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ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(9):
793-800
CN 31-1300/Q |
The
Characterization of Ca2+-calmodulin Independent Phosphorylation of
Myosin Light Chains by a Fragment from Myosin Light Chain Kinase
( Department of Pharmacology, Dalian
Medical University, Dalian 116027, China)
However, this simple on/off system based
on Ca2+-CaM dependent phosphorylation of myosin (CDPM) is not
sufficient to explain all the aspects of smooth muscle contractile activity. It
is known that the rise in [Ca2+]i and CDPM is in a very short time
in physiological conditions, while the tension may keep much longer time
without change in [Ca2+]i[6]. The mechanism of this phenomenon is
still unclear. It’s supposed that other mechanisms different from CDPM by MLCK
may be involved in the force remaining[7－9].
Our previous study suggested that Ca2+-CaM independent
phosphorylation of myosin (CIPM) by MLCK might contribute to the sustained
tension[10]. Recently, what we found new was that a constitutively active MLCK
fragment (MLCKF) was more efficient than intact MLCK to phosphorylate MLC20 in
a Ca2+-CaM independent way. The following experiments are carried
out to reveal the characterization of CIPM by the MLCKF compared with CIPM and
CDPM by MLCK.
1 Materials
and Methods
1.1
Materials
Trypsin, diisopropyl fluorophosphates
(DFP), phenylmethyl sulfonyl fluoride (PMSF),
1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1, 4-diazepine (ML-9) and
dithiothreitol (DTT) were purchased from Sigma. Ethylene glycol bis
(2-aminoethyl ether) tetraacetic acid (EGTA) was purchased from Wako.
Calmodulin (CaM) was generously provided by Prof. K. Kohama, Gumma University,
School of Medicine, Japan. Scp70H Centrifuge was made in Hitachi and UV-120-02
Spectrophotometer was the product of Shimadzu.
1.2
Protein purification
Myosin and MLCK used in the assay were
purified from fresh chicken gizzard smooth muscle and the methods were
described previously[11－15].
1.3 Tryptic
hydrolysis of MLCK
The hydrolysis
of MLCK was carried out according to the methods of references [3,16,17] with
slight modification. Native MLCK was incubated with trypsin [1∶1000
(W/W ) ratio of trypsin∶MLCK] at 3 °C
for 15 min in 1 mmol/L DTT, 0.1 mmol/L PMSF, 20 mmol/L Tris·HCl
(pH 7.4), and 0.5 mmol/L EGTA. The digestion was terminated by the addition of
DFP to the final concentration of 1 mmol/L. The polypeptic mixture of MLCK
digested by trypsin was applied to DEAE-52 chromatographic column and then the
MLCKF about 61 kD was collected according to the band showed in SDS-PAGE.
1.4 Western
blot analysis
Native MLCK,
tryptic fragment of MLCK collected by application of DEAE-52 chromatographic
column and the polypeptic mixture of MLCK digested by trypsin were separated by
SDS-PAGE, and then these proteins were transblotted onto a nitrocellulose
membrane. The membrane blocked with 50 g/L skimmed milk powder was incubated
with primary antibody [rabbit anti-MLCK IgG, 1:500 (V/V)] and
then reacted with horseradish peroxidase-conjugated secondary antibody [goat
anti-rabbit IgG, 1∶1000 (V/V)]. Proteins bound with the anti-MLCK
IgG were detected by means of the peroxidase reaction using
3,3-diaminobenzidine (DAB) as a color substrate[3,17].
1.5 Phosphorylation of myosin light chains
CIPM was
carried out according to the method of references [18, 19] in a 20 mmol/L Tris·HCl
(pH 7.4) buffer containing 1 mmol/L DTT, 5 mmol/L MgCl2, 60 mmol/L KCl, 2
mmol/L EGTA, 2 mmol/L ATP and 4 mmol/L myosin. Various concentrations of MLCKF
or MLCK, different incubation-time, different incubation-temperature, different
concentrations of KCl and different concentrations of MLCK inhibitor ML-9 for
phosphorylation of myosin were described in detail in the corresponding figure
legends. The assay condition for CDPM was same as that for CIPM with the exception
of adding CaCl2 and CaM to a final concentration of 0.1 mmol/L and 5 mg/L
respectively instead of 2 mmol/L EGTA.
