From the Department of Physiology, University of Massachusetts
Medical Center, Worcester, Massachusetts 01655 and
Department of Biochemistry and Cell Biology, Rice
Univesity, Houston, Texas 77005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We coexpressed myosin I heavy chain with three
different calmodulin mutants in which the two
Ca2+-binding sites of the two N-terminal domain
(E12Q), C-terminal domain (E34Q), or all four sites (E1234Q) are
mutated in order to define the importance of these Ca2+
binding sites to the regulation of myosin I
. The calmodulin mutated
at the two Ca2+ binding sites in N-terminal domain and
C-terminal domain lost its lower affinity Ca2+ binding site
and higher affinity Ca2+ binding site, respectively. We
found that, based upon the change in the actin-activated ATPase
activities and actin translocating activities, myosin I
with E12Q
calmodulin has the regulatory characteristics similar to myosin I
containing wild-type calmodulin, while myosin I
with E34Q or E1234Q
calmodulin lose all Ca2+ regulation. While the increase in
myosin I
ATPase activity paralleled the dissociation of 1 mol of
calmodulin from myosin I
heavy chain for both wild type (above
pCa 5) and E12Q calmodulin (above pCa 6), the
Ca2+ level required for the inhibition of
actin-translocating activity of myosin I
was lower than that
required for dissociation of calmodulin, suggesting that the
conformational change induced by the binding of Ca2+ at the
high affinity site but not the dissociation of calmodulin is critical
for the inhibition of the motor activity. Our results suggest that the
regulation of unconventional myosins by Ca2+ is directly
mediated by the Ca2+ binding to calmodulin, and that the
C-terminal pair of Ca2+-binding sites are critical for this
regulation.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Myosins are a large family of molecular motors responsible for
diverse forms of cell contractility and motility, such as muscle contraction, chemotaxis, and phagocytosis (1). Myosin I, one of the 14 classes of this myosin superfamily, is single-headed and unable to form
filaments (1-4). Based on the similarity of their primary structures
in the head domain, myosin Is are further classified into four
subclasses. Myosin I is one of these subclasses (1, 4).
Myosin I was first purified from bovine adrenal gland and brain (5).
Subsequently, cDNAs encoding myosin I
were isolated from bovine
adrenal gland (6), bovine brain (7), and neonatal rat brain (8). Myosin
I
was found in a variety of tissues with the highest expression
levels in heart, lung, adrenal gland, esophagus, and stomach (6, 7, 9,
10). Myosin I
localizes to actin-rich peripheral structures, such as
filopodia and lamellipodia of culture cells (9), and it is thought to
play a role in cytoskeleton rearrangement. Interestingly, myosin I
is also found in hair bundles purified from the bullfrog sacculus,
suggesting that myosin I
may function as an adaptation motor which
regulates the tip link-associated cation selective channels (11,
12).
Studies from both naturally isolated and recombinant myosins I
have shown that calmodulin is associated with myosin I
heavy chain
(5, 13). In contrast to most calmodulin-dependent enzymes, the association of calmodulin with myosin I does not require
Ca2+ binding to calmodulin. Thus, calmodulin functions as a
light chain subunit. Myosin I
, like all the other vertebrate
unconventional myosins, has several repeats of a 24-30-amino acid
sequence called the IQ motif at the neck region between the myosin head
motor domain and the tail domain. This motif has been suggested to
provide the binding site for EF-hand family proteins such as calmodulin (1, 14). All vertebrate unconventional myosins that have been
characterized so far contain calmodulin as light chains. For some
unconventional myosins, other small proteins besides calmodulin have
also been found to function as light chains (15). A common property of
calmodulin targets that contain IQ motif, such as the unconventional
myosins and neuromodulin, is that they have a higher affinity for the
Ca2+-free form of calmodulin (16).
