Smooth muscle myosin is composed of two heavy chains, two 20-kDa
regulatory light chains (LC20) (
)and two 17-kDa essential
light chains (LC17). LC20 can be phosphorylated at Ser
by
Ca
/calmodulin-activated myosin light chain kinase and
the phosphorylation regulates the activity (for review see
Sellers(1991)) and conformation (for review see Trybus(1991)) of smooth
muscle myosin. Dephosphorylated myosin has a low actin-activated ATPase
activity and is unable to support the movement of actin filaments in a
motility assay or to generate superprecipitation. The phosphorylation
of LC20 activates these motor activities. On the other hand, smooth
muscle myosin takes on two different conformations, referred to as the
6 and 10 S forms based on their sedimentation coefficients. The 6 S
myosin assembles into filaments under physiological ionic conditions,
whereas 10 S myosin does not. Dephosphorylated myosin filaments
disassemble into 10 S monomers upon addition of ATP, whereas
phosphorylated myosin remains in filament form.
LC20 has been
removed from smooth muscle myosin by an antibody affinity column
(Trybus and Lowey, 1988) and by gel filtration in the presence of
trifluoperazine (TFP) (Trybus et al., 1994). The removal of
LC20 markedly increased the ATPase activity in the absence of actin,
thus resulting in a decrease in the actin-activated ATPase activity in
comparison with that of native phosphorylated myosin (Trybus et
al., 1994). The ability to move actin filaments in a motility
assay is also decreased by the removal of LC20 (Trybus et al.,
1994). In addition, the LC20-deficient myosin is shown to be unable to
fold into the 10 S form (Trybus and Lowey, 1988). Furthermore, an
expressed heavy meromyosin lacking LC17 is shown to move actin
filaments. The movement is significantly slower than that by control
heavy meromyosin but is phosphorylation-dependent (Trybus, 1994).
However, the effect of the lack of LC17 on the ATPase activity and
conformation of myosin has been unknown. For complete understanding of
phosphorylation-dependent regulation of smooth muscle myosin, it is
also very important to examine whether LC17 is required for the
regulation of actin-activated ATPase activity and of conformation.
It has not been possible to remove LC17 completely from smooth
muscle myosin (Trybus, 1994). Here we show for the first time that
LC17, in addition to LC20, can be completely removed from smooth muscle
myosin without denaturation. Reconstitution experiments with the light
chains showed that LC17 and phosphorylated LC20 initiated
actin-activated ATPase activity and superprecipitation but that
dephosphorylated LC20 inhibited them. For the myosin lacking LC17, not
only the rate of superprecipitation but also the actin-activated ATPase
activity was considerably lower than those for native myosin but were
phosphorylation-dependent, indicative of the nonessential nature of
LC17 for the phosphorylation-dependent regulation of the motor
activities of smooth muscle myosin. However, myosin lacking LC17 could
not fold into the 10 S form, indicating the requirement of LC17 for the
phosphorylation-dependent conformational transition and filament
formation of smooth muscle myosin.
EXPERIMENTAL PROCEDURES
Protein Preparation
Myosin was prepared from
porcine aorta smooth muscle as described by Hasegawa et
al.(1988, 1990), and the prepared myosin was almost completely
dephosphorylated. Phosphorylated myosin was prepared as described
previously (Katoh et al., 1995a). LC20 and phosphorylated LC20
were prepared from aorta myosin as described by Katoh and Morita(1993).
