From the Adolf-Butenandt-Institut, Zellbiologie,
Ludwig-Maximilians-Universität, D-80336
München, Germany
We purified Myr3 (third unconventional myosin
from rat), a mammalian "amoeboid" subclass myosin I, from rat
liver. The heavy chain of purified Myr3 is associated with a single
calmodulin light chain. Myr3 exhibits K/EDTA-ATPase and Mg-ATPase
activity. The Mg-ATPase activity is stimulated by increasing F-actin
concentrations in a complex triphasic manner similar to the Mg-ATPase
activity of myosin I molecules from protozoa. Although purified Myr3
was observed to cross-link actin filaments, it bound in an ATP
regulated manner to F-actin, and no evidence for a
nucleotide-independent high affinity actin binding site that could
explain the triphasic activation pattern was obtained. Micromolar
concentrations of free Ca2+ reversibly inhibit the
Mg-ATPase activity of Myr3 by binding to its light chain calmodulin,
which remains bound to the Myr3 heavy chain irrespective of the free
Ca2+ concentration. Polyclonal antibodies and Fab fragments
directed against the tail domain were found to stimulate the Mg-ATPase activity. A similar stimulation of the Myr3 Mg-ATPase activity is
observed upon proteolytic removal of the very C-terminal SH3 domain.
These results demonstrate that Myr3 is subject to negative regulation
by free calcium and its own tail domain and possibly positive
regulation by a tail-domain binding partner.
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INTRODUCTION |
Myosins are mechanoenzymes that convert chemical energy stored in
the molecule ATP into directed force along actin filaments. They are
heterooligomers consisting of one or two heavy chains and up to six
light chains per heavy chain. The heavy chains exhibit an N-terminal
head region, a light chain binding region, and a C-terminal tail
region. The head region is relatively well conserved in all myosins and
comprises ATP- and actin-binding sites and exhibits actin-activated
ATPase activity (1).
Based on phylogenetic analysis of the head domain sequences myosins can
be divided into more than 10 classes. Class I myosin molecules are
single headed with relatively short tail-domains that neither dimerize
nor form filaments. This class contains numerous members that by
sequence comparison can be grouped into subclasses. Members of the
"amoeboid" subclass were first identified in Acanthamoeba
castellanii (2) and Dictyostelium discoideum (3). More
recently, myosin molecules of this subclass have been identified from
yeast to man (Refs. 4-7 and Knight and Kendrick-Jones, GenBankTM
accession number X70400). So far, only members of this subclass from
A. castellanii and D. discoideum have been characterized biochemically. Their actin-activated Mg-ATPase activity and their ability to produce force is regulated by phosphorylation of a
single serine/threonine residue located in the head region. The
recently identified vertebrate members of this subclass lack this
phosphorylation site. Therefore, their ATPase and motor activities are
likely to be regulated in a different manner. Vertebrate members also
differ in their tail regions from the biochemically characterized protozoan members in that they do not contain an extended GPA/GPQ (glycine, proline, and alanine/glutamine)-rich region. This region was
reported to exhibit actin binding activity (8, 9). Such an
ATP-insensitive actin-binding site was used to explain the peculiar
triphasic Mg-ATPase activation observed as a function of F-actin
concentration. At low concentrations of F-actin, the Mg-ATPase reaches
near maximal velocity. This is followed by a decrease of velocity at
intermediate F-actin concentrations before getting reactivated to
maximal velocity again at high F-actin concentrations (10). Examination
of the actin-activated Mg-ATPase activity of vertebrate amoeboid myosin
I molecules could lead to a further clarification of the mechanism
responsible for this complex triphasic activation kinetics.
The heavy chain of the rat myosin I molecule Myr3, a vertebrate member
of the amoeboid subclass of myosin I molecules, was recently identified
by molecular cloning (7). Based on sequence analysis, the heavy chain
of Myr3 contains a single light chain binding motif. This light chain
binding motif was proposed to bind calmodulin. Myr3 is expressed in
many tissues and cell lines. Due to its localization in cell-cell
contact regions Myr3 was suggested to play a role associated with
cell-cell contacts (7).