Calmodulin is one of the major intracellular Ca2+-sensor proteins, containing four EF-hand type Ca2+-binding loops. The N-terminal pair are linked to the C-terminal pair by a central flexible linker. Among the four Ca2+-binding sites, the C-terminal pair of Ca2+-binding sites have a higher Ca2+-binding affinity than those of the N terminus (17, 18). Ca2+ binding induces structural changes in calmodulin, and it is believed that these Ca2+-induced conformational changes allow calmodulin to activate target enzymes when the cytosolic Ca2+ concentration is elevated (reviewed in Ref. 19). Mutagenesis studies have shown that a conserved glutamic acid residue at the 12th position of each Ca2+-binding loop is critical for Ca2+ binding, and substitution of this conserved glutamic acid with glutamine in each Ca2+-binding site abolishes its Ca2+ binding ability (17, 20).
Both the enzymatic and mechanical activities of vertebrate myosin I
have been shown to be regulated by Ca2+ (5, 13, 21-23),
and this is also true for other unconventional myosins, such as myosin
V (15). A member of another myosin I subclass, intestinal brush-border
myosin I (BBMI),1 has been
extensively characterized in terms of Ca2+ effects. It was
found that BBMI moved actin filaments although the velocity was quite
low (~0.05 µm/s), and the activity was abolished at
Ca2+ concentrations above 5 µM. On the other
hand, actin-activated Mg2+-ATPase activity of BBMI
increased with increasing Ca2+ concentrations (24).
Interestingly, partial dissociation of calmodulin from BBMI was
observed at a Ca2+ concentration of 10 µM.
Similarly, Zhu et al. (13) have shown that one of the three
calmodulin molecules bound to recombinant myosin I dissociated from
the heavy chain at a Ca2+ concentration of 10 µM. While Mg2+-ATPase activity increased
above pCa 6, actin sliding velocity of myosin I
was
abolished at pCa 6 (13). These results suggest that
Ca2+ binding to the calmodulin light chains is critical for
the regulation of vertebrate myosin I motor function. However, it is
unclear whether the dissociation of calmodulin is necessary to stop
myosin I motor activity since the Ca2+ concentration
required for the dissociation of one calmodulin from myosin I
heavy
chain seems to be higher than the Ca2+ concentration for
inhibition of the actin translocating activity (13).
In the present study, we have examined the mechanism by which
Ca2+ and calmodulin regulate myosin I motor function by
coexpressing various calmodulin mutants defective in Ca2+
binding with myosin I
heavy chain and analyzing the actin-activated ATPase activity and motor function of the expressed myosin I
.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of Bovine Myosin I Together with Mutant Calmodulin
in Sf9 Cells--
Expression of bovine myosin I
cDNA
with calmodulin was performed as described previously (13). cDNA
for wild-type calmodulin and calmodulin with the two N-terminal, two
C-terminal, or all four Ca2+-binding sites mutated, termed
E12Q, E34Q, and E1234Q, respectively (25), were subcloned into
pBlueBacM baculovirus transfer vector at the EcoRI site in
the polylinker region. Orientation and accuracy of the subcloning were
examined by DNA sequencing (Sequenase 2.0, U. S. Biochemical
Corp.). Recombinant baculoviruses containing these mutant calmodulin
cDNAs were obtained by blue plaque selection and subsequent steps
of purification and amplification as described in the manual from
Invitrogen, MaxBac Baculovirus Expression System. Sf9 cells were
coinfected with the recombinant viruses of myosin I
heavy chain and
each calmodulin mutant.