LC17 was prepared from aorta myosin according to the method of Grand
and Perry (1983) with modifications (Yoshida and Yagi, 1988). Two LC17
isoforms of aorta myosin were isolated by DE32 (Whatman) ion exchange
chromatography in the presence of 8 M urea (Hasegawa et
al., 1988). Actin was prepared from rabbit skeletal muscle by the
method of Spudich and Watt(1971) and purified by Sephacryl S-300HR
(Pharmacia LKB) gel filtration (Katoh and Morita, 1995). The
concentrations of myosin, actin, LC20, and LC17 were determined from
the absorbance at 280 nm using the absorption coefficients of 0.48,
1.1, 0.20, and 0.31 (mg/ml)
cm
,
respectively. The concentration of isolated heavy chain (LC-deficient
myosin) was determined by the method of Bradford (1976) using myosin as
a standard. The molecular weights used for myosin, actin, LC20, LC17,
and LC-deficient myosin were 500,000, 42,000, 20,000, 17,000, and
426,000, respectively.
Removal and Readdition of Light Chains
Myosin (5
mg/ml) was incubated on ice for 1 h in 0.4 M NaCl, 10 mM EDTA, 2 mM EGTA, 10 mM ATP, 10 mM DTT,
5 mM TFP, 0.05% Triton X-100, and 40 mM imidazole (pH
6.8). Ammonium chloride was added to the solution to 4.5 M,
and the mixture was further incubated on ice for 20 min. The incubated
mixture (0.5 ml) was loaded onto a Sephacryl S-300HR column (1.25
12 cm) equilibrated with 4.6 M ammonium chloride, 5
mM EDTA, 2 mM EGTA, 3 mM ATP, 2 mM DTT, 0.5 mM TFP, 0.05% Triton X-100, and 40 mM imidazole (pH 6.8), and the protein was eluted at 0.9 ml/min at
6-7 °C. The eluted LC-deficient myosin was immediately passed
through a Sephacryl S-300HR spun column at 4 °C to exchange its
buffer solution for 0.3 or 0.4 M NaCl, 2 mM DTT, and
20 mM imidazole (pH 7.0). The obtained LC-deficient myosin was
immediately mixed with LC17 (equimolar mixture of two LC17 isoforms
(Katoh et al., 1995b)) and/or LC20 (2 mol each light chain/mol
head) in 0.3 or 0.4 M NaCl, 5 mM MgCl
, 2
mM DTT, and 20 mM imidazole (pH 7.0) on ice. If
necessary, unbound light chains were removed with a Sephacryl S-300HR
spun column. The structural state (monomeric or oligomeric state) of
LC-deficient myosin or the reconstituted myosin was analyzed by gel
filtration HPLC in 0.4 M NaCl and 10 mM sodium
phosphate (pH 7.0).
Gel Electrophoresis
SDS-polyacrylamide gel
electrophoresis was carried out using 10% slab gels under the
conditions of Porzio and Pearson(1977). The gels were stained with
Coomassie Brilliant Blue G-250 or with silver.
Gel Filtration HPLC
Gel filtration HPLC was
performed at room temperature using a TSKgel G5000PW
column (7.8
300 mm) with a TSK guard column PW
(6.0
40 mm) attached to a Jasco Gulliver series HPLC
system. Elution of proteins was carried out at 0.50 ml/min and
monitored by absorbance at 225 nm.
Conformation Analysis
Myosin conformation (6 or 10
S form) was verified by gel filtration HPLC as described by Trybus and
Lowey (1988). The conformation of dephosphorylated or phosphorylated
myosin was examined at various concentrations of NaCl. Myosin
(0.15-0.19 mg/ml) in 0.15-0.4 M NaCl, 2.6 mM MgCl
, 0.1 mM EGTA, 1 mM DTT, and 10
mM imidazole (pH 7.0) was ultracentrifuged for 10 min at
160,000
g at 4 °C (Hitachi CP-100H
ultracentrifuge) in the presence or the absence of 0.1 mM ATP.
The supernatant was analyzed by gel filtration HPLC at the same NaCl
concentration as above with 1 mM MgCl
, 0.1 mM EGTA, and 10 mM sodium phosphate (pH 7.2) in the presence
or the absence of 20 µM ATP. The retention time of myosin
was plotted against the NaCl concentration (Fig. 1). The profile
of the 6-10 S conformational transition of dephosphorylated
myosin was in agreement with that of gizzard dephosphorylated myosin
shown by Cole and Yount(1992). At 0.24 M NaCl in the presence
of ATP, dephosphorylated myosin was still in the 10 S conformation,
whereas phosphorylated myosin was in the 6 S conformation. On the basis
of these results, the conformation of LC-deficient myosin or the
reconstituted myosin was analyzed at 0.24 M NaCl in the
presence of ATP.