To further define the function of Myr3 we attempted to characterize its
enzymatic properties and their regulation. We now report the
purification of Myr3 from rat liver, the characterization of its
enzymatic activities, and their regulation by actin, free Ca2+, tail binding antibodies, and proteolysis.
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EXPERIMENTAL PROCEDURES |
Purification of Myr3--
Myr3 was purified from rat liver. The
livers of 40 adult, male Sprague-Dawley rats were homogenized in TBS
(150 mM NaCl, 50 mM Tris/HCl, pH 7.4, final
volume: 2 liters) using a tight fitting motor driven Dounce
homogenizer. To minimize proteolysis the protease inhibitors Pefabloc
(50 mg/liter) and aprotinin (7.6 trypsin inhibitor units/liter) were
added to the homogenate, and all purification steps were carried out at
4 °C. The homogenate was centrifuged in a Sorvall GS3 rotor (E. I.
du Pont de Nemours, Bad Homburg, Federal Republic of Germany) at 8500 rpm for 60 min. Ammonium sulfate was added to the supernatant to a
final saturation of 35% and stirred for 20 min. The pellet resulting
after centrifugation (GS3-rotor, 8500 rpm, 30 min) was resuspended in
400 ml of low-salt buffer (50 mM NaCl, 20 mM
Hepes, pH 7.4, 2 mM MgCl2, 0.2 mM
EGTA, 1 mM
-mercaptoethanol) and dialyzed against the
same buffer overnight to obtain an actomyosin pellet. Sometimes the
resuspended pellet was incubated in low-salt buffer for 1 h and
the dialysis step omitted. This shortened procedure reduced the amount
of contaminating proteins in the actomyosin pellet but the actomyosin
pellet was not consistently formed. The actomyosin pellet was collected
by centrifugation (GS3-rotor, 8500 rpm, 45 min) and washed twice with
400 ml of low-salt buffer supplemented with 100 mM KCl.
Myr3 was then extracted from the pellet with sodium pyrophosphate. The
pellet was resuspended in 150 ml of extraction buffer (20 mM sodium pyrophosphate, 100 mM KCl, and 5 mM EDTA in low-salt buffer) and centrifuged in a Beckman
50.2 Ti rotor (Beckman Instruments, Inc., Fullerton, CA) at 45,000 rpm
for 45 min. The resulting extract was concentrated either by
ultrafiltration (Amicon, Witten, Federal Republic of Germany) or
ammonium sulfate precipitation (50% saturation) before loading onto a
gel filtration column. The ammonium sulfate precipitate was directly
resuspended in gel filtration buffer (10 mM Tris/HCl, pH
8.2, 200 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, 10 mM
ATP) and clarified by centrifugation (SS34 rotor, 16,000 rpm, 20 min).
The concentrated extracts were chromatographed over a HiLoad 26/60
Superdex 200 gel filtration column (Amersham Pharmacia Biotech,
Freiburg, Federal Republic of Germany) preequilibrated in gel
filtration buffer. Fractions containing Myr3 as monitored by
immunoblotting were pooled and loaded onto a Mono S HR 5/5 ion-exchange
column (Amersham Pharmacia Biotech) preequilibrated with 10 mM Tris/HCl, pH 8.2, 200 mM NaCl, 2 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA. The column was developed with a linear NaCl salt
gradient (200 mM to 1 M). Myr3 eluted as a
single peak at approximately 850-900 mM NaCl.
ATPase Assay--
Mg-ATPase activity was measured in solutions
containing 15 mM Tris/HCl, pH 8.0, 10 mM
MgCl2, 4 mM EGTA, and ~90 mM NaCl
contributed by the Myr3 Mono S column fractions unless otherwise
stated. Assays were started by the addition of 2 mM ATP
from a concentrated stock solution containing
[
-32P]ATP and incubated at 37 °C for 60 min. The
radioactivity release as Pi from [
-32P]ATP
was measured. Briefly, the reaction was stopped by the addition of 7 volumes of trichloroacetic acid/charcoal (7, 5%/8%). After incubation
for 30 min on ice, the suspension was centrifuged and radioactivity in
the supernatants determined by Cerenkov counting. Phosphate release was
linear with time and less than 20% of the substrate became hydrolyzed.