Purification of Recombinant Myosin I with Mutant
Calmodulin--
The purification of recombinant myosin I
was
performed as previously with slight modifications (13). Briefly, cells
were harvested after 3 days of culture at 28 °C and lysed in the
presence of ATP, Triton X-100, Nonidet P-40, and various protease
inhibitors. The supernatant (150,000 × g for 30 min)
of lysed cells was incubated with 10 mM glucose and 20 units/ml hexokinase at 0 °C for 30 min to completely hydrolyze
residual ATP. F-actin (1 mg/ml) was added to coprecipitate the
expressed myosin I
. The pellet was resuspended with buffer
containing 5 mM MgCl2, 100 mM KCl,
1 mM EGTA, and 25 mM Tris-HCl, pH 7.5, and 1 mM ATP was added to release myosin I
from the myosin
I-actin complex. The sample was ultracentrifuged at 150,000 × g for 30 min to remove F-actin, and the supernatant containing the expressed myosin I
heavy chain and calmodulin was
subject to a DE52 column (1 × 10 cm). The protein was eluted with
a linear gradient (12 ml-12 ml) of 50-250 mM KCl.
Approximately 100 µg of myosin I
can be obtained from 800 ml of
culture.
Expression and Purification of Calmodulin Mutants--
Wild-type
calmodulin and mutant proteins were expressed by infecting Sf9
cells with recombinant virus. Cells were homogenized with 5 volumes of
buffer A (30 mM Tris-HCl, pH 7.5, 8 M urea, 5 mM dithiothreitol, 10 µg/ml leupeptin) for 5 min. After
centrifugation at 35,000 × g for 15 min, 5%
trichloroacetic acid was added to the supernatant. The pellets were
collected and suspended with 8 M urea, and the pH was
adjusted to be neutral. The suspension was dialyzed against buffer B
(30 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol) for 2 h. After insoluble material was removed by centrifugation, the sample was loaded to a DE52 ion exchange column (24 × 3 cm) equilibrated with buffer B. Calmodulin was eluted with a 0-0.4 M NaCl linear gradient. The fractions containing calmodulin
were combined, concentrated, and loaded onto a Sephacryl S200 gel
filtration column (100 × 3 cm). The fractions containing
calmodulin were identified by SDS-polyacrylamide gel electrophroesis
analysis. They were combined and concentrated. The concentrated
calmodulin was dialyzed against buffer C (30 mM Tris-HCl,
pH 7.5, 30 mM NaCl, 1 mM dithiothreitol) and
stored in 80 °C. Bacterially expressed wild type and mutant
calmodulins were prepared as described previously (25).
Fluorimeter Titrations-- Ca2+-induced conformational changes in calmodulins were determined by Ca2+ titrations in the presence of 9-anthroylcholine (9-AC) according to Beckingham with some modifications (20). Purified calmodulin was dialyzed extensively against the titration buffer (0.5 mM CaCl2, 100 mM KCl, and 10 mM MOPS, pH 7.0). 2-ml titration buffer containing 5 µM of such prepared calmodulin and 20 µM of 9-AC bromide (Molecular Probes, Eugene, OR) were transferred to a quartz cuvette, and fluorescence measurements were made as sequential addition of small volume (1 to several microliters) EGTA solutions (20 mM, 200 mM, or 1 M). The amount of EGTA was added so that the concentrations of free Ca2+ in the cuvette were the desired ones (26). The fluorescence intensity was measured by using a Spex Fluorolog fluorimeter (Spex Inc., Edison, NJ) with excitation at 366 nm and emission at 418 nm. The pH of the samples was examined both before and after titration.
ATPase Assays and Other Biochemical Procedures--
The effect
of Ca2+ on actin-activated ATPase activity was assayed in
30 mM KCl, 2 mM MgCl2, 20 mM imidazole-HCl, pH 7.0, with or without 50 µM actin, in the presence of 1 mM EGTA or
various concentrations of Ca2+ (26). All assays were
initiated by adding 100 µM [-32P]ATP
(Amersham Corp.) to the reaction mix. The liberated 32P was
measured as described previously (27) to determine ATPase activity.