Figure 1:
Salt
concentration dependence of the 6-10 S conformational transition
of myosin as analyzed by gel filtration HPLC. Dephosphorylated
(
,
) or phosphorylated myosin (
,
) at 0.15 mg/ml
in 0.15-0.4 M NaCl, 2.6 mM MgCl
,
0.1 mM EGTA, 1 mM DTT, and 10 mM imidazole
(pH 7.0) in the presence (
,
) or the absence (
,
)
of 0.1 mM ATP was ultracentrifuged, and the supernatant (50
µl) was analyzed by gel filtration HPLC (TSKgel
G5000PW
) in the same concentration of NaCl as above, 1
mM MgCl
, 0.1 mM EGTA, and 10 mM sodium phosphate (pH 7.2) in the presence or the absence of 20
µM ATP. The retention time of myosin was plotted against
the NaCl concentration.
Electron Microscopy
Electron microscopy was
performed on a Hitachi H-800 electron microscope operated at 75 kV. For
observation of myosin molecules, myosin samples were diluted to 10
µg/ml in 70% glycerol and 0.4 M ammonium acetate, sprayed
onto freshly cleaved mica, and rotary-shadowed with platinum at an
angle of 6 ° as described previously (Tyler and Branton, 1980;
Winkelmann et al., 1983). For observation of myosin filaments,
10 µl of each myosin sample (80-90 µg/ml) in 0.12 M NaCl, 5 mM MgCl
, 1 mM ATP, 0.1
mM EGTA, 1 mM DTT, and 20 mM imidazole (pH
7.0) was placed on a glow-discharged collodion carbon-coated copper
grid and was negatively stained with 1% uranyl acetate as described by
Trybus and Lowey(1987).
ATPase Assays and Superprecipitation
ATPase
reaction was carried out with 0.12-0.18 µM myosin in
0.1 M NaCl, 6 mM MgCl
, 1 mM ATP,
0.1 mM EGTA, 1 mM DTT, and 20 mM imidazole
(pH 7.0) at 25 °C in the presence and the absence of 20 µM actin. Four aliquots were taken at appropriate time intervals for
colorimetric determination of inorganic phosphate (Chifflet et
al., 1988; Gonzalez-Romo et al., 1992).
Superprecipitation was measured as described previously (Katoh and
Morita, 1995).
RESULTS
Removal and Reconstitution of LC20 and LC17 of Smooth
Muscle Myosin
Porcine aorta myosin was depleted of its LC20 and
LC17 by the method of Trybus et al.(1994) combined with the
method of Wagner and Giniger(1981). Myosin was incubated with TFP in
the presence of EDTA and ATP to dissociate LC20 followed by further
incubation at 0 °C upon addition of ammonium chloride to 4.5 M to dissociate LC17. The myosin heavy chain was separated from the
dissociated light chains by Sephacryl S-300HR gel filtration in the
presence of TFP and ammonium chloride. The isolated heavy chain
(LC-deficient myosin) was essentially free from both LC20 and LC17 (Fig. 2, lane 2) and could rebind stoichiometric
amounts of LC17 and/or LC20 (Fig. 2, lanes 3-5).
