From the Department of Physiology, University of Massachusetts
Medical Center, Worcester, Massachusetts 01655, the
§ Muscle Research Group, Boston Biomedical Research
Institute, Boston, Massachusetts 02114, and the ¶ Institute of
Medical Sciences, University of Tokyo, Minato-ku, Tokyo 108, Japan
The motor function of smooth muscle myosin is
activated by phosphorylation of the regulatory light chain (RLC) at
Ser19. However, the molecular mechanism by which the
phosphorylation activates the motor function is not yet understood. In
the present study, we focused our attention on the role of the central
helix of RLC for regulation. The flexible region at the middle of the central helix (Gly95-Pro98) was substituted or
deleted to various extents, and the effects of the deletion or
substitution on the regulation of the motor activity of myosin were
examined. Deletion of Gly95-Asp97,
Gly95-Thr96, or
Thr96-Asp97 decreased the actin-translocating
activity of myosin a little, but the
phosphorylation-dependent regulation of the motor activity was not disrupted. In contrast, the deletion of
Gly95-Pro98 of RLC completely abolished the
actin translocating activity of phosphorylated myosin. However, the
unregulated myosin long subfragment 1 containing this RLC mutant showed
motor activity the same as that containing the wild type RLC. Since
long subfragment 1 motor activity is unregulated by phosphorylation,
i.e. constitutively active, these results suggest that the
deletion of these residues at the central helix of RLC disrupts the
phosphorylation-mediated activation mechanism but not the motor
function of myosin itself. On the other hand, the elimination of
Pro98 or substitution of
Gly95-Pro98 by Ala resulted in the activation
of actin translocating activity of dephosphorylated myosin, whereas it
did not affect the motor activity of phosphorylated myosin. Together,
these results clearly indicate the importance of the hinge at the
central helix of RLC on the phosphorylation-mediated regulation of
smooth muscle myosin.
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INTRODUCTION |
The motor activity of vertebrate smooth muscle as well as
non-muscle cell myosin is regulated by phosphorylation of the 20,000 dalton light chain (LC20)1 of myosin
(1-5). While the phosphorylation of myosin at LC20 occurs at several
sites which are catalyzed by various protein kinases, the activation
effect is rather specific to the site of phosphorylation, and only the
phosphorylation at serine 19 and/or threonine 18 can activate the motor
activity of myosin (6-8). Recent three-dimensional structural analysis
of the skeletal muscle myosin head domain greatly facilitated the
understanding of the structure-function relationship of the myosin
motor (9). It was revealed that the ATPase active site and actin
binding site, two functionally essential regions of the myosin motor, exist toward the top of the myosin head while the regulatory light chain (i.e. LC20) associates with the myosin heavy chain at
the lower end of myosin head, i.e. adjacent to the head-rod
junction. This structural feature indicates that it is unlikely that
the regulation is achieved by the direct interaction between the motor effector sites and the phosphorylation site. This leads to the hypothesis that the change in the conformation of LC20 induced by
phosphorylation is transmitted to the motor effector sites via
intersubunit communication that would be critical for the regulation of
myosin motor activity (10). While the detail of such a mechanism is not
yet understood, some progress toward this problem has been made. It has
been shown using various probes that phosphorylation of LC20 changes
the conformation at the head-rod junction of myosin (10-13). It was
shown recently that the two-headed structure is critical for the
phosphorylation-mediated regulation of myosin motor activity (14-16).
