J Biol Chem, Vol. 274, Issue 42, 30122-30126, October 15, 1999
Role of the N-terminal Region of the Regulatory Light Chain in
the Dephosphorylation of Myosin by Myosin Light Chain Phosphatase*
Reiko
Ikebe,
Sheila
Reardon,
Toshiaki
Mitsui, and
Mitsuo
Ikebe
From the Department of Physiology, University of Massachusetts
Medical School, Worcester, Massachusetts 01655-0127
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ABSTRACT |
Myosin regulatory light chain (RLC) is
phosphorylated at various sites at its N-terminal region, and
heterotrimeric myosin light chain phosphatase (MLCP) has been assigned
as a physiological phosphatase that dephosphorylates myosin in
vivo. Specificity of MLCP toward the various phosphorylation
sites of RLC was studied, as well as the role of the N-terminal region
of RLC in the dephosphorylation of myosin by MLCP. MLCP
dephosphorylated phosphoserine 19, phosphothreonine 18, and
phosphothreonine 9 efficiently with almost identical rates, whereas it
failed to dephosphorylate phosphorylated serine 1/serine 2. Deletion of
the N-terminal seven amino acid residues of RLC markedly decreased the
dephosphorylation rate of phosphoserine 19 of RLC incorporated in the
myosin molecule, whereas this deletion did not significantly affect the
dephosphorylation rate of isolated RLC. On the other hand, deletion of
only four N-terminal amino acid residues showed no effect on
dephosphorylation of phosphoserine 19 of incorporated RLC. The
inhibition of dephosphorylation by deletion of the seven N-terminal
residues was also found with the catalytic subunit of MLCP.
Phosphorylation at serine 1/serine 2 and threonine 9 did not influence
the dephosphorylation rate of serine 19 and threonine 18 by MLCP. These
results suggest that the N-terminal region of RLC plays an important
role in substrate recognition of MLCP.
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INTRODUCTION |
The motor function of conventional myosins expressed in smooth
muscle as well as nonmuscle cells is regulated by phosphorylation of
the regulatory light chain subunit (1-5). A
calmodulin-dependent myosin light chain-specific protein
kinase, myosin light chain kinase
(MLCK),1 phosphorylates
serine 19 and threonine 18 of the regulatory light chain (RLC), and the
phosphorylation of these sites activates the motor activity of myosin
(1-5). Serine 19 is the preferred site and is important for the
activation of the actomyosin contractile apparatus under physiological
conditions. Other protein kinases can phosphorylate RLC at serine 19 in vitro (5, 6), and they may play a role in activation of
myosin-based contractile events under certain conditions. On the other
hand, protein kinase C (7, 8) and cdc2 kinase (9) phosphorylate serine
1/serine 2 and threonine 9 of RLC. Phosphorylation of these sites does not activate myosin motor activity, but rather inhibits it because of a
decrease in affinity for actin (7-9).
One of the enzymes which catalyzes dephosphorylation of the RLC of
myosin is called myosin light chain phosphatase (MLCP). Serine/threonine protein phosphatases can be classified into several types based upon their substrate specificity, sensitivity to
inhibitors, and the requirement of divalent cations for their
activation (10-12), i.e. types 1, 2A, 2B, and 2C. A number
of myosin phosphatases have been prepared from various smooth muscle
tissues. Pato and co-workers (13, 14) purified four distinct protein
phosphatases from avian gizzards, i.e. SMP-I, SMP-II,
SMP-III, and SMP-IV. SMP-I and SMP-II dephosphorylated isolated myosin
regulatory light chain, but not intact myosin (13), and are classified
as PP2A and PP2C, respectively. SMP-III and SMP-IV dephosphorylated
intact myosin, and the characterization of these enzymes indicates that they are similar to type I phosphatases (14, 15). However, they are not
inhibited by protein phosphatase inhibitor 2 (14, 15), which is one of
the characteristics of type I phosphatase (10).
It has been shown that myosin preparations contain significant myosin
phosphatase activity, suggesting that MLCP associates with myosin. MLCP
purified from a smooth muscle myosin preparation (16) is a type I
phosphatase based upon biochemical characterization. MLCP prepared from
the actomyosin fraction of gizzard smooth muscle is composed of three
subunits, 130, 37, and 20 kDa, of which the 37 kDa is the catalytic
subunit (17). Similar phosphatases were subsequently purified by other
groups (18, 19). Because these smooth muscle MLCPs show an affinity to
myosin, it is thought that these phosphatases are responsible for the
dephosphorylation of myosin in situ. Consistent with this
notion, it was shown that the major myosin phosphatase in skinned
smooth muscle is the type I phosphatase.
