(Received for publication, September 25, 1995; and in revised form, October 23, 1995)
From the
Regulatory light chain (RLC) phosphorylation is necessary to
activate smooth muscle myosin, unlike constitutively active striated
muscle myosins. Here we show that an actin-binding surface loop located
at the 50/20-kDa junction contributes to this fundamental difference
between myosins. Substitution of the native actin-binding loop of
smooth muscle heavy meromyosin (HMM) with that from either skeletal or
-cardiac myosin caused the chimeric HMMs to become unregulated
like the myosin from which the loop was derived. Dephosphorylated
chimeric HMMs gained the ability to move actin in a motility assay and
had 50-70% of the actin-activated ATPase activity of
phosphorylated wild-type HMM. Direct binding measurements showed that
the affinity of HMM for actin in the presence of MgATP was unaffected
by loop substitution; thus the rate of a step other than binding is
increased. Phosphorylation of the chimeras did not lead to a higher V
than obtained for wild-type HMM. In the
absence of actin, a foreign loop did not affect nucleotide trapping.
Native regulated molecules have thus evolved a loop sequence which
prevents rapid product release by actin when the RLC is
dephosphorylated, thereby allowing activity to be controlled by RLC
phosphorylation.
Myosins differ from each other in how fast they can move actin,
the rates of their actin-activated ATPases, and whether or not these
activities are regulated. It has been recently suggested that a
divergent surface loop at the actin-binding interface
``tunes'' the rate of phosphate release and thus sets the
maximum velocity (V) for ATPase activity. A
second loop, at the 25/50-kDa interface near the ATP-binding site, was
proposed to control the rate of ADP release and thus be responsible for
determining the velocity at which different myosins move actin
(Spudich, 1994). The actin-activated ATPase activity of a chimeric Dictyostelium myosin containing the actin-binding loop from
skeletal muscle myosin was 5-fold higher than wild-type Dictyostelium myosin, providing experimental evidence in
support of the first part of this hypothesis (Uyeda et al.,
1994). This study did not show if such a generalization would hold true
for other myosin motors nor was the question of heavy chain sequences
involved in regulation of activity addressed.
The molecular step
that is controlled by light chain phosphorylation in smooth muscle
myosin is phosphate release from the active site (Sellers, 1985). It
has been recently proposed that myosin is a ``back door''
enzyme, whereby the cleaved phosphate leaves via a cleft in the 50-kDa
domain, instead of through the nucleotide-binding pocket from which it
entered. Binding of actin is suggested to promote movement of the
highly conserved P-loop near the active site so that phosphate can
leave (Yount et al., 1995). The interaction of actin with
myosin probably involves several steps; the first is thought to be a
weak interaction between the N terminus of actin and the 50/20-kDa
junction of myosin, followed by stronger interactions involving
hydrophobic residues in the upper and lower 50-kDa domains (Rayment et al., 1993b; Holmes, 1995). Mutation of regions involved in
actin binding might therefore be expected to affect regulation of
ATPase activity. Here we show that smooth muscle HMM ()can
be turned into an unregulated, constitutively active molecule simply by
replacing its native actin-binding loop (ABL) at the 50/20-kDa junction
with that from an unregulated striated muscle myosin.
Actin-activated Mg-ATPase activity
was measured as a function of actin concentration in 10 mM
imidazole hydrochloride, pH 7.0, 8 mM KCl, 1 mM MgCl
, 1 mM EGTA, 1 mM
dithiothreitol, 2 mM MgATP (37 °C, 20 µg/ml HMM).
Inorganic phosphate was determined colorimetrically (White, 1982).
Binding of mutant and WT-HMMs to actin in the presence of MgATP was
determined by a sedimentation assay. A longer tail-length WT-HMM
construct of 1329 amino acids was used (Trybus, 1994) to allow the
mutant and WT-HMMs to be distinguished in a single gel lane. SABL-HMM
and long WT-HMM (0.6 µM heads) were mixed with varying
concentrations of actin in the buffer used for ATPase measurements. One
mM MgATP was added, and the sample was spun at 350,000 g for 20 min. Equal volumes of the supernatant and pelleted
fractions were run on 9% SDS gels, and the percent of HMM bound was
determined by densitometry. Control experiments showed that in the
absence of actin essentially all the protein was in the supernatant and
that in the absence of MgATP all the protein bound to actin.