Furthermore, it was found that the chimeric myosin which consisted of
the skeletal globular motor domain, smooth muscle light
chain-associated domain and S2, is completely regulated by
phosphorylation (17), suggesting that the regulatory domain solely
confers the phosphorylation-dependent regulation. Based on
these findings, it is plausible that the interaction between the two
myosin heads at the C-terminal regulatory domain of S1 is altered by
phosphorylation that is critical for regulation. The question is how
the phosphorylation changes the conformation of RLC (regulatory light
chain), thus activating the myosin motor activity. Using myosin
molecules containing the chimeric or mutated LC20, it was revealed
that: 1) the negative charge introduced by the phosphate moiety is
important for the activation of myosin motor activity because the
substitution of Ser19/Thr18 of LC20 by the
acidic residues partially mimicked the phosphorylation-induced activation of myosin motor activity (18-19); and 2) the C-terminal domain of LC20 is critical for both heavy chain binding and
phosphorylation-dependent regulation because an N-terminal
smooth RLC/C-terminal skeletal RLC chimera failed to activate myosin
motor activity by phosphorylation (20), the deletion of the C-terminal
residues diminished the binding to the heavy chain (21-22), and the
deletion or substitution of the C-terminal residues of LC20 disrupted
the phosphorylation-dependent activation of smooth muscle
myosin (21). These results suggest that the phosphorylation at the
N-terminal region of RLC influences the conformation at the RLC-heavy
chain interface at the C-terminal domain of RLC and thus achieves the
regulation of myosin motor function. For calmodulin, it has been
suggested that there is a cross-talk between the N- and C-terminal
domains, and the long central helix which connects the two domains
plays a role on the cross-talk. Since three-dimensional structure of
RLC is homologous to calmodulin (9), it is plausible that the long
central helix of RLC also plays a role on the cross-talk between the N-
and C-terminal domains of RLC, thus involved in regulation of smooth muscle myosin motor activity.
In the present study, we produced a series of mutant LC20s in which the
hinge region at the middle of the long central helix of LC20 is
modified. The motor activity and the phosphorylation dependence of
myosin containing these mutant LC20s were examined. The results clearly
indicate that the hinge region at the central helix of LC20 plays a
critical role for the phosphorylation-induced regulation of myosin
motor activity.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction enzymes and modifying enzymes were
purchased from New England Biolabs (Beverly, MA). The PT7-7
Escherichia coli expression vector containing the T7
promotor (23) was provided by Dr. S. Tabor (Harvard Medical School).
Smooth muscle myosin was prepared from frozen turkey gizzards as
described (24). Actin was prepared from rabbit skeletal muscle acetone
powder according to Spudich and Watt (25). Smooth muscle myosin light chain kinase was prepared from frozen turkey gizzards (26). Recombinant
calmodulin was expressed in E. coli and prepared as follows.
Calmodulin cDNA from Xenopus oocytes (27) in the
pTNcoI2 vector was kindly supplied by Dr. C. Klee (National
Institutes of Health). Two oligonucleotide primers, 5'-GAA ATA CTT CAT
ATG GCT GAC CAA CTG ACA G-3', containing an NdeI site as
well as an initiation codon, and 5'-TAC CGC AAG CGA CAG GCC G-3',
downstream of the polylinker region of the vector, were synthesized and
used for polymerase chain reaction amplification of calmodulin
cDNA. The amplified cDNA was digested with NdeI and
HindIII and subcloned into PT7-7 using unique
NdeI and HindIII sites at the polylinker region.
BL21(DE3) was transformed with the calmodulin expression vector and
then cultured in LB medium supplemented with 50 µg/ml ampicillin
overnight at 37 °C. 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside was added to the
liquid culture, and the cells were harvested 10 h later. The
packed cells (3 g) were sonicated in buffer containing 8 M
urea, 5 mM DTT, 10 µg/ml leupeptin, and 30 mM
Tris-HCl, pH 7.5. The sample was centrifuged at 35,000 × g for 15 min at 4 °C, and 5% trichloroacetic acid was
added to the supernatant. The precipitates were collected by
centrifugation (27,000 × g for 10 min), dissolved with
8 M urea and Tris base, and dialyzed against buffer A (1 mM DTT, 30 mM Tris-HCl, pH 7.5). After
centrifugation at 27,000 × g for 10 min, the
supernatant was subjected to DE52 ion-exchange chromatography. After
the sample was loaded, the column (3 cm × 24 cm) was washed with
buffer A and the recombinant calmodulin was eluted with a linear NaCl
gradient from 0 to 500 mM. The fractions containing
calmodulin were determined by SDS-PAGE analysis, concentrated, and
subjected to Sephacryl S200 gel filtration chromatography (3 cm × 90 cm) in buffer containing 100 mM NaCl, 1 mM
DTT, and 30 mM Tris-HCl, pH 7.5. The fractions containing calmodulin were collected, dialyzed against buffer B (30 mM
KCl, 1 mM DTT, and 30 mM Tris-HCl, pH 7.5), and
stored at -80 °C.