Whereas the isolated RLC can be dephosphorylated by several types of
protein phosphatases, only the myosin-associated phosphatase efficiently dephosphorylates intact myosin (16-19) but types 2A and 2C
(13) phosphatases do not. This suggests that there may be a specific
substrate recognition mechanism for dephosphorylation of myosin by
MLCP. In the present paper, we studied the role of the N-terminal
residues of RLC in dephosphorylation of smooth muscle myosin. The site
specificity of the myosin-associated MLCP was also studied using RLC
phosphorylated at various sites.
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MATERIALS AND METHODS |
Preparation of Proteins and Construction of RLC Expression
Vector--
Smooth muscle myosin (20) and myosin light chain kinase
(21) were prepared from turkey gizzards as described. Myosin was washed
several times with high MgCl2 (30 mM)
containing buffer to remove residual MLCP activity. HMM and S1 were
prepared from gizzard myosin as described (22). Calmodulin was prepared
from frozen bull testes according to Walsh et al. (23). MLCP
was prepared from turkey gizzard as described (17). The catalytic subunit of smooth muscle MLCP was prepared as described (16). cDNA
of smooth muscle myosin RLC was obtained from a chicken gizzard 
gt11
cDNA library and subcloned into a PT7-7 Escherichia
coli expression vector (24) as described (25). Truncation of RLC cDNA was done using a PCR-based method as described previously (25). The expression of recombinant RLC and its mutants in the E. coli strain BL21 (DE3) was performed according to Kamisoyama et al. (26). The expressed RLC in E. coli cells
was extracted in urea-containing buffer and purified with a series of
liquid chromatography steps according to the method of Ikebe et
al. (25).
Protein Biochemical Procedures--
Phosphorylation of RLC at
serine 19 was achieved by incubating RLC (4 mg/ml) with 2 µg/ml MLCK
and 5 µg/ml calmodulin in buffer containing 0.5 mM
[
-32P]ATP, 150 mM KCl, 1 mM
MgCl2, 0.1 mM CaCl2, 1 mM DTT, and 30 mM Tris-HCl, pH 7.5 (buffer A),
at 25 °C for 20 min. The extent of phosphorylation was 0.9 mol of
phosphate/mol of RLC, and no phosphorylation at threonine residues was
detected based upon phosphoamino acid analysis, indicating that only
serine 19 was phosphorylated. Phosphorylation at both serine 19 and
threonine 18 was achieved by incubating RLC (4 mg/ml) with 50 µg/ml
MLCK and 30 µg/ml calmodulin in modified buffer A containing 1 mM [-32P]ATP and 30 mM KCl at
25 °C for 40 min. Incorporation of 1.8 mol of phosphate/mol of RLC
was obtained, and an equal amount of phosphoserine and phosphothreonine
was detected using phosphoamino acid analysis, indicating that both
serine 19 and threonine 18 were phosphorylated (27). Phosphorylation of
RLC (2 mg/ml) at serine1/serine2 and threonine 9 by PKC was done by
incubating with a solution containing 3 µg/ml PKC, 100 ng/ml phorbol
12-myristate 13-acetate, 0.1 mg/ml phosphatidylserine, 1 mM
[-32P]ATP, 30 mM KCl, 1 mM
MgCl2, and 30 mM Tris-HCl, pH 7.5 at 24°C for
50 min. Two mol of phosphate/RLC was incorporated, and both serine and
threonine were phosphorylated as judged by phosphoamino acid analysis
(Ref. 7, also see Fig. 6). The phosphorylated RLCs were precipitated
with 5% trichloroacetic acid and dissolved in and dialyzed against
buffer B (30 mM KCl, 1 mM DTT, and 30 mM Tris-HCl, pH 7.5).
RLC-deficient gizzard myosin was prepared according to the method of
Trybus et al. (28) with modification (29). Phosphorylated RLC (2 molar excess) was added to RLC-deficient myosin in buffer C (30 mM KCl, 1 mM DTT, 2 mM
MgCl2, and 30 mM Tris-HCl, pH 7.5) at 0 °C.