1.6 Phosphorylation determination
After
phosphorylation of MLC20 in both Ca2+-dependent and Ca2+-independent
ways, solid urea and sample solution which contained bromophenol blue and
glycerol were added to reaction mixture. 10% Gly-PAGE was used to measure the
extent of phosphorylation of MLC20 and Scoin Image Software, a densitometry
Software gotten from Scion Co. Ltd. was applied to measure the relative
phosphorylation extent of MLC20.
1.7 Measurement of myosin Mg2+-ATPase
activity
The myosin Mg2+-ATPase
activities of CIPM by MLCKF, CDPM by MLCK, CIPM by MLCK and dephosphorylated
myosin were measured according to the method of references[20－22].
The assays were carried out with 4 mmol/L myosin, 2 mmol/L MLCKF or MLCK at 25 °C
for 20 min.
1.8 Other procedures
A polyclonal
antibody against MLCK was obtained by injecting MLCK to rabbit together with
complete Freuds adjvant (purchased from Sigma).
Protein
concentrations were determined by the method of Bradford[23] using bovine serum
albumin as the standard.
The results of
experiments are expressed as x±s and Student's
t-test was used to evaluate the significance of differences.
2.1 The hydrolysis of MLCK and Western blot
analysis
Fig.1(A) showed
that intact MLCK purified from chicken gizzard smooth muscle was about 108 kD
(Lane 2); trypsin-digested MLCKF obtained by application of DEAE-52
chromatographic column was approximately 61kD (Lane 3). Tryptic proteolysis of
MLCK which was stopped by addition of DFP to the final concentration of 1mmol/L
produced polypeptides as follows (Lane 4): big fragment about 61 kD and some
small fragments from 17 kD to 31 kD. All of the molecular weights were
evaluated compared to molecular weight standards (Lane 1).
the antibody of MLCK recognized not only intact
MLCK(Lane 1) but also all of the tryptic MLCKFs (Lane 2, 3). This indicated
that these fragments were all the products of digested MLCK.
Fig.1 The hydrolysis of MLCK and
Western blot analysis
(A)
Proteins separated on SDS-PAGE. 1, molecular weight standard; 2, purified
intact MLCK; 3, trypsin-digested MLCKF collected by application of DEAE-52
chromatographic column; 4, proteolytic mixture of MLCK digested by trypsin. (B)
The proteins described above which were transbloted from SDS-PAGE onto a
nitrocellulose membrane. The MLCK antibodies recognized intact MLCK (Lane 1), trypsin-digested
MLCKF collected by application of DEAE-52 chromatographic column (Lane 2) and
all of the mixed fragments following the tryptic proteolysis of MLCK (Lane 3).
2.2.1
The
comparison between CIPM by MLCKF, CDPM by MLCK and CIPM by MLCK in different
concentrations To find out the differences
between CIPM by MLCKF, CDPM by MLCK and CIPM by MLCK, 0.00002 mmol/L to 2
mmol/L MLCKF and MLCK were selected for the assay. The results showed that the
extent of MLC20 phosphorylation in CIPM by different concentrations of MLCKF
[Fig.2(C), (D)] was obviously lower than that of CDPM by MLCK [Fig.2(A), (D)],
but was higher than that of CIPM by MLCK [Fig.2(B), (D)]. The differences were
statistically significant (**P<0.01, #P<0.05, ##P<0.01). These results
indicated that CIPM by MLCKF was less efficient than CDPM by MLCK, but was more
efficient than CIPM by MLCK.
2.2.2 The
comparison between CIPM by MLCKF, CDPM by MLCK and CIPM by MLCK at different
incubation-time To determine whether the
incubation-time influences CIPM by MLCKF, CDPM by MLCK and CIPM by MLCK or not,
20 min to 80 min were selected for examining MLC20 phosphorylation. The results
showed that with the increase of incubation-time, CDPM by MLCK [Fig.3(A), (D)]
showed an apparently declined extent of MLC20 phosphorylation(**P<0.01), but
CIPM by MLCKF [Fig.3(C), (D)] and CIPM by MLCK [Fig.3(B), (D)] showed no
tendency of declined MLC20 phosphorylation (#P>0.05). This proved that CIPM
by MLCKF and CIPM by MLCK were less influenced by the prolonging of
incubation-time than CDPM by MLCK.
