The Journal of Cell Biology, Volume 154, Number 3, August 6, 2001 611–617
The Rockefeller University Press, 0021-9525/2001/08/611/7 $5.00
Myosin light chain kinase binding to a unique
site on F-actin revealed by three-dimensional
James T. Stull,
and William Lehman
Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA
Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX
Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA
osin regulatory light chains by the catalytic COOH-
terminal half of myosin light chain kinase (MLCK)
activates myosin II in smooth and nonmuscle cells. In addi-
tion, MLCK binds to thin filaments in situ
vitro via a specific repeat motif in its NH
stoichiometry of one MLCK per three actin monomers.
We have investigated the structural basis of MLCK–actin
interactions by negative staining and helical reconstruc-
tion. F-actin was decorated with a peptide containing the
-terminal 147 residues of MLCK (MLCK-147) that binds
to F-actin with high affinity. MLCK-147 caused formation of
–calmodulin-dependent phosphorylation of my-
and F-actin in
terminus at a
F-actin rafts, and single filaments within rafts were used for
structural analysis. Three-dimensional reconstructions showed
MLCK density on the extreme periphery of subdomain-1 of
each actin monomer forming a bridge to the periphery of
subdomain-4 of the azimuthally adjacent actin. Fitting the
reconstruction to the atomic model of F-actin revealed inter-
action of MLCK-147 close to the COOH terminus of the first
actin and near residues 228–232 of the second. This
unique location enables MLCK to bind to actin without
interfering with the binding of any other key actin-bind-
ing proteins, including myosin, tropomyosin, caldesmon,
Signal transduction pathways, linking extracellular stimuli to
intracellular processes, are essential for regulation of all cells.
Their effective operation requires complex spatial and tem-
poral organization that couples the component steps together.
Phosphorylation–dephosphorylation pathways, for example,
represent major cell control mechanisms, and generally the
effector kinases and phosphatases and their protein targets are
discretely compartmentalized in cells (Perry, 1983; Cohen,
2000). Myosin II activity, for example in smooth muscle and
nonmuscle cells, is regulated by phosphorylation, and the
kinase responsible is anchored on actin filaments.
Myosin regulatory light chains are phosphorylated by
–calmodulin-dependent myosin light chain kinase
(MLCK)* and dephosphorylated by a distinct form of phos-
phatase type 1 (for reviews see Kamm and Stull, 1985, 2001;
Sellers and Adelstein, 1986; Erdödi et al., 1996; Somlyo and
Somlyo, 2000). There is no troponin in smooth muscle, and
here, as in nonmuscle cells, Ca
calmodulin, as cytoplasmic Ca
cell stimulation. The Ca –calmodulin complex then binds
to MLCK, leading to its activation, light chain phosphory-
lation, increased actin-activated myosin ATPase activity,
cross-bridge cycling on thin filaments, and contraction.
Both MLCK and phosphatase are modulated by phosphory-
lation and dephosphorylation, reactions mediated by signaling
networks (Stull et al., 1996, 1997; Somlyo and Somlyo,
2000; MacDonald et al., 2001).
There are two genes that express a skeletal muscle specific
MLCK and a smooth muscle MLCK, respectively. The
smooth muscle MLCK is ubiquitously expressed (Herring et
al., 2000) and consists of short and long isoforms due to
binds to its target protein,
concentration increases after
Address correspondence to William Lehman, Department of Physiology
and Biophysics, Boston University School of Medicine, 715 Albany St.,
Boston, MA 02118-2526. Tel.: (617) 638-4397. Fax: (617) 638-4273.
L. Smith’s current address is Department of Biological Chemistry, Alabama
State University, Montgomery, AL 36101.
Key words: actin; electron microscopy; myosin; myosin light chain kinase;
*Abbreviations used in this paper: 3-D, three-dimensional; MLCK, myo-
sin light chain kinase; MLCK-147; peptide containing the NH2-termi-
nal 147 residues of MLCK.
612 The Journal of Cell Biology
Volume 154, 2001
multiple promoters in the gene (Kamm and Stull, 2001).
Short MLCK is present in smooth muscle cells at
(Hartshorne, 1987; Tansey et
molar ratio to myosin of 1:10 and to actin in filaments of
1:100. The short MLCK isoform found in smooth muscle
and some nonmuscle cells possesses an NH
binding extension (Kanoh et al., 1993; Gallagher and Stull,
1997; Ye et al., 1997), not present in skeletal muscle MLCK
(Nunnally and Stull, 1984; Lin et al., 1997), that localizes
the kinase to thin filaments, restricting its cellular mobility
al., 1994) in an approximate
(Guerriero et al., 1981; Dabrowska et al., 1982; Sellers and
Pato, 1984). The long MLCK is the same as the short
MLCK with additional actin-binding motifs and structural
modules at the NH
terminus. The long MLCK has a
greater affinity for F-actin and is found primarily in non-
muscle cells (Kamm and Stull, 2001), where myosin II–
based motility is also regulated by light chain phosphoryla-
tion. The resulting compartmentalization of MLCK may be
involved in defining contractile regions in both smooth
muscle and nonmuscle cells.
-terminal extensions contain a novel high-affin-
ity actin-binding sequence, DFRXXL, which repeats three
times in short MLCK, leading to binding saturation of one
MLCK to three actin monomers along F-actin in vitro
(Smith et al., 1999; Smith and Stull, 2000). Mutating the
binding motifs significantly decreases association of MLCK
with myofilaments in vitro and with actin-containing fila-
ments in intact smooth muscle cells (Smith et al.., 1999).