Figure 1: A, 12% acrylamide SDS gel of turkey gizzard myosin prepared from tissue (lane 1), expressed WT-HMM (lane 2), SABL-HMM (lane 3), and CABL-HMM (lane 4). Arrowheads mark the position of the myosin HCs, the 20-kDa RLC, and the 17-kDa essential light chain (ELC). B, equal binding of SABL-HMM and long WT-HMM to actin in the presence of MgATP. Supernatant (S) and pelleted (P) fractions following sedimentation of both HMMs with 125 µM actin and 1 mM MgATP (lanes 1, 2) are shown. Controls in the absence of MgATP show that all the protein can bind to actin (lanes 3 and 4). See Fig. 2D for a plot of percent bound as a function of actin concentration. C, immunoblot of a glycerol/charge gel shows the state of phosphorylation of samples used in steady-state ATPase assays. Dephosphorylated and phosphorylated WT-HMM (lanes 1 and 2) and dephosphorylated and phosphorylated SABL-HMM (lanes 3 and 4) are shown. Arrowheads mark the position of dephosphorylated (deP) and phosphorylated (P) RLC. The band with the fastest mobility in lane 4 is RLC, which was phosphorylated at both Thr-18 and Ser-19.
Figure 2:
Actin-activated ATPase activity and
binding of mutant and WT-HMMs. A-C, phosphorylated HMM (filled circles) and dephosphorylated HMM (open
circles) are shown. V and K
values, respectively, are as follows: A, 3.1±.34 s
and 14 µM for dephosphorylated SABL-HMM, 2.6 ± 0.12 s
and <1 µM for phosphorylated SABL-HMM; B, 2.0 ± 0.45 s
and 24 µM for dephosphorylated CABL-HMM, 3.7 ± 0.68 s
and 13 µM for phosphorylated CABL-HMM; C,
4.2 ± 1.1 s
and 57 µM for
phosphorylated WT-HMM. Measurements were made on five independent
preparations of SABL-HMM, two of CABL-HMM, and seven of WT-HMM. Data
were fitted using the equation, V = V
*[actin]/(K
+ [actin]). D, percent WT-HMM (open squares) and SABL-HMM (filled squares) bound to
actin in the presence of MgATP as a function of actin concentration.
See Fig. 1B for an example of the raw
data.
The actin-activated ATPase activity of
dephosphorylated and phosphorylated WT-HMM, SABL-HMM, and CABL-HMM were
determined (Fig. 2, A-C). The extrapolated V values obtained from the illustrated best fit
curves for the phosphorylated species are: 2.6 ± 0.12
s
for SABL-HMM, 3.7 ± 0.68 s
for phosphorylated CABL-HMM, and 4.2 ± 1.1 s
for phosphorylated WT-HMM. These ATPase activities do not reflect
the higher activity of the myosin from which the loop sequence was
derived.
Surprisingly, the major effect of the loop mutations was to
activate the dephosphorylated species. The V of
dephosphorylated SABL-HMM (3.1 ± 0.34 s
) was
virtually identical to its phosphorylated counterpart, while
dephosphorylated CABL-HMM had a V
of 2.0
± 0.45 s
, only 2-fold less than
phosphorylated CABL-HMM. Immunoblots of the samples used for the ATPase
assay showed that the levels of phosphorylation in the
phosphatase-treated samples were negligible (Fig. 1C).
The dephosphorylated chimeric mutants had a 3-4-fold lower K for actin than phosphorylated WT-HMM (see Fig. 2legend for values). Phosphorylation decreased the K
further to <1 µM for
phosphorylated SABL-HMM. Thus at low actin concentrations, the apparent
effect of the ABL mutations is to increase activity, but this trend
does not persist when the values are extrapolated to V
.
Figure 3:
Release of P in a single
turnover from dephosphorylated SABL-HMM. Data were obtained at 25
mM KCl (filled circles) and 0.6 M KCl (open circles). Rate constants were obtained using the best
fit equation of the form, y = N
*e
,
where N
is moles of phosphate bound per mol of HMM
head at t = 0, k is the rate constant, and t is time.
Figure 4:
Velocity of actin movement by
phosphorylated and dephosphorylated HMM. All values are mean velocities
± S.D. for three independent preparations of SABL-HMM, two of
CABL-HMM, and four of WT-HMM. For each preparation, 12 filaments
were measured to obtain an average velocity for each condition.
Dephosphorylated (deP) WT-HMM did not show any measurable
motility.
The chimeric substitutions caused a decrease in the velocity of actin filament movement by the phosphorylated HMM species, from 1.1 µm/s with WT-HMM to 0.34 µm/s with SABL-HMM and 0.80 µm/s with CABL-HMM. The motility with SABL-HMM was about 2-fold lower than would be expected based on the ATPase measurements, which were 60-70% of phosphorylated WT-HMM.
The sequence of the ABL at the 50/20-kDa junction in the
myosin head is highly divergent, suggesting that it could confer
isoform-specific properties onto myosin. Here we show that the native
sequence of the smooth muscle myosin ABL is required for regulation by
light chain phosphorylation. Replacement of the native ABL with that
from unregulated fast skeletal (SABL-HMM) or -cardiac myosin
(CABL-HMM) caused dephosphorylated HMM, which is normally in the
``off'' state, to proceed through its ATPase cycle with at
least 50% the rate of phosphorylated WT-HMM and move actin in a
motility assay.