Fig.2 The comparison between CIPM by MLCKF,
CDPM by MLCK and CIPM by MLCK in different concentrations (x±s, n=6)
The
assay was carried out with 4 mmol/L myosin and various concentrations of MLCKF
and MLCK at 25 °C for 20 min. (A), (B) and (C)
represent glycerol electrophoresis results of CDPM by MLCK, CIPM by MLCK and
CIPM by MLCKF, respectively. a0, b0 and c0 represent dephosphorylation
controls; a1 and b1, a2 and b2, a3 and b3, a4 and b4 were added with 2 mmol/L,
0.02 mmol/L, 0.0002 mmol/L and 0.00002 mmol/L MLCK, respectively. c1, c2, c3 and
c4 were added with the same concentrations of MLCKF, respectively. LC20
represents dephosphorylated 20 kD myosin light chains; p-LC20 represents
mono-phosphorylated 20 kD myosin light chains; pp-LC20 represents
di-phosphorylated 20 kD myosin light chains. LC17 represents 17 kD myosin
essential light chains. The extent of MLC20 phosphorylation of CDPM by MLCK
(filled rhombus), CIPM by MLCKF (filled squares) and CIPM by MLCK (filled
triangles) were plotted against the concentrations of MLCKF and MLCK (D). **P<0.01
vs. CDPM by MLCK, #P<0.05, ##P<0.01 vs CIPM by MLCK.
Fig.3 The comparison between
CIPM by MLCKF, CDPM by MLCK and CIPM by MLCK at different incubation-time (x±s,
n=6)
Samples
were incubated with 4 mmol/L myosin, 2 mmol/L MLCKF and MLCK at 25 °C for different incubation-time. (A), (B)
and (C) represent glycerol electrophoresis results of CDPM by MLCK, CIPM by
MLCK and CIPM by MLCKF, respectively. Four different incubation-time, i.e., a1,
b1 and c1=20 min, a2, b2 and c2=40 min, a3, b3 and c3=60 min, a4, b4 and c4=80
min were selected for the assay. a0, b0 and c0 represent dephosphorylation
controls; The extent of MLC20 phosphorylation of CDPM by MLCK (filled rhombus),
CIPM by MLCKF (filled squares) and CIPM by MLCK (filled triangles) were plotted
against the incubation-time (D). **P<0.01, #P>0.05 vs. the corresponding
controls at 20 min.
2.2.3 The
comparison between CIPM by MLCKF, CDPM by MLCK and CIPM by MLCK at different
incubation-temperature To
observe the influences of incubation-temperature on CIPM by MLCKF, CDPM by MLCK
and CIPM by MLCK, 15 ℃, 25 ℃, 35 ℃, 45
℃ were chosen
for determining MLC20 phosphorylation. It was shown that with the increase of
incubation-temperature, CDPM by MLCK [Fig.4(A), (D)] showed a significantly
declined tendency of MLC20 phosphorylation (**P<0.01); While CIPM by
MLCKF [Fig.4(C), (D)] and CIPM by MLCK [Fig.4(B), (D)] didn't show apparent
declined extent of MLC20 phosphorylation(#P>0.05). These
results suggested that CIPM by MLCKF and CIPM by MLCK were more sustained and
less influenced by the change of incubation-temperature than CDPM by MLCK.
The
assay was carried out with 4 mmol/L myosin, 2 mmol/L MLCKF and MLCK for 20 min
at different incubation temperatures. (A), (B) and (C) represent glycerol
electrophoresis results of CDPM by MLCK, CIPM by MLCK and CIPM by MLCKF,
respectively. Four different incubation temperatures, i.e., a1, b1 and c1=15 ℃,
a2, b2 and c2=25 ℃, a3, b3 and c3=35 ℃,
a4, b4 and c4=45 ℃ were chosen for the assay. a0, b0 and c0
represent dephosphorylation controls. The extent of MLC20 phosphorylation of
CDPM by MLCK (filled rhombus), CIPM by MLCKF (filled squares), CIPM by MLCK
(filled triangles) were plotted against the incubation temperatures (D). **P<0.01,
#P>0.05 vs. the corresponding controls at 25 ℃.