All activities were determined in triplicates and activities in the
absence of Myr3 were subtracted. Free Ca2+ concentrations
were adjusted according to calculations with the program Calcium V2.1
(created by K.-J. Foehr and W. Warchol). Myr3 antibodies in TBS, 2 mM NaN3 were preincubated with Myr3 for 20 min
prior to the addition to the reaction mixture. K/EDTA-ATPase activity
was assayed in a buffer containing 0.5 M KCl, 2 mM EDTA, and 15 mM Tris/HCl, pH 8.0, as
described above.
Actin Binding Assay--
Actin was purified from rabbit skeletal
muscle as described (11) and stored as G-actin at
70 °C. Actin was
polymerized in a buffer of 100 mM KCl, 2 mM
MgCl2, 20 mM Hepes, pH 7.4, 0.5 mM
-mercaptoethanol, and 2 mM NaN3. F-actin
binding assays were performed as described previously (12) except that
bovine serum albumin (1 mg/ml) was added to the assay mixture and
centrifuge tubes were precoated with 5% non-fat dry milk.
Electron Microscopy--
F-actin (50 µg/ml) incubated either
with different amounts of purified Myr3 or buffer alone was adsorbed
for 30 s on copper grids coated with a glow-discharged carbon
film. Grids were negatively stained with 1% uranyl acetate and
examined with a Philips CM10 electron microscope at an accelerating
voltage of 60 kV.
Amino Acid Sequence Determination--
Purified Myr3 was
separated by SDS-PAGE1 and
stained with Coomassie Blue. The light chain was excised from the gel
and digested with endoproteinase Lys-C. Digested peptides were
separated by reversed-phase HPLC on a Superspher 60 RP select B column
(Merck, Darmstadt, Federal Republic of Germany) and sequenced by
N-terminal degradation using an automated sequencer (Porton 3600, Beckman Instruments).
Miscellaneous Techniques--
Polyclonal antibodies against Myr3
were raised and purified as described (7). Fab fragments were produced
by papain digestion as described by Wallimann and Szent-Györgyi
(13). A C-terminally truncated fragment of Myr3 was obtained by limited
proteolysis of purified Myr3 with mercuripapain. The proteolysis was
stopped by the addition of 5 mM iodoacetamide. Protein
concentrations were determined using the Bradford colorimetric assay
(14). Immunoblotting was performed according to Towbin et
al. (15). Primary antibodies were diluted in 5% non-fat dry milk
to 2 µg/ml. Alkaline phosphatase-coupled secondary antibody was
diluted in 5% non-fat dry milk and 0.1% Triton X-100. The phosphatase
reaction was visualized by the ProtoBlot system (Promega Corp.,
Madison, WI). Gel electrophoresis was performed according to Laemmli
(16). For quantitative protein analysis, Coomassie-stained SDS gels were scanned and analyzed using the Image Master gel quantification system (Amersham Pharmacia Biotech).
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RESULTS |
Purification of Myr3--
To characterize the biochemical
properties of Myr3 we established a purification scheme for Myr3 from
rat liver. Rat liver was chosen as starting material, because it
contains high levels of Myr3, and the proteolytic breakdown of Myr3 in
liver homogenate is less pronounced than in homogenates from other
tissues. Myr3 was readily soluble at salt concentrations of 150 mM (>80%) and could be precipitated from the liver
extract with ammonium sulfate reaching between 25 and 35% saturation.
Since the bulk of proteins precipitated only at higher ammonium sulfate
concentrations, the precipitation of Myr3 at 35% saturation
represented a useful first purification step. Myr3 was further purified
by the formation of an actomyosin pellet from which it could be
quantitatively extracted by sodium pyrophosphate. The use of sodium
pyrophospate as opposed to Mg-ATP reduced the amount of conventional
myosin present in the extract. Myr3 was further purified by gel
filtration and cation-exchange column chromatography. Purified Myr3
eluted from a Mono S cation-exchange column as a single sharp peak at relatively high salt concentrations of about 850-900 mM
(Figs. 1A and 10A).