In Vitro Motility Assay-- In vitro motility assays were performed as described previously (28). A larger nitrocellulose-coated coverslip (24 × 30 mm) was covered by smaller coverslip (18 × 18 mm), each edge of which was applied with 0.1 g of silicon grease (Dow Corning, MI), to create a fluid-filled flow cell. After 60 µl of myosin I-containing solution was applied to the flow cell, unbound myosin I was washed away with 180 µl of buffer A (400 mM KCl, 25 mM HEPES, pH 7.5, 4 mM MgCl2, and 10 mM dithiothreitol). Then the unoccupied nitrocellulose surface was coated with 0.5 mg/ml bovine serum albumin in buffer B (30 mM KCl, 20 mM HEPES, 1 mM EGTA, pH 7.5). The flow cell was washed with buffer B, then rhodamine-phalloidin (Molecular Probes Inc., Eugene, OR) labeled actin filaments in the motility buffer (50 mM KCl, 5 mM MgCl2, 25 mM imidazole, 1 mM EGTA, 1% 2-mercaptoethanol, 0.5% methylcellulose, 4.5 mg/ml glucose, 216 µg/ml glucose oxidase, 36 µg/ml catalase) with various concentrations of Ca2+ were introduced onto the myosin-coated coverslip. After 120 µl of motility buffer were perfused to wash out unbound actin filaments, motility buffer containing Mg2+-ATP was applied to initiate the reaction. Movements of fluorescent actin filaments were observed using an inverted fluorescence microscope (Diaphot, Nikon) with a SIT camera (VE 1000 SIT, DAGE MTI) and a video cassette recorder. The actin sliding velocity was determined as described previously (13).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three different calmodulin Ca2+ binding site mutants, termed E12Q, E34Q, and E1234Q (25), were used in this study. In each protein, particular Ca2+-binding sites were inactivated by mutation of a conserved glutamate residue (at position 12 of the Ca2+-binding loops) to glutamine. In E12Q the two N-terminal sites were mutated, in E34Q the two C-terminal sites were mutated, and in E1234Q, all four sites carried the mutation.
Wild-type calmodulin and each of the calmodulin mutants was coexpressed
with myosin I heavy chain in insect Sf9 cells, and the
expressed myosin I
was isolated. All of the calmodulin mutants copurified with myosin I
heavy chain, suggesting that the mutations do not affect the binding of calmodulin to myosin I
heavy chain. The
ability of Ca2+ to increase electrophoretic mobility, a
characteristic of wild-type calmodulin, was examined for each of these
three mutants (Fig. 1). Wild type
calmodulin migrated with an apparent molecular mass of 16 kDa in 5 mM Ca2+ but with an apparent molecular mass of
21 kDa in the presence of 1 mM EGTA. E12Q calmodulin mutant
migrated at 22 kDa in EGTA but at 19 kDa in the presence
Ca2+. On the other hand, E34Q migrated at 21 and 18 kDa in
the absence and presence of Ca2+, respectively. The
mobility shift by Ca2+ was abolished with E1234Q calmodulin
mutant, which migrated at 21 kDa under both conditions. These results
confirm that the calmodulin light chain associated with the myosin I
heavy chain in each preparation is indeed the expressed recombinant
calmodulin mutant, and not endogenous calmodulin. They also suggest
that the effect of mutating the two N-terminal Ca2+ binding
sites on the conformational change of calmodulin is different from that
of mutating the two C-terminal sites.
|
Ca2+-induced conformational changes in the calmodulin
mutants were further monitored as a function of Ca2+
concentration by use of the reporter molecule 9-AC bromide. The Ca2+-induced appearance of hydrophobic sites on calmodulin
is revealed by the enhanced fluorescence of 9-AC upon binding to these
sites, and this technique has been used previously to examine the
Ca2+ binding and conformational properties of
Ca2+ binding site mutants of calmodulin (20). The 9-AC
fluorescence enhancement for wild type calmodulin as a function of
Ca2+ concentration is shown in Fig.