Production of the Mutant LC20--
The mutant cDNAs were
made by the site-directed mutagenesis strategy as described previously
using the LC20 expression vector pT7-LCW as a template (18, 21).
PT7-LCW does not contain a tag sequence, to avoid having artificial
amino acid residues in the expressed recombinant LC20. After confirming
the mutation by direct sequencing analysis, the mutant LC20 expression
vectors were transferred to BL21(DE3). The expression and purification of the mutant LC20s was done as described previously (21).
Incorporation of the Mutant LC20s into Myosin--
The
regulatory light chain was removed from myosin according to Trybus
et al. (22) with the following modification. Myosin (5 mg/ml) was incubated with buffer C containing 0.4 M NaCl, 5 mM EDTA, 2 mM EGTA, 3 mM
NaN3, 10 mM DTT, 5 mM TFP, 0.1%
Triton X-100, 1.5 mM ATP, and 20 mM
imidazole-HCl, pH 7.0, for 1 h at 0 °C and then applied to a
Sepharose CL4B column (3 × 90 cm) equilibrated with buffer D
containing 0.4 M NaCl, 5 mM EDTA, 2 mM EGTA, 3 mM NaN3, 0.5 mM ATP, 1 mM DTT, and 20 mM
imidazole-HCl, pH7.0. The myosin-containing fractions were combined and
a three-fold molar excess of LC20 was added to them. The mixture was
dialyzed for 90 min against buffer F containing 0.4 M NaCl,
1 mM EGTA, 5 mM MgCl2, 0.2 mM ATP, 1 mM DTT, and 10 mM
imidazole-HCl, pH 7.0. The mixture was further dialyzed for 2 h
against buffer G containing 15 mM MgCl2, 2 mM DTT, and 20 mM Tris-HCl, pH 7.5, and then
centrifuged for 2 min at 10,000 × g. The precipitates
were washed twice with buffer D and then dissolved with buffer G (0.3 M KCl, 5 mM DTT, and 50 mM
Tris-HCl, pH 7.5). To prepare myosin containing phosphorylated LC20,
the purified LC20s were thiophosphorylated before adding to the
LC20-deficient myosin. The extent of phosphorylation was monitored by
urea-gel electrophoresis (29), and complete phosphorylation of LC20 was
confirmed (not shown).
Expression of the Recombinant Truncated Smooth Muscle
Myosin--
The baculovirus transfer vectors of smooth muscle heavy
chain and light chains were produced as described (16). Recombinant baculoviruses for the heavy chain and the light chains were produced according to the protocols described by O'Reilly et al.
(30). To express truncated smooth muscle myosin, Sf9
insect cells were coinfected with three separate viruses expressing the
heavy chain and two light chains. The recombinant smooth muscle myosin
was purified as described previously (16).
Determination of Myosin Motor Function--
Actin-activated
ATPase activity was measured at 25 °C in an assay mixture containing
0.1 mg/ml myosin, 10 mM MgCl2, 30 mM KCl, 1 mM EGTA, 0.2 mM ATP, and
30 mM Tris-HCl, pH 7.5 with or without 5 mg/ml F-actin. The
ATPase activity of myosin or actomyosin was determined by measuring the
liberated 32P as described previously (24). SDS-PAGE was
carried out on 7.5-20% polyacrylamide gradient slab gels by using the
discontinuous buffer system of Laemmli (31).