Fig.5 The effects of different
concentrations of KCl on CIPM by MLCKF, CDPM by MLCK and CIPM by MLCK (x±s, n=6)
The
assay was carried out with 4 mmol/L myosin, 2 mmol/L MLCKF and MLCK at 25 ℃
for 20 min. (A), (B) and (C) represent glycerol electrophoresis results of CDPM
by MLCK, CIPM by MLCK and CIPM by MLCKF, respectively. Four different
concentrations of KCl, i.e., a1, b1 and c1 = 60 mmol/L KCl; a2, b2 and c2= 120
mmol/L KCl; a3, b3 and c3=240 mmol/L KCl; a4, b4 and c4 = 360 mmol/L KCl were
chosen for the assay. a0, b0 and c0 represent dephosphorylation controls. The
extent of MLC20 phosphorylation of CDPM by MLCK (filled rhombus), CIPM by MLCKF
(filled squares) and CIPM by MLCK (filled triangles) were plotted against the
concentrations of KCl (D). **P<0.01, #P>0.05 vs.
the corresponding controls with 60 mmol/L KCl.
ML-9 is a MLCK inhibitor. To observe
whether the inhibitory effects of ML-9 on CIPM by MLCKF, CDPM by MLCK and CIPM
by MLCK were same or not, 0.1 mmol/L and 0.2 mmol/L ML-9 were selected and
MLC20 phosphorylation was determined for 20 min and 40 min, respectively. It
was observed that, with 0.1 mmol/L ML-9, the effect of MLCK on CDPM [Fig.6(A),
(D1), (D2)] was significantly inhibited (*P<0.01); in contrast, no
inhibitory effects on CIPM by MLCKF [Fig.6(C), (D1), (D2)] and CIPM by MLCK
[Fig.6(B), (D1), (D2)] were observed in the presence of 0.1 mmol/L ML-9
(#P>0.05). With the increase of ML-9 to 0.2 mmol/L, the inhibitory effects
of ML-9 on CIPM by MLCKF [Fig.6(C), (D1), (D2)] and CIPM by MLCK [Fig.6(B),
(D1), (D2)] appeared (**P<0.01). These results suggested that CIPM by MLCKF
and CIPM by MLCK were more stable and less sensitive to ML-9 than CDPM by MLCK.
Fig.6 The effects of different
concentrations of ML-9 on CIPM by MLCKF, CDPM by MLCK and CIPM by MLCK (x±s, n=6)
The
assay was carried out with 4 mmol/L myosin, 2 mmol/L MLCKF and MLCK at 25 ℃
for 20 min. (A), (B) and (C) represent glycerol electrophoresis results of CDPM
by MLCK, CIPM by MLCK and CIPM by MLCKF, respectively. Among (A), (B) and (C),
different concentrations of ML-9 were selected, i.e., a1, a2, b1, b2, c1 and c2
= 0 mmol/L ML-9, a3, a4, b3, b4, c3 and c4 = 0.1 mmol/L ML-9, a5, a6, b5, b6,
c5 and c6 = 0.2 mmol/L ML-9; Two different incubation-time were chosen i.e.,
a1, a3, a5, b1, b3, b5, c1, c3 and c5 =20 min; a2, a4, a6, b2, b4, b6, c2, c4,
and c6=40 min. a0, b0 and c0 represent dephosphorylation controls. The extent
of MLC20 phosphorylation of CDPM by MLCK (filled rhombus), CIPM by MLCKF
(filled squares), CIPM by MLCK (filled triangles) were plotted against the concentrations
of ML-9 (D1, incubation for 20 min; D2, incubation for 40 min). **P<0.01,
#P>0.05 vs. the corresponding controls without ML-9.
2.2.6 The
comparison of myosin Mg2+-ATPase activities between CIPM by MLCKF,
CDPM by MLCK, CIPM by MLCK and dephosphorylated myosin
It was shown in
Fig.7 that myosin Mg2+-ATPase activitity of CIPM by MLCKF (Column 3)
was lower than that of CDPM by MLCK (Column 4)(**P<0.01), but was
higher than those of CIPM by MLCK (Column 2) and dephosphorylated myosin
(Column 1) (##P<0.01). This indicated that CIPM by MLCKF was less efficient
than CDPM by MLCK but was more efficient than CIPM by MLCK and dephosphorylated
myosin to stimulate myosin Mg2+-ATPase activity.