After 10 min, the myosin was centrifuged for 2 min at 10,000 × g. The pellets were suspended with buffer C and centrifuged again. This step was repeated three times. The pellets were then dissolved with 0.4 M KCl, 5 mM DTT, and 30 mM Tris-HCl, pH 7.5, and used for experiments. The obtained
myosin contained phosphorylated RLC as judged by SDS-polyacrylamide gel
electrophoresis analysis followed by autoradiography (not shown). The
extent of phosphorylation of the obtained myosin was stable at least
for 6 h at 25 °C.
Phosphoamino acid analysis was done as described previously (27). The
extent of phosphorylation of RLC was determined as described (27).
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RESULTS |
Dephosphorylation of Myosin by Smooth Muscle MLCP--
Smooth
muscle myosin containing various truncated RLCs (Fig.
1) was prepared as described under
"Materials and Methods." Dephosphorylation of native smooth muscle
myosin phosphorylated at serine 19 of RLC by smooth muscle MLCP was
described by single exponential kinetics (Fig.
2). The dephosphorylation time course of
myosin incorporating exogenous serine 19 phosphorylated RLC was
identical to that of native myosin, suggesting that the exogenous RLC
was properly incorporated into myosin. The rate of dephosphorylation was somewhat reduced with NDL-4 RLC-incorporated myosin, but the decrease was not significant. On the other hand, when the N-terminal seven residues of RLC were deleted (NDL-7), the dephosphorylation by
smooth muscle MLCP was markedly and significantly inhibited. The rate
of dephosphorylation was 1/10 of that of myosin incorporating wild type
RLC. Further deletion of three amino acid residues did not affect the
rate of dephosphorylation. These results suggest that
Ala5-Lys6-Ala7 is critical for
dephosphorylation of RLC at serine 19 by MLCP.
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Fig. 1.
N-terminal amino acid sequence of RLC
deletion mutants and the phosphorylation sites by various protein
kinases. The phosphorylation sites are indicated by bold
letters. * and ** denote the phosphorylation sites by PKC/cdc
2kinase and MLCK, respectively.
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Fig. 2.
Dephosphorylation of myosin containing RLC
truncation mutants that are phosphorylated at serine 19 by smooth
muscle MLCP. Reaction was initiated by the addition of MLCP (0.4 µg/ml) to the reaction buffer containing 75 mM KCl, 10 mM MgCl2, 1 mM EGTA, and 30 mM Tris-HCl, pH 7.5, at 25 °C. An aliquot was removed at
various times, and the reaction was stopped by the addition of 5% TCA.
The phosphorylation level of myosin was measured as described (21).
Solid lines represent single exponential decay curve of the
dephosphorylation of wild type RLC and NDL-7, respectively.  , wild
type RLC;  , NDL-4;  , NDL-7;  , NDL-10;  , native
myosin.
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Dephosphorylation of Isolated RLC--
Because many protein
phosphatases can dephosphorylate isolated RLC but not intact myosin, we
wished to assay whether or not the effect of deletion of the N-terminal
amino acid residues observed above is also found with isolated RLC as a
substrate. To address this notion, the dephosphorylation reaction by
smooth muscle MLCP was carried out with isolated truncated mutants of
RLC. As shown in Fig. 3, the rate of
dephosphorylation was not markedly affected by the deletion of any
N-terminal residues of RLC. The rates of dephosphorylation of NDL-4 and
NDL-7 were slightly lower than that of wild type RLC, but the decrease
was not significant (Fig. 3). These results indicate that the deletion
of Ala5-Lys6-Ala7 diminished
dephosphorylation of serine 19 of RLC only in the presence of myosin
heavy chain but not in its absence. These results also suggest that
myosin heavy chain plays a role in the smooth muscle MLCP-RLC
interaction.
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Fig. 3.
Dephosphorylation of the serine 19 phosphorylated isolated mutant RLCs by smooth muscle MLCP.
Reaction was done as described in the legend to Fig. 2. A solid
line represents single exponential decay curve of
dephosphorylation of wild type RLC. , wild type RLC; , NDL-4;
, NDL-10.
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Dephosphorylation of Myosin by the Catalytic Subunit of Smooth
Muscle MLCP--
Smooth muscle MLCP is composed of three subunits,
i.e. the myosin binding subunit, catalytic subunit, and
small subunit (17-19). It was shown that phosphatase activity against
myosin is enhanced in the presence of the myosin binding subunit (30),
suggesting a difference in the nature of the dephosphorylation reaction
between apoenzyme and holoenzyme. To examine whether the decrease in
phosphatase activity of smooth muscle MLCP by the deletion of the
N-terminal region of myosin-incorporated RLC is because of the function
of the myosin binding subunit or because of the characteristics of the
catalytic subunit of smooth muscle MLCP, we used catalytic subunit of
smooth muscle MLCP to dephosphorylate truncated phosphorylated RLC
incorporated into myosin. As shown in Fig.