Attempts to concentrate and/or desalt purified Myr3 consistently
failed, because Myr3 exhibited a strong tendency to adhere to plastic
surfaces. Therefore, we directly used the fractions from the Mono S
column in most of our assays. The described purification protocol
yielded between 150 and 300 µg of purified Myr3 from 40 rat
livers.

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Fig. 1.
Calmodulin is the single light chain of
Myr3. A, purified Myr3 and authentic calmodulin were
separated on SDS-PAGE and stained with Coomassie Brilliant Blue. The
position of Myr3 heavy chain (myr3 HC) and light chain
(LC) as well as the position of authentic calmodulin
(CaM) are indicated. B, purified Myr3 was boiled
for 3 min and centrifuged. The supernatant (myr3 LC) and
authentic calmodulin (CaM) were separated on SDS-PAGE in the
presence (C) or absence (E) of free
Ca2+. The two arrowheads on the right
indicate the relative positions of authentic calmodulin in the presence
(lower arrowhead) and absence (higher arrowhead)
of free Ca2+. C, comparison of the peptide
sequences obtained from the Myr3 light chain and calmodulin
(numbers refer to the position of the first displayed amino
acid in the calmodulin sequence) from rat. The first lysine residue of
the peptide sequences is deduced from the specificity of the Lys-C
protease, which was used to cleave the light chain of Myr3.
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Myr3 Contains a Single Calmodulin Light Chain--
Together with
the Myr3 heavy chain a protein of 17 kDa was copurified (Fig.
1A). The stoichiometry of the 17-kDa protein to the Myr3
heavy chain was determined to be 0.82 ± 0.03 by gel densitometry. This number is in good agreement with the prediction of a single light
chain which binds to the single "IQ motif" in the neck region of
the Myr3 heavy chain. We previously reported that calmodulin was
present in Myr3 heavy chain immunoprecipitates and that calmodulin bound to the Myr3 heavy chain in a gel-overlay assay (7). Indeed, the
17-kDa light chain comigrated with authentic calmodulin on SDS gels
(Fig. 1A). Furthermore, the Myr3 light chain was resistant to boiling and demonstrated a calcium-dependent shift in
electrophoretic mobility, two properties that characterize authentic
calmodulin (Fig. 1B). Additional proof for the identity of
the Myr3 light chain with calmodulin was obtained by peptide
sequencing. Two peptide sequences derived from the Myr3 light chain (9 and 16 amino acid residues in length) were identical to corresponding sequences in rat calmodulin (Fig. 1C). By all these
criteria, the Myr3 light chain is identical to calmodulin.
ATPase Activity--
To analyze myosin-like activities of purified
Myr3, K/EDTA-ATPase and the physiologically relevant actin-activated
Mg-ATPase activities were determined. Myr3 exhibited a K/EDTA-ATPase
activity of 722 ± 22 nmol/min/mg and a Mg-ATPase activity that
was stimulated by F-actin (Table I).
Both, basal and actin-activated Mg-ATPase, activities of Myr3 increased
with increasing salt concentrations (Fig.
2, Table I). However, up to a salt
concentration of approximately 200 mM NaCl the slope of the
actin-activated Mg-ATPase activity was smaller than the slope of the
basal Mg-ATPase activity. At this salt concentration, there was no
longer any actin activation of the Mg-ATPase detectable. Upon
increasing the salt concentration further, the Mg-ATPase activity
increased irrespective of the presence or absence of F-actin (Fig. 2).
This salt-dependent increase in Mg-ATPase activity of Myr3
was reversible as Myr3 was diluted into the assay mixtures from a
buffer containing 900 mM NaCl. Addition of exogenous
calmodulin to the assay mixtures did not influence the Myr3 ATPase
activities.
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Table I
Enzymatic activities of purified myr3
The K/EDTA-ATPase activity was determined in the presence of 500 mM KCl and 2 mM EDTA. The Mg-ATPase activity in
the absence or presence of F-actin was determined in assay mixtures
containing either 12 mM KCl, 90 mM NaCl, or 590 mM NaCl, respectively.
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Fig. 2.
Increasing salt concentrations raise the
Mg-ATPase activity of Myr3 in the absence and presence of F-actin.
The Mg-ATPase activity of Myr3 was determined at increasing salt
concentrations in the absence (closed circles, broken line)
and presence (open circles, solid line) of F-actin. The
inset shows a more extended range of NaCl
concentrations.