Actin-translocating velocity was measured by an in vitro
motility assay as described previously (16). The Student's
t test was used for statistical comparison of mean values. A
value of p < .01 was considered to be significant.
Analytical Ultracentrifugation--
Sedimentation velocity
analysis was performed at 20 °C on a Beckman model-E analytical
ultracentrifuge. Sedimentation patterns were acquired with the on-line
Rayleigh system (32) and converted into concentration versus
radius every 20 s. The camera lens was focused at the 2/3 plane of
the cell equipped with sapphire windows. The apparent sedimentation
coefficient distribution function were computed as described by
Stafford (33, 34).
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RESULTS |
Hybridization of Smooth Muscle Myosin with Exogenous LC20--
The
hybrid myosin containing various mutant LC20 was prepared by adding
exogenous LC20 to the LC20-deficient myosin. The exogenous LC20 was
properly bound to the LC20-deficient myosin at the LC20 binding site
based on the following findings. 1) The stoichiometry of the heavy
chain and LC20 in the reconstituted myosin was identical to that of
naturally isolated smooth muscle myosin based upon gel densitometry
analysis (data not shown, see also Ref. 16). 2) The reconstituted
myosin with wild type LC20 showed phosphorylation-dependent actin-activated ATPase activity and actin-translocating activity, which
were indistinguishable to naturally isolated myosin (Table I, see Fig. 2).
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Table I
Actin-activated ATPase activity of smooth muscle myosin containing
various mutant RLC
The activities were measured with three independent preparations of
myosin. All data are presented as mean activity ± S.D. ATPase
activity was measured as described under "Experimental
Procedures."
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Six mutant LC20s were produced in which the four amino acid residues
corresponding to the hinge region at the middle of the central helix of
LC20 were deleted or substituted to various extents (Fig.
1). These mutant LC20s were introduced to
the LC20-deficient myosin as described above. All mutant LC20s bound
stoichiometrically to myosin heavy chain as judged by gel densitometry
(not shown). To produce phosphorylated myosin, LC20s were first
thiophosphorylated with ATP
S in the presence of
Ca2+/calmodulin/MLC kinase to avoid possible
dephosphorylation during the preparations and then introduced to the
LC20-deficient myosin. For all LC20 mutants tested in this study, the
rate of phosphorylation was indistinguishable from that of wild type
LC20 (data not shown). The LC20s were completely phosphorylated as
judged by the mobility shift due to the phosphate group in urea-gel
electrophoresis (not shown, see also Ref. 21).

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Fig. 1.
Amino acid sequence and three-dimensional
structure of the regulatory light chain. A, the amino acid
sequence of smooth muscle regulatory light chain (50). The residues in
the central helix region are boxed, and the phosphorylation
sites that are involved in activation of myosin motor activity are
indicated by *. B, the amino acid sequence of various
regulatory light chain mutants at the hinge region of the central
helix. Mutation is indicated by bold letters.
C, the three-dimensional structure of the chicken skeletal
regulatory light chain showing the position of the central hinge at the
middle of the central helix. The central hinge is indicated in
red. Sequence number is based upon smooth RLC
sequence.
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Actin-activated ATPase Activity of Smooth Muscle Myosin Containing
LC20 Mutants--
The actin-activated ATPase activity of myosins was
determined in the presence of 5 mg/ml F-actin, in which the extent of
actin activation was saturated (maximum ATPase activity). The
actin-activated ATPase activity of myosin, containing phosphorylated
P in which Pro98 was deleted, and GTDP/AAAA, in which
all four residues were substituted by Ala, were practically the same as
that of myosin containing wild type LC20 (Table I), whereas the myosins
containing other phosphorylated LC20 mutants showed lower ATPase
activity (Table I). The decrease in the activity was moderate for
TD
and
GT and more extensive for
GTDP and
GTD. These results
suggest that the hinge at the central helix of LC20 is involved in the
phosphorylation-induced activation mechanism of the actomyosin ATPase
activity. On the other hand, the actin-activated ATPase activity of
dephosphorylated myosin containing mutant LC20s was higher than that of
myosin containing wild type LC20, particularly for
GTDP and
P
(Table I).