4, dephosphorylation of myosin by the
catalytic subunit of smooth muscle MLCP was also significantly
attenuated by deletion of the N-terminal residues. The rate of
dephosphorylation of NDL-7-containing myosin was 9% of the rate of
wild type RLC-containing myosin. This was also confirmed using a
different myosin fragment which contained truncated RLC generated by a
brief tryptic proteolysis of myosin which cleaves off the N-terminal 16 residues of RLC (31). Myosin was first phosphorylated with MLCK, then
subjected to tryptic digestion and used as a substrate for smooth
muscle MLCP holoenzyme and apoenzyme as described under "Materials
and Methods." The rate of dephosphorylation of myosin incorporating
the truncated RLC which lacked the N-terminal 16 residues was
significantly decreased (not shown).
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Fig. 4.
Dephosphorylation of the serine 19/threonine
18 phosphorylated myosin by the catalytic subunit of smooth muscle
MLCP. Reaction was done as described in the legend to Fig. 2,
except the catalytic subunit of MLCP (1 µg/ml) was used. , myosin
containing wild type RLC; , myosin containing NDL-10.
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Dephosphorylation of Myosin Phosphorylated at Various Sites on
RLC--
It is known that RLC of smooth muscle and nonmuscle myosins
are phosphorylated at various sites in the N-terminal region (7, 8, 32)
(see Fig. 1). Serine 19 and threonine 18 are phosphorylated by MLCK
(32). On the other hand, serine 1/serine 2 and threonine 9 are
phosphorylated by PKC (7, 8) and cdc2 kinases (9). Myosin
phosphorylated at these different sites was prepared (see "Materials
and Methods"), and the rate of dephosphorylation by smooth muscle
MLCP was determined. The dephosphorylation of myosin phosphorylated at
both serine 19 and threonine 18 showed a single exponential decay
curve, and the rate of dephosphorylation was identical to that for
dephosphorylation of myosin phosphorylated at serine 19 alone (Fig.
5). This suggests that the rate of
dephosphorylation of serine 19 and threonine 18 of RLC by smooth muscle
MLCP is indistinguishable. Consistent with this notion, phosphoamino
acid analysis revealed that both phosphoserine and phosphothreonine were decreased to the same extent after 10 min of the dephosphorylation reaction where approximately 70% of the total incorporated phosphate was removed by the phosphatase (Fig. 6).
On the other hand, the dephosphorylation of myosin phosphorylated at
PKC sites (i.e. serine 1/serine 2 and threonine 9) by smooth
muscle MLCP showed dual phases. Myosin was initially dephosphorylated
by smooth muscle MLCP with a rate similar to that for the
dephosphorylation of the serine 19/threonine 18 sites but then became
resistant to dephosphorylation by MLCP. To determine the sites
resistant to smooth muscle MLCP, a sample was taken at 30 min after the
addition of smooth muscle MLCP and subjected to phosphoamino acid
analysis. As shown in Fig. 6, both phosphoserine and phosphothreonine
were detected before MLCP addition, whereas only phosphoserine was observed at 30 min after the addition of MLCP, indicating that phosphorylation at serine 1/serine 2 is resistant to dephosphorylation by smooth muscle MLCP. This difference in the MLCP susceptibility of
serine and threonine sites was also found using isolated RLC as a
substrate (not shown).
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Fig. 5.
Dephosphorylation by smooth muscle MLCP of
myosin phosphorylated at various sites by MLCK and PKC. Reaction
was done as described in the legend to Fig. 2. Myosin containing RLC
phosphorylated at serine 19 (), serine 19 + threonine 18 (), and
serine 1/serine 2 + threonine 9 (.
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Fig. 6.
Phosphoamino acid analysis of the
phosphorylated RLC. P-Ser, phosphoserine;
P-Thr, phosphothreonine. The dephosphorylation reaction in
Fig. 5 was stopped at 10 min (lane 3) or 30 min (lane
5) by the addition of 5% TCA. The pellets were collected by
centrifugation at 5000 × g for 5 min and washed with 6 N HCl for several times. Hydrolysis and thin layer chromatography were
done as described (27). Lane 1, myosin phosphorylated at
serine 19 before addition of MLCP; lane 2, myosin
phosphorylated at serine 19 and threonine 18 before addition of MLCP;
lane 3, myosin phosphorylated at serine 19 and threonine 18 at 10 min after addition of MLCP; lane 4, myosin
phosphorylated at serine 1/serine2 + threonine 9 before addition of
MLCP; lane 5, myosin phosphorylated at serine 1/serine2 + threonine 9 at 30 min after addition of MLCP.