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Next we determined the effect of the actin concentration on the
Mg-ATPase activity of Myr3. As shown in Fig.
3, the Mg-ATPase activity of Myr3 was
activated by F-actin at low concentrations of salt (12 and 35 mM KCl, respectively) in a triphasic manner. Measurements
under standard conditions (90 mM NaCl) revealed no qualitative differences except for a higher basal activity and a
smaller activation by F-actin due to the higher salt concentration. The
Mg-ATPase activity increased up to a concentration of ~1
µM actin, then decreased up to a concentration of ~3.5
µM actin and finally increased again until it reached a
plateau at actin concentrations higher than 7 µM.

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Fig. 3.
Mg-ATPase activity of Myr3 as a function of
actin concentration. Purified Myr3 was incubated with different
concentrations of F-actin, and the Mg-ATPase activity was determined.
Open and closed circles represent data from two
different preparations. These assays were performed in the presence of
12 (open circles) and 35 mM KCl (closed
circles). Data points were fitted by combining the
Michaelis-Menten equation with a polynomial function (solid
line). The Michaelis-Menten term is indicated by the broken
line.
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Myr3 Can Cross-link F-actin, and It Binds in an ATP-regulated
Manner to F-actin--
As mentioned above, the triphasic F-actin
activation of the Mg-ATPase of amoeboid myosin I molecules from
protozoa is explained by a mechanism that involves cross-linking of
F-actin. To test whether Myr3 can cross-link actin filaments, we
examined mixtures of F-actin and purified Myr3 by electron microscopy
(Fig. 4). Bundles of cross-linked F-actin
were observed in the presence of purified Myr3. This finding indicates
that Myr3 is able to cross-link actin filaments, although it lacks a
"GPA" region in its tail domain. To determine directly whether Myr3
contains an ATP-independent high affinity actin-binding site, which
could explain the triphasic activation of its ATPase activity by
increasing F-actin concentrations, we performed actin cosedimentation
experiments at low Myr3 concentrations. Myr3 was found to specifically
cosediment with actin filaments in the absence, but not in the
presence, of ATP (Fig. 5), indicating
that Myr3 does not contain a nucleotide-independent high affinity actin
binding site.

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Fig. 4.
Myr3 is able to cross-link F-actin.
F-actin (50 µg/ml) was incubated together with purified Myr3 in the
presence of 200-250 mM salt and viewed by negative stain
in the electron microscope. Large bundles of F-actin laterally
cross-linked by Myr3 were noticed. The inset shows two actin
filaments bundled by Myr3 at a higher magnification. Bars:
50 nm.
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Fig. 5.
Binding of purified Myr3 to F-actin. The
F-actin binding of Myr3 was determined at 150 mM salt by
cosedimentation in the absence ( ) or presence (+) of actin (18.7 µM) and ATP (2 mM), respectively.
Supernatants (S) and pellets (P) were analyzed
for their content of Myr3 by immunoblotting with antibody Tü
58.
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Ca2+ Negatively Regulates the Mg-ATPase Activity of
Myr3 by Binding to Its Light Chain Calmodulin--
Since we have shown
that the Ca2+-binding protein calmodulin serves as the
single light chain of Myr3, we hypothesized that the activity of Myr3
might be regulated by Ca2+. Therefore, we examined the
effect of free Ca2+ ions on its Mg-ATPase activity (Fig.
6A). Addition of micromolar free calcium ions reduced both the basal and actin-activated Mg-ATPase activity of Myr3 by a factor of 2-3. This inhibitory effect of free
calcium on the Mg-ATPase activity of Myr3 was reversible. Chelation of
free calcium ions with EGTA after a 20-min incubation of purified Myr3
with free Ca2+ increased the Mg-ATPase activity again to
the initial level (Fig. 6A). A detailed analysis of the free
calcium concentration needed to inhibit the Myr3 Mg-ATPase activity
revealed a marked inhibition for both the basal and actin-activated
Mg-ATPase activity between a concentration range of 0.1-1
µM free Ca2+ (Fig. 6B). This
concentration range exactly coincides with the affinity of calmodulin
for calcium, strongly supporting the notion that the observed
inhibition is due to the binding of Ca2+ to the Myr3 light
chain calmodulin. This Ca2+-dependent
inhibition of the Myr3 Mg-ATPase activity could be explained either by
an allosteric effect or by dissociation of calmodulin from the Myr3
heavy chain. To discriminate between these two possibilities, we
performed an actin cosedimentation assay in the absence and presence of
micromolar free Ca2+ (Fig.