Figure 2:
Characterization of LC-deficient and
reconstituted myosins by SDS-polyacrylamide gel electrophoresis. The
LC-deficient myosin was mixed with LC17 and/or LC20 (2 mol each light
chain/mol head) in 0.4 M NaCl, 5 mM MgCl
,
2 mM DTT, and 20 mM imidazole (pH 7.0). After unbound
light chains were removed by a Sephacryl S-300HR spun column, the
myosin samples were analyzed by SDS-polyacrylamide gel electrophoresis. Lane 1, Native myosin; lane 2, LC-deficient myosin; lane 3, myosin reconstituted with LC17; lane 4,
myosin reconstituted with LC20; lane 5, myosin reconstituted
with LC17 and LC20. HC, myosin heavy
chain.
Characterization of LC-deficient and Reconstituted
Myosins
The structural states of LC-deficient and reconstituted
myosins in high salt conditions were examined by electron microscopy.
The rotary-shadowed images of the LC-deficient myosin and LC-deficient
myosin reconstituted with LC17 alone showed oligomers in addition to
monomers (not shown) as reported for LC-deficient skeletal muscle
myosin (Lowey et al., 1993). The heads in monomeric
LC-deficient myosin appeared to be more rounded and shorter than in
native myosin and the two heads in some LC-deficient myosin monomers
appeared to cohere. In contrast, the myosins reconstituted with LC20
alone and with both LC20 and LC17 were observed to be predominantly in
monomers, and the shapes of their heads appeared to be
indistinguishable from that of native myosin (Fig. 3). The
observation that the lack of LC20 resulted in the aggregation of myosin
molecules was supported by gel filtration HPLC in 0.4 M NaCl
(data not shown).
Figure 3:
Characterization of reconstituted myosin
by electron microscopy. Native myosin (A), LC-deficient myosin
reconstituted with LC20 (B), or with LC17 and LC20 (C) was rotary-shadowed with platinum. Scale bar, 0.1
µm.
Conformations of LC-deficient and Reconstituted
Myosins
The conformations of LC-deficient and reconstituted
myosins were examined by gel filtration HPLC in 0.24 M NaCl in
the presence of ATP as described under ``Experimental
Procedures.'' Dephosphorylated and phosphorylated myosins remained
soluble, existing predominantly as 10 and 6 S monomers, respectively,
and could be separated by gel filtration HPLC ( Fig. 1and Fig. 4, A and B). Most of the LC-deficient
myosin and myosins reconstituted with LC17 alone and with
dephosphorylated or phosphorylated LC20 alone were pelleted by
ultracentrifugation prior to the gel filtration HPLC. These pelleted
myosins would be filamentous and unable to fold. Electron microscopy of
these myosin samples without ultracentrifugation actually showed the
filaments and their aggregates (not shown). The monomers of these
myosin species remaining in the supernatant were mainly in the 6 S form (Fig. 4, D-F), but the monomer conformation of
LC-deficient myosin was uncertain because of the low concentration of
monomers (Fig. 4C). A part of the myosin reconstituted
with dephosphorylated LC20 alone was eluted at the time for 10 S myosin (Fig. 4E). However, the 10 S peak fraction showed the
LC17 and LC20 bands in approximately equimolar ratio on SDS gel stained
with silver (data not shown). The 10 S peak might be due to a very
small amount of LC17 remained in the LC-deficient myosin preparation.
The myosins reconstituted with both LC17 and dephosphorylated or
phosphorylated LC20 were mostly soluble and predominantly in the 10 and
6 S forms, respectively (Fig. 4, G and H), as
observed for native myosin (Fig. 4, A and B).
Therefore, these results suggest that both LC17 and LC20 are required
for the formation of the 10 S conformation and thus for the
phosphorylation-dependent 6-10 S conformational transition of
smooth muscle myosin.
Figure 4:
Conformation of reconstituted myosin
analyzed by gel filtration HPLC. Native dephosphorylated (A)
and phosphorylated myosin (B), LC-deficient myosin (C), LC-deficient myosin reconstituted with LC17 (D),
dephosphorylated LC20 (E), phosphorylated LC20 (F),
LC17 and dephosphorylated LC20 (G), or LC17 and phosphorylated
LC20 (H) was analyzed by gel filtration HPLC in 0.24 M NaCl, 1 mM MgCl
, 20 µM ATP, 0.1
mM EGTA, and 10 mM sodium phosphate (pH 7.2) as
described in the legend to Fig. 1.