How can a loop at the opposite end of the head from
the RLC affect regulation? Loss of regulation implies that inorganic
phosphate can no longer be trapped at the active site in the presence
of actin. The ABL straddles the cleft that separates the upper and
lower 50-kDa domains (Rayment et al., 1993a) and may be
instrumental in initiating a multistep process that leads to cleft
closure and release of phosphate through the ``back door''
(Yount et al., 1995). Thus there is a fairly direct line of
communication between the ABL and the phosphate-binding site at the
base of the cleft in the 50-kDa domain, and it is reasonable that
changing the way actin interacts with a given ABL could inhibit or
facilitate cleft closure and hence affect regulation. Continuity
between the ABL, the nucleotide-binding site, and the RLC is maintained
through the 20-kDa portion of the HC, which traverses the length of the
head; the ABL is adjacent to the N terminus of the 20-kDa segment,
which continues past the nucleotide site, and ends in a long
-helix to which the LCs bind (Rayment et al., 1993a). The
balance between two lines of communication, RLC
active site
ABL, therefore determines whether or not phosphate is released.
Spudich and colleagues (Uyeda et al., 1994) suggested that
the 50/20-kDa loop affects the rate-limiting step of myosin's
ATPase cycle, based on the observation that a chimeric phosphorylated Dictyostelium myosin with a fast skeletal ABL had a 5-fold
higher actin-activated ATPase than WT myosin. Because the
actin-activated ATPase activity of phosphorylated smooth muscle SABL or
CABL-HMM was not enhanced, this role for the loop is not a general
feature of all myosin motors. The ABL mutations decreased the K for actin, resulting in an apparent activation
at low actin concentrations, but this increase does not persist at high
actin concentrations.
Activation of the dephosphorylated chimeras
was not due to an increased affinity for actin in the presence of
MgATP. This result implies that the foreign loops increase the rate of
a transition other than binding. The molecular step is probably the
same as that activated by RLC phosphorylation in the native molecule,
which is phosphate release or a step just preceding release but
following hydrolysis (Sellers et al., 1982). The similarity in
the V values for the dephosphorylated and
phosphorylated chimeras argues for this interpretation.
Introduction
of the ABL loop into smooth muscle HMM must also affect the rate of
other kinetic transitions in the ATPase cycle. The K for SABL-HMM is significantly lower than for phosphorylated
WT-HMM. The leftward shift in K
upon
phosphorylation of SABL-HMM is consistent with the observation that
phosphorylation increases binding to actin (Sellers et al.,
1982). Since more than one elementary rate constant contributes to K
in a model-dependent way (Taylor, 1979), we
cannot speculate at present as to which step is altered by the
introduction of chimeric ABLs. The reduced rate of motility with
SABL-HMM implies that ADP release (Siemankowski et al., 1985)
may also be affected by the skeletal loop sequence.
Regulation of activity in smooth muscle myosin also occurs in the absence of actin. The most striking example of this is the ``trapping'' of nucleotide at the active site when myosin adopts the folded monomeric conformation (Cross et al. 1986), but chymotryptic HMM (Sellers, 1985) and dephosphorylated myosin filaments (Trybus, 1989) also show a strong inhibition of product release. Dephosphorylated SABL-HMM retained the ability to trap products, showing that foreign loops facilitate phosphate release only in the presence of actin. The ABL is likely to be disordered and flexible since it is not seen in the crystal structure (Rayment et al., 1993a) and can be readily proteolyzed (Mornet et al., 1981). When it interacts with actin, the ABL probably adopts a more ordered structure and thereby increases its influence on the active site.
Nineteen of the 26
residues in the actin-binding loops from myosins regulated by light
chain phosphorylation are identical. Only one charged residue is not
conserved between smooth (Yanagisawa et al., 1987; Babij et al., 1991) and non-muscle myosin (Shohet et al.,
1989), suggesting that interactions involving these residues are
necessary to preserve regulation. Two features differ between the loops
from regulated and unregulated myosins. 1) The net positive charge of
the loop from smooth muscle myosin ABL (+2) is less than that from
-cardiac or skeletal muscle myosin (both +3), although this
charge difference did not cause an increase in binding affinity to
actin, and 2) the smooth ABL is longer (26 residues) than that from
either
-cardiac (19 residues) or from skeletal muscle myosin (16
residues). CABL-HMM retained a somewhat higher degree of regulation
than SABL-HMM, but it remains to be established if the length of the
ABL is the critical factor in accounting for this trend.
These results suggest that one difference between regulated and unregulated myosins involves a highly divergent surface loop. The sequence of the actin-binding loop from smooth muscle myosin has been conserved so that its binding to actin does not overcome the inhibition of product release imposed by a dephosphorylated RLC. Regulation is thus an intricate mechanism of how two spatially distant sites, the RLC and the actomyosin interface, communicate with the active site.