphorylation was not found. In a few cases,
labeled proteins of about 28 and 20 kDa were detected. The amount of
radioactivity in the 20-kDa region, which would include the myosin
light chain, ranged from undetectable to a maximum of 15% of the total
counts. This would indicate an upper limit of about 0.5 µM for the amount of thiophosphorylated myosin light chain resulting
from ATP
S pretreatment. Since the myosin light chain concentration
is
50-100 µM(18) , the fraction of
myosin light chains thiophosphorylated is very small (<1%).
S]ATP
S. PanelA, total
muscle homogenate on SDS gel (7.5-20% acrylamide); from left, Coomassie Blue-stained gel, PhosphorImager autoradiogram
of the same gel, and a scan of total
S counts. PanelB, total muscle homogenate on low porosity SDS gel (same
order as in panelA). PanelC,
Western blot using mAb to the 130-kDa subunit of myosin light chain
phosphatase on low porosity SDS gel; from left, isolated
chicken gizzard 130-kDa subunit, total muscle homogenate, and
PhosphorImager autoradiogram of the total homogenate lane. Panels
D-F, Western blots of IEF-SDS gels using the mAb to the
130-kDa phosphatase subunit. PanelD, total muscle
homogenate of a control sample (not treated with ATP
S). PanelE, total muscle homogenate of an ATP
S-treated
sample. PanelF, PhosphorImager autoradiogram of
whole muscle homogenate. The inset shows the stained portion
of the Western blot, along with a scan of the radioactivity in the same
portion of the autoradiogram.
S-labeled) cross-reacts with the mAb (Fig. 4C). Its mass is slightly higher than the gizzard
M130, which is also shown. The Western blots of a control muscle and an
ATP
S-treated muscle run on two-dimensional gels are shown in Fig. 4(D and E). The control sample, which was
frozen in relaxing solution with no exposure to ATP
S, showed a
simple staining pattern with a major component at pI 5.9-6.0.
Following ATP
S treatment, several components were seen. They
extended toward more acidic values over a pH range of 5.5-6.0,
and at least four isoforms were apparent. An autoradiogram of a Western
blot of a two-dimensional gel is shown in Fig. 4F. The inset shows the stained portion of the blot along with the
autoradiographic scan of the same area. There are several
S-labeled components that are superimposable on the
staining pattern of the Western blot.
-toxin-permeabilized
smooth muscle. These results suggest that phosphorylation and
dephosphorylation of this subunit may play a role in the regulation of
smooth muscle contraction.
S, guanosine
5`-3-O-(thio)triphosphate; ATP
S, adenosine
5`-O-(thiotriphosphate); DIDS,
4,4`-diisothiocyanatostilbene-2,2`-disulfonic acid; mAb, monoclonal
antibody.
We thank S. U. Mooers and S. R. Narayan for expert
technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.