7). This experiment allows for the
separation of free calmodulin from calmodulin bound to the Myr3 heavy
chain. Comparable amounts of calmodulin were found to cosediment with
Myr3 and F-actin irrespective of the free calcium concentration (Fig.
7). This result demonstrates that calmodulin remains bound to the Myr3
heavy chain irrespective of the free Ca2+ concentration and
hence that the regulation of the Myr3 Mg-ATPase by Ca2+ is
of allosteric nature. In further support of this notion, addition of
exogenous calmodulin had no effect on the Mg-ATPase activity of Myr3
(data not shown).

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Fig. 6.
Free Ca2+ in the micromolar range
inhibits the ATPase activity of Myr3. A, Mg-ATPase
activity of Myr3 was determined either in the absence (black
bars) or presence (gray bars) of F-actin. Activities
were measured in the presence of EGTA (Mg) and 3 µM free Ca2+ (Mg/Ca). To assess
the reversibility of the Ca2+ inhibition, Myr3 was
preincubated for 20 min in 3 µM free Ca2+
before chelating free Ca2+ with EGTA
(Mg/Ca+EGTA). B, purified Myr3 was incubated in
various free Ca2+ concentrations ranging from nanomolar to
millimolar using a Ca/EGTA buffer system. The ATPase activity was
measured in the absence (closed circles, broken line) or
presence (open circles, solid line) of F-actin (8 µM).
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Fig. 7.
Association of calmodulin with Myr3 in the
absence and presence of free Ca2+. Myr3 was incubated
in the absence of free Ca2+ (4 mM EGTA)
(lane 1) and in the presence of free Ca2+ (3 µM Ca2+) (lane 2), respectively.
To determine the amount of calmodulin bound to Myr3, Myr3 was
cosedimented with F-actin. The pellets were analyzed by SDS-PAGE
followed by Coomassie Blue staining. Bovine serum albumin was added to
the assay to prevent unspecific binding and adhesion to the tube walls.
HC, Myr3 heavy chain; LC, light chain
(calmodulin).
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The Myr3 Tail Domain Inhibits the Mg-ATPase Activity of the Myr3
Head Domain--
As a substitute for a physiological Myr3 tail binding
partner, the antibody FML 6, which was raised against a fusion protein of the Myr3 tail domain (encompassing amino acids 821-1107; Ref. 7),
was tested for potential regulatory effects on the Mg-ATPase of the
Myr3 head domain. Interestingly, the antibody FML 6 caused in a
concentration-dependent manner an increase of both basal and actin-activated Mg-ATPase activity (Fig.
8A). The basal Mg-ATPase activity was raised to values that were comparable to the
actin-activated Mg-ATPase activity. The actin-activated Mg-ATPase
activity was also increased, but to a somewhat smaller degree. The
observed activation of the Myr3 Mg-ATPase activity by antibody FML 6 was not simply a result of cross-linking of Myr3 molecules by the antibodies. Monovalent Fab fragments also stimulated the Myr3 Mg-ATPase
activity and that to a similar extent as the FML 6 IgG antibodies (Fig.
9). This activation of the Myr3 Mg-ATPase
activity was not observed with control antibodies (data not shown) and the previously described polyclonal antibody Tü 58 (7) that recognizes a region in the myosin head domain of Myr3 (amino acids 326-342) corresponding to the so called ordered loop in the
conventional myosin head (amino acids 400-416; Ref. 34). Antibody
Tü 58 specifically inhibited the F-actin activation of the Myr3
Mg-ATPase (Fig. 8B). Additive effects were observed when the
two antibodies, Tü 58 and FML 6, were added simultaneously.