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Effect of PKC Phosphorylation on the Dephosphorylation of MLCK
Sites--
The results described above indicate that the N-terminal
region of RLC is important for the dephosphorylation of RLC at serine 19/threonine 18 when it is incorporated into myosin heavy chain. Because PKC and cdc2 kinases phosphorylate RLC in the N-terminal region, it is of interest to examine whether or not the phosphorylation of RLC by these kinases can affect the dephosphorylation of serine 19/threonine 18. RLCs were first phosphorylated by PKC under conditions incorporating 2.0 mol of phosphate/mol of RLC with nonradioactive ATP
as a substrate. RLCs were then precipitated with 5% TCA, dissolved, and dialyzed against neutral pH buffer. RLC was then phosphorylated with MLCK using radioactive ATP to incorporate 32P into
both serine 19 and threonine 18. RLCs were then hybridized with
RLC-deficient myosin as described under "Materials and Methods." As
shown in Fig. 7, the dephosphorylation of
serine 19/threonine 18 by smooth muscle MLCP was not significantly
affected by phosphorylation at serine 1/serine 2 and threonine 9. The
rate was decreased by 18% because of serine 1/serine 2 and threonine 9 phosphorylation. To determine the effect of phosphorylation at MLCK
sites on the dephosphorylation of PKC sites, RLC was first
phosphorylated by MLCK at serine 19 and threonine 18 with
nonradioactive ATP and then phosphorylated with radioactive ATP by PKC.
The phosphorylated RLC was incorporated into myosin heavy chain and
subjected to the MLCP-catalyzed dephosphorylation reaction. The time
course of dephosphorylation of RLC at PKC sites was unaffected by MLCK phosphorylation (not shown).
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Fig. 7.
Effect of phosphorylation at PKC sites on
dephosphorylation of serine 19/threonine 18. RLC was first
phosphorylated by PKC as described under "Materials and Methods"
with nonradioactive ATP. Phosphorylated RLC was precipitated with 5%
TCA and then dissolved and dialyzed against 50 mM Tris-HCl,
pH 7.5, 50 mM KCl, 1 mM DTT. RLC phosphorylated
by PKC was then phosphorylated at serine 19 by MLCK with
[-32P]ATP. The phosphorylated RLC was incorporated
into myosin as described under "Materials and Methods." The
dephosphorylation reaction was done as described in the legend to Fig.
2. Semilogarithmic plots of time course data are shown. , control
myosin; , myosin phosphorylated at serine 1/serine 2 + threonine 9 with nonradioactive ATP.
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DISCUSSION |
While isolated RLC can be dephosphorylated by various
serine/threonine protein phosphatases (1-5), not many protein
phosphatases dephosphorylate RLC incorporated into the myosin molecule.
A type 1 serine/threonine protein phosphatase has been identified as the physiological myosin light chain phosphatase in smooth muscle (16-19); therefore, we studied the nature of myosin dephosphorylation catalyzed by this myosin light chain phosphatase as presented in this
paper. It was shown that the dephosphorylation of myosin at serine
19/threonine 18 of RLC is significantly influenced by the presence of
the N-terminal amino acid residues of RLC. The critical residues are
Ala5-Lys6-Ala7 because the deletion
of seven N-terminal amino acid residues markedly attenuates smooth
muscle MLCP-induced dephosphorylation while the deletion of four
N-terminal amino acid residues does not. An interesting result is that
the decrease in smooth muscle MLCP-dependent
dephosphorylation is found only when RLC associates with myosin heavy
chain. A possible scenario to account for this finding would be that
the N-terminal region of RLC is involved in the interaction of RLC with
smooth muscle MLCP and that the serine 19/threonine 18 of RLC are
somewhat occluded from the smooth muscle MLCP catalytic site in the
presence of the heavy chain. It is plausible that the interaction of
smooth muscle MLCP with the N-terminal region of RLC increases the
accessibility of the MLCP catalytic site to the phosphate moieties of
phosphorylated RLC.