2. A single transition is detected, with
midpoint at a Ca2+ concentration (107) that
is lower than the dissociation constant for the high affinity sites on
calmodulin. This finding probably reflects increased overall affinity
for Ca2+ and increased cooperativity of Ca2+
binding induced by 9-AC. The hydrophobic reporter
1-anilino-8-naphtalene sulfonate has been shown previously to increase
the affinity of calmodulin for Ca2+ (29). The curve for the
E12Q (see Fig. 2) is very similar to the wild type curve at low
Ca2+ concentrations but shows no increase in fluorescence
enhancement at Ca2+ concentrations above pCa 6. This is consistent with induction of a conformational change as a
result of Ca2+ binding to the two intact C-terminal sites
present on this protein followed by absence of Ca2+ binding
and the associated conformational change in the N-terminal domain. In
contrast, the E34Q mutant shows no fluorescence enhancement at low
Ca2+ and relatively minor enhancement of fluorescence with
a midpoint at about pCa 6 as Ca2+ levels are
increased. Thus a major conformational change normally associated with
C-terminal high affinity sites is lost in the E34Q mutant leaving a
smaller conformational change associated Ca2+ binding in
the intact N-terminal domain. The sum of the fluorescence changes for
E12Q and E34Q equals the changes for the wild type calmodulin (Fig. 2).
The E1234Q mutant showed no change in 9-AC fluorescence throughout the
entire pCa range tested (data not shown).
|
The actin-activated Mg2+-ATPase activity of myosin is
coupled to actomyosin cross-bridge turnover. In order to examine the
effect of Ca2+ binding at the N- and C-terminal sites of
calmodulin on myosin I mechanoenzymatic function, actin-activated
Mg2+-ATPase activity of myosin I
containing the mutant
calmodulins was measured as a function of Ca2+ (Fig.
3). For all assays, the timecourse of
Pi liberation was determined and the activity was estimated
from the slope of the Pi release timecourse. Unlike myosin
I
-containing wild-type calmodulin, for which Mg2+-ATPase
activity increased in both the absence and presence of F-actin with
Ca2+ concentration above pCa 6 (Fig.
3A), myosin I
-containing mutant calmodulin showed
different Ca2+ dependencies. The ATPase activity of E12Q
myosin I
started to increase below pCa 6 and reached
maximum at pCa 6 (Fig. 3B). On the other hand,
the ATPase activities of myosin I
containing both E34Q and E1234Q
mutants did not show any Ca2+ dependence (Fig. 3,
C and D). The activity of myosin I
containing these two mutant calmodulins was similar to that of myosin I
with
wild-type calmodulin in EGTA, suggesting that Ca2+ binding
to calmodulin in the C-terminal domain induced the enhancement of
ATPase activity. These data suggest that the
Ca2+-dependence of actin-activated ATPase activity of
myosin I
is mediated through Ca2+ binding to calmodulin
and that the two C-terminal Ca2+-binding sites (high
affinity sites) have a more important role in the Ca2+
sensitivity of myosin I
ATPase activity.
|
To access the effects of mutating the N- and C-terminal
Ca2+-binding sites of calmodulin on myosin I motor
activity, actin sliding velocity was measured by the in
vitro motility assay system (Table
I). Wild-type, E12Q, E34Q, and E1234Q
myosin I
were all able to translocate actin filament at a rate about
0.3 µm/s in the presence of EGTA. The value agrees well with the one
reported in the previous study (13). While switching motility buffer from 1 mM EGTA to 1 or 10 µM Ca2+
abolished the actin filament movement for wild-type and E12Q myosin
I
, it had little effect on the motility activity of E34Q and E1234Q
myosin I
. These results suggest that the Ca2+ regulation
of motor activity of myosin I
is also mediated through the binding
of Ca2+ to calmodulin. They further demonstrate that the
two C-terminal Ca2+-binding sites but not the two
N-terminal sites are critical for this regulation.
|
The effects of Ca2+ on the binding of the various
calmodulins to myosin I heavy chain were also examined (Fig.