Fig.7 The relative Mg2+-ATPase
activities of CIPM by MLCKF, CDPM by MLCK, CIPM by MLCK and dephosphorylated
myosin (x±s, n=6)
1,
dephosphorylatd myosin; 2, CIPM by MLCK; 3, CIPM by MLCKF; 4, CDPM by MLCK.
Fig.7 represents the relative Mg2+-ATPase activities of
dephosphorylated myosin (Column 1), CIPM by MLCK (Column 2), CIPM by MLCKF
(Column 3) and CDPM by MLCK (Column 4). The assay was carried out with 4 mmol/L
myosin, 2 mmol/L MLCKF and MLCK at 25 ℃ for 20 min. It was designed
that the Mg2+-ATPase activity of dephosphorylated myosin was 100%.
The others were relative value comparing to the Mg2+-ATPase activity
of dephosphorylated myosin. **P<0.01 vs. CDPM by MLCK, ##P<0.01
vs. CIPM by MLCK and dephosphorylated myosin.
3 Discussion
It was
previously thought that contractile activity of smooth muscle is controlled
primarily by the reversible CDPM by MLCK. However, this simple view is not
sufficient to explain all the aspects of smooth muscle contractile activity,
especially as concerns the sustained tension, termed tonic contraction[7,24－26].
Coirault et al.[8] and Hai et al.[27－29] suggested
that dephosphorylated myosin which attend to form a special slow cycling
crossbridges, termed "latch bridges", involve in tonic contraction of
smooth muscle. Two facts encouraged us to make the further investigation. One
was that our previous study suggested that CIPM by MLCK may contribute to the
tonic contraction[10]; the other was that Weber et al.[3] described that the
MLCKF prepared by tryptic digestion of MLCK had a specific activity to
phosphorylate MLC20 both in the presence and in the absence of Ca2+.
Though our previous study suggested that in the presence of Ca2+,
CDPM by MLCKF was more efficient than CDPM by MLCK(data not shown), but the characterization
of CIPM by MLCKF in the absence of Ca2+ still remained unclear. To
further explore the characterization of CIPM by MLCKF, we prepared the
constitutively active MLCK fragment (MLCKF) according to the methods of Weber
et al.[3] and used it in our assay. It was found that CIPM by MLCKF was more
efficient than CIPM by MLCK and was less efficient than CDPM by MLCK in
phosphorylating MLC20 and stimulating myosin Mg2+-ATPase activity;
both CIPM by MLCKF and CIPM by MLCK were less influenced by the rise of
incubation-temperature, the prolonging of incubation-time, the increase of
ionic strength of KCl and less sensitive to MLCK inhibitor ML-9 than CDPM by
MLCK. These results may be useful to further investigate the mechanism of
sustained tension characterized by less energy consumption.
As to the enhancement of the activity of MLCKF in phosphorylating MLC20, a
reasonable explanation might be as follows. Previous studies on the proteolysis
of MLCK showed that the autoinhibitory domain at the C-terminal of MLCK might
be removed by tryptic cleavage, but the catalytic domain might be
retained[3,17,30]. We digested MLCK with trypsin according to the methods of
above researchers and obtained the tryptic MLCKF which had approximately the
same molecular weight (about 61 kD) as the tryptic MLCKF reported previously
(61 kD). Western blot has demonstrated that the tryptic MLCKF we obtained was
homogenous with intact MLCK. Therefore, we could not rule out the possibility
that, in our study, the enhancement of the activity of trypsin-digested MLCKF
in phosphorylating MLC20 was due to the removal of C-terminal of MLCK which
contained autoinhibitory domain. To confirm this possibility, it is worth to
make further investigation to identify the amino acid sequence of the MLCKF.
We also found that during the isolation of MLCK in the absence of PMSF, part of
MLCK was automatically cleaved into a constitutively active MLCKF (about 61
kD), which had the same specific activity in phosphorylating MLC20 and the same
molecular weight as the tryptic MLCKF we obtained (61 kD). Western blot
demonstrated that the automatically proteolytic MLCKF and trypsin-digested
MLCKF were all homogenous.
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Received: February 28, 2003 Accepted:
June 3, 2003
This work was supported by a grant from
the National Natural Science Foundation of China ( No. 30070203)
*Corresponding author: Tel,
86-411-4720652; Fax, 86-411-4673573; e-mail, [email protected]
or [email protected]