Filament Formation of LC-deficient and Reconstituted
Myosins
The filament formation of the LC-deficient and
reconstituted myosins was examined in 0.12 M NaCl in the
presence of ATP. These myosin samples were negatively stained and
observed by electron microscopy (data not shown). The LC-deficient
myosin and myosin reconstituted with LC17 alone both showed large
aggregates of filaments as previously shown for the LC20-deficient
gizzard myosin (Trybus and Lowey, 1988). The myosin reconstituted with
LC20 alone also showed filaments and their aggregates irrespective of
the phosphorylation state of LC20, but the aggregates appeared to be
smaller and looser than those of LC-deficient myosin and myosin
reconstituted with LC17 alone. The myosin reconstituted with both LC17
and dephosphorylated LC20 showed few, but slightly more, filaments than
native dephosphorylated myosin. The myosin reconstituted with both LC17
and phosphorylated LC20 showed many filaments like native
phosphorylated myosin. These results suggest that both light chains are
required for the phosphorylation-dependent filament formation of smooth
muscle myosin.
ATPase Activities of LC-deficient and Reconstituted
Myosins
The ATPase activities of the LC-deficient and
reconstituted myosins were measured in the absence and the presence of
actin (Table 1). The activities of the LC-deficient myosin in the
absence and the presence of actin were both as low as those of native
dephosphorylated myosin and were increased 4-5-fold upon
reconstitution with LC17 alone, indicating that LC17 could activate the
ATPase activity. The reconstitution with dephosphorylated LC20 in
addition to LC17 decreased the ATPase activity to of that of myosin
reconstituted with LC17 alone, irrespective of the presence of actin.
When dephosphorylated LC20 was replaced by phosphorylated LC20, the
ATPase activity in the absence of actin was still one-half of that of
myosin having only LC17 but increased 2-fold in the presence of actin;
the levels of activities were comparable with those of native
phosphorylated myosin. Therefore, dephosphorylated LC20 strongly
inhibited the ATPase activity that had been activated by LC17,
irrespective of the presence of actin. Phosphorylated LC20 also
inhibited the ATPase activity in the absence of actin but activated it
in the presence of actin. Similar effects of LC20 were also observed in
the absence of LC17. However, the level of ATPase activity in the
presence of actin for myosin having only phosphorylated LC20, as well
as that for myosin having only LC17, was considerably lower than those
for myosin having both light chains and for native phosphorylated
myosin. The activating effects of LC17 and phosphorylated LC20 seemed
to be additive for the ATPase activity in the presence of actin,
indicating a requirement of both light chains for the generation of
full activity. These results suggest that LC17 acts as an activator and
dephosphorylated and phosphorylated LC20, a repressor and an activator,
respectively, of the actin-activated ATPase of smooth muscle myosin.
The reconstitution with both light chains fully restored the
phosphorylation-dependent regulation of the actin-activated ATPase
activity (Table 1). When the LC-deficient myosin was
reconstituted with LC20 alone, the actin-activated ATPase activity of
the reconstituted myosin was phosphorylation-dependent. Therefore, LC17
may not be essential for the phosphorylation-dependent regulation of
actin-activated ATPase activity. However, the degree of regulation for
myosin lacking LC17 (4.5-fold activation by phosphorylation) was
considerably lower than that for myosin having both LC20 and LC17
(20-fold activation by phosphorylation). This was mainly due to the low
activity of myosin having only phosphorylated LC20 in the presence of
actin, and the activity was complemented by LC17.
Superprecipitations of Acto-LC-deficient and
Acto-reconstituted Myosins
Superprecipitation time courses for
the LC-deficient and reconstituted myosins were examined.