Previously, it was found that the dephosphorylation of myosin at serine
19 by smooth muscle MLCP is significantly affected by the conformation
of myosin, i.e. a folded conformation of myosin is highly
resistant to dephosphorylation by MLCP (33). It was suggested that the
myosin tail interacts with the N-terminal region of RLC, thus
stabilizing the folded conformation, because the mutation of the basic
residues at the N-terminal region of RLC abolishes the folded
conformation (25). That result is consistent with the present finding
that the N-terminal region of RLC plays a role in the substrate
recognition of smooth muscle MLCP. It is plausible that in the folded
conformation smooth muscle MLCP cannot access the substrate site
because of obstruction by the myosin tail binding to the N-terminal
region of RLC.
It is known that the rates of phosphorylation of serine 19 and
threonine 18 by MLCK are significantly different from each other. In
contrast, the rate of dephosphorylation of these sites by smooth muscle
MLCP is practically the same. The results suggest that the difference
in the phosphorylation level at serine 19 and threonine 18 in cells
reflects the difference in the phosphorylation rate of these sites but
not by the dephosphorylation process. On the other hand, the
dephosphorylation of PKC/cdc 2 kinase sites showed a marked difference
in their susceptibility to smooth muscle MLCP. It has been known that
PKC phosphorylates threonine 9 several times faster than the serine
site in vitro (7), whereas phorbol ester, an activator of
PKC, induces only serine phosphorylation but not threonine
phosphorylation in cells (34). The present result provides a clear
answer to this apparent discrepancy. The phosphorylation at threonine 9 would be rapidly dephosphorylated by MLCP in cells but the
phosphorylated serine 1/serine 2 would be resistant to
dephosphorylation by MLCP. Other protein phosphatases may slowly
dephosphorylate the serine 1/serine2 sites in cells because a
spontaneously active aorta phosphatase was shown to dephosphorylate
both threonine and serine sites with the same rate constant (7).
However, the fact that serine 1/serine 2 sites remained phosphorylated
in cells after phorbol ester stimulation provides further evidence that
MLCP is the physiological myosin light chain phosphatase. The slow
dephosphorylation of the serine 1/serine 2 sites was previously shown
with crude gizzard phosphatase (7) so the present results are
consistent with the earlier results.
The time course of dephosphorylation of myosin at serine 19 by smooth
muscle MLCP is explained by a single rate constant. Furthermore, the
rate of dephosphorylation of serine 19 is virtually the same for HMM
and S1 (not shown). These results indicate that the dephosphorylation
process of RLC at serine 19 is random and independent relative to the
other head of myosin. In phosphorylation reactions, it has been
reported that myosin is phosphorylated sequentially by MLCK,
i.e. phosphorylation of the first head is faster than that
of the second head (35-38), although this is still controversial. If
one accepts the sequential phosphorylation of myosin by MLCK, it would
be expected that significantly higher MLCK activity would be required
for phosphorylation of the second head and that the majority of
phosphorylated myosin population at lower overall levels of RLC
phosphorylation in cells would be singly phosphorylated myosin. To
date, it is controversial whether or not the motor activity of
phosphorylated myosin head is dependent on the phosphorylation of the
other head of myosin, but the actomyosin contractile activity as a
function of overall myosin phosphorylation in cells could be complex.
While the N-terminal region of RLC is important for determining the
dephosphorylation rate of myosin at serine 19 by smooth muscle MLCP,
phosphorylation at this region, i.e. serine 1/serine2 and
threonine 9, failed to influence the dephosphorylation rate of myosin
at serine 19. This is in contrast to the MLCK reaction in which the
rate of phosphorylation at serine 19 is decreased by phosphorylation at
serine1/serine 2 and threonine 9 (7, 8). This difference might be
because of a difference in the manner of substrate recognition between
the two enzymes because MLCK requires basic residues at the N-terminal
side of the phosphorylation sites. This charge interaction may not be
critical for the MLCP reaction.
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FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL41776, HL60381, and HL61426.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Physiology,
University of Massachusetts Medical School, 55 Lake Ave. North,
Worcester, MA 01655-0127. Tel.: 508-856-1954; Fax: 508-856-4600.
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ABBREVIATIONS |
The abbreviations used are:
MLCK, myosin light
chain kinase;
DTT, dithiothreitol;
RLC, regulatory light chain of
myosin;
PKC, protein kinase C;
MLCP, myosin light chain phosphatase;
TCA, trichloroacetic acid: HMM, hevymero myosin;
SI, myosin subfragment
1.
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Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.
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