Actin Sliding Velocity--
The effect of the mutation of LC20 at
the hinge of the central helix on myosin motor function was evaluated
more directly by using an in vitro motility assay. Myosin
containing wild type LC20 showed completely
phosphorylation-dependent actin translocating activity, and an
actin sliding velocity of 0.67 ± 0.04 µm/s at 25 °C was obtained
for phosphorylated LC20, which is comparable with the value previously
obtained for naturally isolated smooth muscle myosin (16). While myosin
containing GTDP/AAAA,
P, or
TD showed virtually the same
phosphorylation-activated actin- translocating activity, the activity
was significantly lower for the myosins containing
GTD and
GT
(Fig. 2). The most striking effect was
observed for
GTDP-containing myosin in which actin-translocating activity was completely abolished (Fig. 2). There are two possibilities to account for this observation. First, the deletion of the four residues at the hinge of LC20, i.e.
GTDP, disrupts the
motor function of myosin. Second, the mutation does not abolish the motor activity itself but disrupts the
phosphorylation-dependent activation mechanism. To distinguish
between these two possibilities, we utilized a smooth muscle myosin
mutant having phosphorylation-independent, constitutively active motor
activity. Previously, we produced truncated mutants of smooth muscle
myosin containing various lengths of the S2 portion and showed that the
monomeric myosins have phosphorylation-independent motor activity while
the dimeric (double-headed) myosins have the
phosphorylation-dependent activity (16). Fig.
3 shows the actin translocating activity
of the 108-kDa truncated smooth muscle myosin (a monomeric myosin). The
108-kDa myosin containing
GTDP showed motor activity
indistinguishable from that of the myosin containing wild type LC20.
Virtually the same results were obtained for the 108-kDa myosin
containing phosphorylated
GTDP and phosphorylated wild type LC20
(not shown). The results clearly indicate that the deletion of the four
residues at the hinge of the central helix of LC20 disrupts the
phosphorylation-induced activation process of smooth muscle myosin
motor activity but does not diminish the myosin motor activity itself
(Fig. 3). The result suggests that
GTDP cannot support the activation by phosphorylation when bound to
myosin heavy chain and is thus trapped in the off state. To further
evaluate this possibility, in vitro motility assay was
performed using the mixture of phosphorylated myosin and
GTDP-containing myosin (Fig. 4). The
significant reduction of the velocity was not found until the majority
myosin was a
GTDP-containing one. Previously, Cuda et al.
(35) reported that more than 60% of dephosphorylated myosin is
required until significant reduction of the velocity occurs. The
present results are consistent with the earlier observation and suggest
that
GTDP light chain traps phosphorylated myosin in the off state.
It should be noted that the observed actin translocating velocity for
the 108-kDa myosin (single-headed) was approximately 40% of that of
the full-length myosin (Fig. 3). This is consistent with the previous
finding (16) and suggests that the mechano-chemical activity is
affected by the interaction between the two heads. Another important
finding is that the myosins containing GTDP/AAAA or
P showed
significant phosphorylation-independent actin translocating activity
(Fig. 5). The actin sliding velocity of the dephosphorylated myosins containing these two mutants was approximately 30% of that of the
phosphorylated myosins. Myosin containing the dephosphorylated GTDP/AAAA or
P light chain supported continuous actin filament movement and majority of the filaments moved. This result suggests that
the inhibitory activity of dephosphorylated LC20 is diminished by
mutation at the hinge of the central helix of LC20.

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Fig. 2.