Superprecipitation was not observed up to 60 min for the LC-deficient
myosin, myosin reconstituted with dephosphorylated LC20 alone, that
with both LC17 and dephosphorylated LC20, or for native
dephosphorylated myosin (Fig. 5, a, c, e, and g). When the LC-deficient myosin was
reconstituted with both LC17 and phosphorylated LC20, superprecipitaion
occurred quickly after the addition of ATP, as did superprecipitation
of acto-native phosphorylated myosin, although the maximum level of
superprecipitation for the reconstituted myosin was somewhat lower than
that for native phosphorylated myosin (Fig. 5, b and h). When the LC-deficient myosin was reconstituted with
phosphorylated LC20 alone, superprecipitation occurred but was
considerably slow in comparison with that for native phosphorylated
myosin, although the maximum level was even higher than that for native
phosphorylated myosin (Fig. 5, f). The results indicate
that superprecipitation of actomyosin can be regulated by
phosphorylation even if the myosin is depleted of LC17. For the myosin
reconstituted with LC17 alone, superprecipitation was observed, but the
development was very slow (Fig. 5, d), consistent with
a low motility for LC20-deficient myosin (Trybus et al.,
1994). The results suggest that superprecipitation is activated by LC17
alone and by phosphorylated LC20 alone but that both light chains are
required for full activation.
Figure 5:
Superprecipitation of acto-reconstituted
myosin. Native dephosphorylated (a) or phosphorylated myosin (b), LC-deficient myosin (c), LC-deficient myosin
reconstituted with LC17 (d), dephosphorylated LC20 (e), phosphorylated LC20 (f), LC17 and
dephosphorylated LC20 (g), or LC17 and phosphorylated LC20 (h) at 0.24 µM with 5.2 µM actin in
0.1 M NaCl, 5 mM MgCl
, 0.1 mM EGTA, 1 mM DTT, and 20 mM imidazole (pH 7.0) was
mixed with 1 mM ATP at 25 °C. The reaction was monitored
by absorbance at 660 nm.
DISCUSSION
Removal of LC20 and LC17 from Smooth Muscle
Myosin
The removal of LC20 from smooth muscle myosin has been
reported (Trybus and Lowey, 1988; Trybus et al., 1994), but
the removal of LC17 has not until now been achieved. On the other hand,
the removal of both regulatory and essential light chains from skeletal
muscle myosin has been done by gel filtration in the presence of 4.7 M ammonium chloride (Lowey et al., 1993), although
this method does not dissociate the light chains from smooth muscle
myosin (Lowey and Trybus, 1995). The stronger interaction of regulatory
light chain with the heavy chain in smooth muscle myosin than in
skeletal muscle myosin (Trybus and Chatman, 1993) may have prevented
the dissociation of the light chains from smooth muscle myosin. In
fact, we could dissociate LC17 from smooth muscle myosin in a high
concentration of ammonium chloride after the dissociation of LC20 by
TFP. The results are consistent with the observations that the
essential light chain exchange in skeletal muscle myosin subfragment 1
lacking regulatory light chain occurs more readily than in myosin
(Waller and Lowey, 1985) and that the essential light chain exchange in
desensitized scallop myosin is easier than in intact myosin (Ashiba and
Szent-Gyorgyi, 1985).
Roles of LC20 and LC17 in Conformational Transition and
Filament Formation of Smooth Muscle Myosin
The lack of either
LC20 or LC17 made smooth muscle myosin unable to fold into the 10 S
form, resulting in the formation of filaments in the presence of ATP (Fig. 4). The impairment of the ability to fold into the 10 S
form for myosin lacking LC20 or both light chains might have been due
to cohesion of its two heads, as observed for myosin whose two heads
were chemically cross-linked (Katoh and Morita, 1995). However, this
was not the case for the reconstituted myosin with dephosphorylated
LC20 alone because such cohesion of the two heads was not observed for
this myosin by electron microscopy. Not only LC20 but also LC17 might
be required for the formation of the native structure of the neck
region, which is suggested to be involved in the folding into the 10 S
form (Katoh et al., 1995a). On the other hand, the 6-10
S conformational transition is modulated by ATP (Fig. 1),
suggesting that signals of the binding of ATP to the active site of
myosin somehow transmitted to the neck region resulted in the 10 S
conformation. Both LC17 and LC20 subunits may mediate such
transmission.