ATPase measurements in the presence of both antibodies demonstrated an
increase in the basal Mg-ATPase activity but no longer any activation
by F-actin (data not shown). These results suggest that the two
antibodies were affecting two independent regulatory mechanisms and
they demonstrate the specificity of the activation of the Mg-ATPase by
the Myr3 tail antibodies. Interestingly, a similar activation of the
Myr3 Mg-ATPase activity as with the tail binding antibodies was noticed
upon proteolytic cleavage of the very C terminus of the Myr3 tail
domain (Fig. 10). Limited digestion of
Myr3 with mercuripapain produced a Myr3 fragment truncated at its C
terminus by approximately 10 kDa (Fig. 10A). A similar
degradation product of Myr3 is also observed in tissue homogenates as
described previously (7). This C-terminally truncated Myr3 molecule
exhibited a clearly increased Mg-ATPase activity as compared with the
intact Myr3 molecule (Fig. 10B), indicating that the
C-terminal tail domain provides some inhibitory constraint for the
Mg-ATPase activity located in the head domain.

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Fig. 8.
Myr3 antibodies modulate its Mg-ATPase
activity. Myr3 Mg-ATPase activity was determined in the presence
of increasing amounts of either the tail antibody FML 6 (A)
or the head antibody Tü 58 (B). The relative
activation was calculated by setting the basal activity to zero and the
actin-activated activity to 100%. Open and closed
circles represent the activation in the presence or absence of
F-actin, respectively.
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Fig. 9.
Activation of Myr3 Mg-ATPase activity by tail
binding Fab fragments. Myr3 Mg-ATPase activity was determined in
the absence ( Fab) and presence of 30 µg of Fab fragments derived
from FML 6 total IgG (+ Fab).
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Fig. 10.
Activation of Myr3 Mg-ATPase activity by
C-terminal truncation of the tail domain. Myr3 (lane 1)
and Myr3 truncated at its C terminus by limited digestion with
mercuripapain (lane 2) were separated on SDS-PAGE and
stained with Coomassie Blue (A). The corresponding aliquots
were used for determination of Mg-ATPase activity (B).
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DISCUSSION |
The establishment of a purification procedure for the rat myosin I
molecule Myr3 allowed us to initiate its biochemical characterization. Preparations of purified Myr3 contained in addition to the heavy chain
as predicted from its sequence a single light chain in a 1:1
stoichiometry. This light chain was identified as calmodulin based on
its electrophoretic mobility, heat stability, and partial peptide
sequence. Calmodulin has been found to be associated with other
vertebrate myosin I molecules (12, 17-19) but not with amoeboid myosin
I molecules from protozoa, which are the closest homologues of Myr3
(20, 21). This difference in the identity of the light chain between
Myr3 and other amoeboid myosin I molecules might reflect a difference
in regulatory properties. This notion is supported by the fact that
Myr3 does not contain the regulatory phosphorylation site present in
the head domain of amoeboid myosin I molecules from protozoa.
Myr3 displayed K/EDTA-ATPase and actin-activated Mg-ATPase activities
that are characteristic of bona fide myosins. The
K/EDTA-ATPase activity of Myr3 was higher than for other vertebrate
myosin I molecules such as chicken brush border myosin I (17, 22), myr1
(23), or myr2 (23-25), but lower than for myosin I molecules from
protozoa (20). The actin-activated Mg-ATPase activity of Myr3 was
comparable with that of other myosin I molecules from vertebrates (17,
25) and the dephosphorylated myosin I molecules from protozoa (20).
However, it was considerably lower as compared with the actin-activated
Mg-ATPase activity of the phosphorylated myosin I molecules from
protozoa.