Sliding velocity of actin filaments on smooth
muscle myosins containing various phosphorylated mutant RLCs.
Actin movement was observed in 30 mM KCl, 5 mM
MgCl2, 25 mM imidazole-HCl, pH 7.5, 1 mM EGTA, 1% 2-mercaptoethanol, 0.5% methylcellulose, 4.5 µg/ml glucose, 216 µg/ml glucose oxidase, 36 µg/ml catalase, and
2 mM ATP at 25 °C. Measurements were made with three
independent preparations, and 11-21 actin filaments were measured to
obtain an average velocity for each preparation. All values are mean
velocity ± S.D.
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Fig. 3.
Sliding velocity of actin filaments on smooth
muscle long S1 containing either wild RLC or GTDP. Actin
movement was observed as described in Fig. 3, except a monoclonal
antibody (mm9) which recognizes the S2 portion
(Ala873-Ser944) of myosin was used to bind the
long S1 to the nitrocellulose surface as described previously
(16).
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Fig. 4.
Sliding velocity of actin filaments for
mixtures of myosins containing phosphorylated wild type RLC and
GTDP. Actin movement was observed as described in Fig. 3. The
velocities were normalized with respect to that obtained with myosin
containing phosphorylated wild type light chain (0.61 ± 0.06 µm/s).
, phosphorylated myosin mixed with dephosphorylated myosin; ,
phosphorylated myosin mixed with myosin containing phosphorylated
GTDP RLC; , phosphorylated myosin mixed with myosin containing
dephosphorylated GTDP RLC.
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Fig. 5.
Activation of sliding velocity of actin
filaments on smooth muscle dephosphorylated myosin by disruption of the
hinge at the central helix of RLC. Measurements were made with
three independent preparations, and 11-20 actin filaments were
measured to obtain an average velocity for each preparation. All values
are mean velocity ± S.D.
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The Effect of the Mutation at the Hinge of the Central Helix of
LC20 on Myosin Conformation--
It has been known that smooth
muscle/non-muscle myosins exist in two interchangeable conformations, a
folded conformation and an extended conformation (36-38) that is
directly related to the filament forming ability of myosin (39, 40).
The equilibrium between the two conformations is sensitive to ionic
strength (41) and phosphorylation of LC20 (38, 41-43). The
conformational transition of myosins containing various mutant LC20s
was examined using analytical ultracentrifugation. Sedimentation
patterns for myosin were measured at 0.2 and 0.4 M KCl
(Fig. 6). The sedimentation pattern of
myosins containing various mutant LC20s revealed a single symmetric
peak under both conditions. The sedimentation coefficient
(s20,w value) of myosin containing
dephosphorylated wild type LC20 increased from 6.81 to 9.89 with
decreasing KCl concentration, which is consistent with the previous
reports (36, 39, 41) indicating that myosin forms an extended and a
folded conformation (36-38) at 0.4 M KCl and 0.2 M KCl, respectively. While the sedimentation coefficient of
myosins containing various LC20 mutants did not differ from each other
at 0.4 M KCl, the values at 0.2 M KCl were
significantly different among the myosins containing different LC20
mutants. The sedimentation velocities of the myosins containing
dephosphorylated
GTDP,
P, and GTDP/AAAA were 6.6 ± 0.05 s,7.3 ± 0.05 s, and 6.5 ± 0.05 s, which indicated that myosin having these mutant LC20s
failed to form a folded conformation. On the other hand, the
sedimentation constants of the myosins containing
GT and
TD were
similar to that of the myosin having wild type LC20 (Fig. 6).

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Fig. 6.
Apparent sedimentation coefficient
distributions for myosins having various mutant LC20s.
Sedimentation velocity was determined in solution containing 1 mM MgCl2, 1 mM ATP, and 30 mM Tris-HCl, pH 7.5, 0.5 mg/ml myosin containing various
LC20 mutants, and either 0.2 M KCl (A) or 0.4 M KCl (B). The velocity run was carried out at
56,000 rpm at 20 °C.