Roles of LC20 and LC17 in Motor Activities of Smooth
Muscle Myosin
The removal of regulatory and/or essential light
chains from skeletal muscle myosin does not significantly decrease the
actin-activated ATPase activity (Pastra-Landis and Lowey, 1986; Lowey et al., 1993). On the other hand, the lack of these light
chains decreases the motility of skeletal muscle myosin, but the
isolated heavy chain still moves actin filaments (Lowey et
al., 1993). Skeletal muscle myosin is not regulated and is always
in the ``on'' state that corresponds to the phosphorylated
state in smooth muscle myosin. In comparison with phosphorylated smooth
muscle myosin, the isolated smooth muscle myosin heavy chain showed the
actin-activated ATPase activity of less than (Table 1) and no
superprecipitation (Fig. 5). Therefore, the skeletal muscle
myosin heavy chain may be in the active state, whereas the smooth
muscle myosin heavy chain, in contrast, may be in the inactive state.
Both LC17 and phosphorylated LC20 are required for smooth muscle myosin
to be in the active state ( Table 1and Fig. 5). The role
of skeletal myosin light chains is suggested to stabilize the light
chain-binding domain as the lever arm for efficient coupling of ATP
hydrolysis to motility (Lowey et al., 1993; Waller et
al., 1995). In contrast, LC17 and phosphorylated LC20 activate not
only superprecipitation but also the actin-activated ATPase activity
for smooth muscle myosin. Therefore, the smooth muscle myosin light
chains have specific functional roles in the generation of motor
activities, in addition to the structural role that is suggested for
skeletal muscle myosin.On the other hand, LC17 may not be essential
for the phosphorylation-dependent regulation of motor activities ( Table 1and Fig. 5), in agreement with the
phosphorylation-dependent motility of an expressed gizzard heavy
meromyosin lacking LC17 (Trybus, 1994). However, the levels of the
motor activities of myosin having only phosphorylated LC20 were still
considerably lower than those of myosin having LC17 and phosphorylated
LC20 ( Table 1and Fig. 5). Therefore, LC17 may be required
for full activation and thus for full regulation of the motor
activities of smooth muscle myosin.
The three-dimensional structure
of the head of skeletal muscle myosin shows that a 10 nm stretch of the
-helical heavy chain segment to which light chains are bound
extends near the ATPase and actin-binding sites (Rayment et
al., 1993). As the head of smooth muscle myosin probably has a
homologous structure, the phosphorylation site may be spatially apart
from the ATPase and actin-binding sites. The signals of LC20
phosphorylation should be transmitted to the active site through the
light chain-binding domain in each head or through interactions between
two heads. In either case, our results suggest that the transmission
may be essentially mediated by LC20 to generate the
phosphorylation-dependent regulation of motor activities in smooth
muscle myosin.
In contrast, the regulation of 6-10 S
conformational transition through phosphorylation requires both light
chains as described above. The phosphorylation-dependent regulation of
motor activities is not directly coupled with that of the
conformational transition of smooth muscle myosin. Such an uncoupling
between regulations in the activity and conformation is also reported
by Ikebe et al.(1994).
Interactions among both light chains
and the heavy chain may be required for full regulation of the
conformation and motor activities of smooth muscle myosin as suggested
for Ca
-dependent regulation of scallop myosin (Xie et al., 1994). To elucidate the mechanisms of the
phosphorylation-dependent regulation and thus of the intramolecular
signal transmission in smooth muscle myosin, more detailed studies are
undoubtedly required, and the method of removing light chains presented
in this paper could be an aid for the purpose.