Myr3, like the amoeboid myosin I molecules from protozoa, exhibited a
peculiar triphasic activation of its Mg-ATPase by F-actin. For the
myosin I molecules from protozoa this complex kinetic behavior has been
proposed to be due to a second ATP-insensitive high affinity F-actin
binding site in their tail domains (26, 27). An ATP-insensitive F-actin
binding site has been mapped to the GPA/GPQ-rich region in these myosin
I molecules (8, 9, 28). The tail domain of Myr3 lacks such a
GPA/GPQ-rich region. Nevertheless, in electron micrographs we observed
bundling of actin filaments by Myr3. Similar bundling activity has also been reported for brush border myosin I (17, 29), myr1, and myr2 (23),
which also lack a GPA/GPQ-rich region. Therefore, in vitro
bundling of actin filaments by myosin I molecules does not necessarily
require the presence of a GPA/GPQ-rich region. On the other hand,
bundling does not automatically mean complex triphasic activation
kinetics, because brush border myosin I and myr2 exhibit simple
hyperbolic activation kinetics (17, 25, 30). At present, we do not know
whether cross-linking of actin filaments by Myr3 is due to a
nucleotide-insensitive actin binding site or rather self-association.
This question is of relevance for understanding the mechanism of its
complex activation by F-actin. However, in accordance with the lack of
a GPA/GPQ-rich region in Myr3, we have not obtained any evidence for a
high affinity ATP-insensitive F-actin binding site in actin binding
experiments performed with purified Myr3. Therefore, alternative
mechanisms for explaining the complex triphasic kinetics cannot be
excluded.
Surprisingly, the ATPase activity of Myr3 was found to be negatively
regulated by micromolar free Ca2+ concentrations. This is
exactly the opposite of what has been reported for other vertebrate
myosin molecules, which contain calmodulin molecules associated as
light chains. Micromolar free Ca2+ concentrations have been
shown to activate the Mg-ATPase activities of brush border myosin I,
myr1, myosin I
, and myosin V (25, 30-32). In brush border myosin I
and myosin V elevation of free Ca2+ concentrations also led
to a partial dissociation of calmodulin light chains (30, 32). However,
we demonstrated that calmodulin is associated with the Myr3 heavy chain
irrespective of the free Ca2+ concentration and, therefore,
conclude that binding of Ca2+ to the light chain calmodulin
regulates the Myr3 Mg-ATPase activity allosterically. This conclusion
is supported by the lack of any modulation of the Mg-ATPase by an
excess of exogenously added calmodulin.
Binding of the antibody FML 6 or of monovalent Fab fragments to the
tail-domain of Myr3 caused an increase in its Mg-ATPase activity. To
explain how the binding of an antibody to the tail-domain of a myosin
can affect its ATPase activity, further experimentation will be needed.
However, one clue comes from the observation that a C-terminally
truncated Myr3 fragment also exhibits increased Mg-ATPase activity.
Therefore, it seems likely that both the tail binding antibody FML 6 and the truncation at the very C terminus of the tail domain neutralize
an inhibitory constraint imposed by the tail domain. This inhibitory
constraint might be imposed by the C-terminal SH3 domain that is
missing in the truncated Myr3 molecule (7). This SH3 domain could bind
intramolecularly to proline-rich motifs present in the Myr3 tail domain
and thereby inhibit Mg-ATPase activity. An inhibitory function for SH3
domains has already several precedents in protein kinases (33).
We reported previously that intact Myr3 in lung homogenates did not
bind significantly to F-actin, whereas a C-terminally truncated Myr3
degradation product did bind to F-actin in an ATP-regulated manner (7).
We now report that intact purified Myr3 binds in an ATP-regulated
manner to F-actin. These seemingly contradictory findings might either
be explained by removal of a factor(s) during purification interacting
with or modifying the C terminus of Myr3 or by disruption of inhibitory
intramolecular interactions. These previously reported results,
however, are in agreement with our present findings of an inhibitory
function of the Myr3 tail domain.
Our results suggest that caution should be exercised when comparing
data obtained from in vitro motility assays in which the tail domains have been immobilized with data obtained in solution. The
mode of immobilization of the tail domain might critically affect the
motor activity. Furthermore, these results suggest that in
vivo binding partners of myosin tail domains might regulate the
motor activity and suggest a novel approach for identifying myosin tail
binding partners.
We thank Rainer T. Müller and Georg
Kalhammer for assistance and continuous support during the whole
progress of this work. We are indebted to Dr. Heinz Schwarz for his
help in analyzing Myr3-F-actin complexes by negative stain electron
microscopy and to Dr. Susanne Beck for her help in determining the
Myr3-calmodulin stoichiometry. We also acknowledge the support and
encouragement of Dr. Manfred Schliwa.