4). Purified myosin I
containing each
calmodulin mutant was coprecipitated with F-actin at various
Ca2+ concentrations, and the precipitated myosin I
and
calmodulin were subjected to SDS-polyacrylamide gel electrophroesis
followed by densitometry analysis to quantify the stoichiometry of the bound calmodulin. It is known that 3 mol of calmodulin bind to 1 mol of
myosin I
heavy chain (13). For wild-type calmodulin, one of the
three molecules of bound calmodulin was dissociated from the heavy
chain above pCa 5 (Fig. 4A). On the other hand, for E12Q myosin I
, the dissociation of calmodulin was observed at
lower Ca2+ (i.e. pCa 6) (Fig.
4B). In contrast, Ca2+ had no effect on the
binding of E34Q calmodulin, i.e. all three calmodulin
molecules were associated with the heavy chain even at pCa 4 (Fig. 4C). As expected, the binding of E1234Q mutant calmodulin to the heavy chain showed no Ca2+
sensitivity (Fig. 4D).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have coexpressed myosin I heavy chain with
three different calmodulin mutants, in which the conserved critical glutamic acid residue at the 12th position of the two N-terminal, two
C-terminal, or all four of the Ca2+ binding loops were
substituted by glutamine. The importance of the carbonyl side chain of
this glutamic acid to the Ca2+ coordination system was
revealed by crystallographic studies of calmodulin (30). As expected,
the Ca2+-binding abilities of the mutated sites are
completely abolished, based upon the Ca2+ binding induced
conformational changes probed by the fluorescent hydrophobic reporter
molecule, 9-AC. The results are consistent with the earlier study in
which the conserved glutamic acid residue in each individual
Ca2+-binding site was mutated (20).
The fluorescence titration results show that the two C-terminal Ca2+-binding sites have a higher affinity for Ca2+ than the two N-terminal sites. The different electrophoretic mobility of calmodulin in the presence of EGTA as compared with Ca2+ also reflects conformational changes upon Ca2+ occupation. The obtained results suggest that the conformational change caused by Ca2+ binding to the two N-terminal sites (E34Q) is different from that caused by Ca2+ binding to the two C-terminal sites (E12Q); occupation of the two C-terminal Ca2+-binding sites has a greater impact on the overall conformational change. This is consistent with the previous findings that the Ca2+ binding at the C-terminal sites of calmodulin induces a larger conformational change (31). Recent structural studies suggest that the C-terminal domain of calmodulin exists in a semi-open conformation in contrast to the close conformation of the N-terminal domain in the absence of Ca2+, and it changes to an open conformation upon Ca2+ binding (32). This may explain the difference of the mobility shift between E12Q and E34Q.
The present results clearly indicate that the effects of
Ca2+ on the properties of myosin I molecule,
i.e. the change in actin-activated ATPase activity, actin
sliding motor activity, and the dissociation of calmodulin light chains
at high Ca2+ concentration are mediated through calmodulin,
and that its two high affinity C-terminal Ca2+-binding
sites are critical for the regulatory effect of Ca2+.
For myosin I associated with E12Q, the effects of Ca2+
on actin-activated ATPase activity, motor activity, and the calmodulin dissociation are similar to those for myosin I
associated with wild-type calmodulin. On the other hand, the disruption of the Ca2+-binding sites in the C-terminal domain of calmodulin
abolishes the Ca2+ dependence of the ATPase activity, the
motor activity of myosin I
, and the dissociation of 1 mol of
calmodulin from the heavy chain at above pCa 6. These
results suggest that Ca2+ binding to the C-terminal domain
of calmodulin, i.e. high affinity Ca2+ binding
sites, is responsible for the dissociation of one calmodulin molecule
from myosin I
heavy chain. The increase in ATPase activity parallels
the dissociation of one molecule of calmodulin according to the
Ca2+ dependence data (Fig. 4), thus it is reasonable to
conclude that the dissociation of calmodulin increases myosin I
ATPase activity. It should be noted, however, actin independent ATPase
activity but not actin dependent activity increases with
Ca2+. Similar result has also been found for conventional
myosin in which the dissociation of regulatory light chain increases
basal myosin ATPase activity (33). It should be noted that while the deletion of the Ca2+ binding at the N-terminal domain of
calmodulin did not prevent the dissociation of 1 mol of calmodulin from
the heavy chain, it shifted the Ca2+ required for
calmodulin dissociation to lower concentration. This result suggests
that there is a cross-talk between the N-terminal and C-terminal
domains and that deletion of the Ca2+ binding ability at
the N-terminal domain of calmodulin affects the conformational change
at the C-terminal domain of calmodulin. This is consistent with the
earlier finding that conformation of the Tyr-138 in the C-terminal
domain of calmodulin is significantly influenced by a change in the
Ca2+ binding properties of the N-terminal domain (25).