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DISCUSSION |
While it is well known that the phosphorylation at serine 19 of
LC20 activates smooth muscle or non-muscle myosin motor activity, it is
not clear how the phosphorylation can activate the motor activity at a
molecular level. According to the three-dimensional structural analysis
of skeletal muscle S1, the regulatory light chain consists of two
domains that are segregated by a long central
-helix (9). We
hypothesized that the central long
-helix of LC20 plays a role in
the phosphorylation-mediated regulation mechanism. Particularly, we
focused our attention on the middle of the central helix where the
helix is distorted (Fig. 1). To verify our hypothesis, we produced six
mutant LC20s in which the four amino acid residues (Gly-Thr-Asp-Pro)
corresponding to the hinge of the long central
-helix of LC20 were
modified (Fig. 1). We found that the change in the structure of the
hinge region significantly altered the motor function of smooth muscle
myosin. The most notable findings were: 1) the deletion of all four
residues (
GTDP) abolished the activation effect of phosphorylation
on myosin motor activity; and 2) two mutant LC20s in which the four residues were substituted for Ala (GTDP/AAAA) or a proline residue in
the hinge was deleted (
P) conferred the phosphorylation-independent motor activity on smooth muscle myosin, i.e. the inhibitory
function of dephosphorylated LC20 was diminished. These findings
clearly show that the hinge region of the central helix of LC20 is
critical for phosphorylation-mediated regulation of smooth muscle
myosin. It is expected that the deletion or substitution of the proline residue at the hinge would eliminate the distortion at the middle of
the long
-helix; therefore, the results obtained in the present work
suggest that the flexed nature of the central shaft of the LC20 is
important for phosphorylation-mediated regulation of myosin motor
activity. Previously, it was reported that the C-terminal domain of
LC20 is responsible for the binding of LC20 to the heavy chain (21).
Furthermore, the deletion of the C-terminal residues of LC20 abolishes
the phosphorylation-mediated regulation of smooth muscle myosin despite
the fact that the phosphorylation site is located in the N-terminal
domain (21, 22). This raised the hypothesis that there is an
interaction between the N- and the C-terminal domains of LC20, which is
a critical component for the regulation of smooth muscle myosin motor
function (21). The present results are consistent with this notion and
suggest that the flexed region in the central helix plays a critical
role in the phosphorylation-dependent interaction between
the N- and the C-terminal domains.
LC20 shares considerable structural homology with calmodulin and
troponin C (9, 44, 45) in which the two domains are linked with a long
central connecting
-helix. It has been shown that the deletion of
several amino acid residues at the central helix of calmodulin
significantly disrupts the activation of target enzymes by calmodulin
(46), suggesting the importance of the central connecting linker region
in calmodulin function. The importance of the central helix of
calmodulin in its function is supported by analyzing the
three-dimensional structure of the calmodulin/MLC kinase peptide
complex. Upon formation of the ternary complex with the calmodulin
binding peptide in the presence of Ca2+, the long central
helix of calmodulin is disrupted into two helices connected by a
flexible loop (47, 48). In the case of myosin, the N-terminal domain of
LC20 wraps around the C terminus of the S1 heavy chain while the
C-terminal domain of LC20 interacts with the long helix portion of the
S1 heavy chain (9). Therefore, it is plausible that the change in the
distortion at the hinge of the central helix of LC20 would influence
the bend at the C-terminal end of S1 heavy chain. In scallop myosin, in
which the myosin function is regulated by the Ca2+ binding
to the essential light chain, a change in the bend at the C-terminal
end of S1 by Ca2+ binding is observed (49). Consistent with
this idea, the substitution of LC20 by
GTDP, in which the flex at
the hinge region of the central helix is disrupted, abolishes the
formation of a folded structure (Fig. 6) that is likely to be related
to the bend at the C-terminal end of S1.