Clearly, the binding of Ca2+ to calmodulin at the
C-terminal sites is critical for the inhibition of actin translocating
activity of myosin I by Ca2+, since this
Ca2+-induced inhibition of the motility was not observed
with myosin I
containing E34Q or E1234Q calmodulin (Table I).
However, it is more complicated to determine whether or not the
dissociation of the calmodulin molecule from myosin I
heavy chain is
critical for the inhibition of the motor activity. Thus, although the
wild-type myosin I
still binds all three calmodulin light chains at
pCa 6, its motor activity is completely inhibited at this
Ca2+ concentration. One possible explanation is that
although the C-terminal domain of the one calmodulin molecule is
dissociated from myosin I
heavy chain at pCa 6, the
N-terminal domain is still associated with the heavy chain at this
Ca2+ concentration, and further conformational change
induced by Ca2+ binding at the N-terminal low affinity
sites of this molecule is necessary for the complete dissociation (Fig.
5). Presumably, this incomplete
association of calmodulin with myosin I
is no longer able to support
motor activity. For the E12Q mutant, the conformational change induced
by the binding of Ca2+ to the higher affinity sites may be
sufficient to dissociate calmodulin from myosin I
heavy chain. It
should be noted, however, Ca2+ binding to calmodulin
dissociates only one of the three bound calmodulin from myosin I
heavy chain. According to the amino acid sequence, myosin I
has
three IQ motifs, one of which is not a completely matched IQ motif,
IQXXXRGXXXR (one-letter amino acid code;
X is any amino acid residue) (6). It is plausible that the
calmodulin bound to the incomplete IQ motif is dissociated from myosin
I
when Ca2+ binds to the C-terminal domain.
Alternatively, the conformational change in all three calmodulin upon
Ca2+ binding to the C-terminal domain results in the
inhibition of motility and the additional conformational change upon
the Ca2+ binding at the N-terminal low affinity sites
destabilizes the association of one of the bound calmodulin to the
heavy chain presumably due to steric hindrance. Further studies are
needed to clarify the reason why only one molecule of calmodulin is
dissociated from myosin I
.
|
Studies reported here with mutant calmodulin show that motor function
of myosin I is regulated by Ca2+ binding to the high
affinity sites of calmodulin light chains. This regulatory mechanism
may also apply to those of other unconventional myosins which contain
calmodulin as their light chains.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Yvon Delville (Neuroanatomy Laboratory of Behavioral Neuroscience in the Department of Psychiatry at the Univeristy of Massachusetts Medical Center) for letting us use the densitometry equipment, Vivek Rao for preparation of bacterially expressed mutant calmodulins, and Dr. Shinya Saito for fluorescence measurements.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants AR 41653, HL 47530, and HL 56218 (to M. I.) and National Institutes of Health Grant GM 49155 and R. A. Welch Foundation Grant C-1119 (to K. B.).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 Center, Worcester, MA 01655. Tel.: 508-856-6698; Fax: 508-856-4600; E-mail: Mitsuo.Ikebe{at}ummed.edu.
The abbreviations used are: BBMI, brush-border myosin I; MOPS, 4-morpholinepropanesulfonic acid; 9-AC, 9-anthroylcholine.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|