The full-length short kinase is an elongated molecule, and
intervening PEVK, Ig-like, and fibronectin-like repeats be-
-terminal and more COOH-terminal catalytic
domains are thought to provide sufficient extension for the
catalytic core to reach thick filaments abutting thin fila-
ments (Stull et al., 1996; Lin et al., 1999; Smith and Stull,
2000), thus allowing effective light chain phosphorylation of
cross-bridges. A COOH-terminal low-affinity myosin-bind-
ing fragment in MLCK (Sellers and Pato, 1984) may direct
the kinase towards myosin filaments, increasing the proba-
bility of myosin phosphorylation during activity, whereas
terminus binds with sufficient high affinity to an-
chor the kinase to actin filaments. Additionally, since
MLCK apparently is not dissociated from thin filaments by
or Ca –calmodulin (Lin et al., 1999), the enzyme will
localize close to thin and thick filaments both at the onset
and during contractile activity.
Actin, the core of the thin filament, serves as a molecular
track for the myosin cross-bridge motor. Tropomyosin, also
present on thin filaments, ensures that on–off switching of
myosin ATPase and consequently contractility occurs coop-
(a) Rabbit skeletal muscle F-actin alone (two examples). (b) Skeletal
muscle F-actin–MLCK-147 (five examples). Note the increased di-
ameter of the decorated F-actin. Bar, 50 nm.
Electron micrographs of negatively stained filaments.
tions) of 3-D reconstructions. (a) F-actin
control. Because adjacent actin mono-
mers are staggered by ?1/2 ? 55 Å,
sectioning the reconstruction through
the widest part of one actin (monomer
on the right) results in sectioning through
a narrower part of the adjacent one (on
the left); outer and inner domains are
marked Ao and Ai on each. (b) F-actin–
MLCK-147; note the extra density (arrow)
due to MLCK on the extreme periphery
of the map bridging inner and outer
domains of the two actin monomers
sectioned. (c) Maps in a and b were
compared, and the difference between
the two (filled black region) was super-
imposed on a copy of the F-actin map. The difference densities, attributable to the bound MLCK, were statistically significant at ?99.95%
confidence levels. This analysis also showed that the protein-free pore between the MLCK density and F-actin (* in b; cross-hatching in c) was
negatively significant, as if decorated filaments drew in extra stain in this region. Sections shown are at the same axial position and have the
same relative orientation. The average phase residual (? ? SD), a measurement of the geometrical agreement among filaments generating re-
constructions, was 54.0 ? 5.9? for the F-actin–MLCK-147 data. The average up–down phase residual (?? ? SD), a measure of filament
polarity, was 23.7 ? 8.5?. The two values were comparable to those previously obtained.
Transverse sections (z-sec-
Myosin light chain kinase binding to F-actin |
Hatch et al. 613
eratively, narrowing the Ca
for regulation (Lehman et al., 2000). Smooth muscle and
nonmuscle thin filaments also contain the protein caldesmon
and smooth muscle filaments calponin, both often implicated
in the regulation of thin filaments but of uncertain function
despite extensive biochemical characterization (Marston and
Huber, 1996; Chalovich et al., 1998; Marston et al., 1998;
Winder et al., 1998). Structural information on the interac-
tions of each of these proteins with F-actin has been extracted
from three-dimensional (3-D) reconstructions (Vibert et al.,
1993; Whittaker et al., 1995; Hodgkinson et al., 1997a,b;
Lehman et al., 1997; Volkmann et al., 2000). Together, these
proteins and myosin cover and/or move over much of the
outer face of actin, leaving little room free for extra binding
proteins, hence raising the possibility of a structural competi-
tion and functional clash with actin-bound MLCK. In the
current report, the interactions of MLCK on F-actin were ex-
amined structurally. The site of MLCK binding was deter-
mined by electron microscopy and 3-D reconstruction to as-
concentration range required
sess if MLCK is specifically and uniquely localized on
F-actin. We found that MLCK associates between laterally
adjacent actin monomers on F-actin, over an unoccupied
patch far from the myosin-binding site and from sites occu-
pied by tropomyosin, caldesmon, and calponin on actin.
Hence, MLCK binding should not interfere sterically with
myosin cross-bridge cycling, cooperative activation by tro-
pomyosin, or the operation of caldesmon or calponin.
Results and discussion
Electron microscopy of F-actin–MLCK-147 complexes
F-actin was complexed with peptide containing the NH
terminal 147 residues of MLCK (MLCK-147) under condi-
tions that should saturate filaments with the protein. Elec-
tron micrographs of negatively stained filaments showed
that the MLCK peptide caused extensive cross-linking of
F-actin into loosely packed bundles and rafts. Only unbun-
dled filaments within aggregates or those splaying off later-
ally were analyzed. Actin substructure, although evident, was
frequently obscured by the binding of the MLCK-147 on
the surface of filaments (Fig. 1), which also caused them to
appear wider than pure F-actin. Globular structures were oc-
casionally seen projecting from filaments but details of the
shape, orientation, and periodicity of the MLCK peptide
were not discernable. To detect the MLCK binding and de-
termine its position on F-actin, image processing and 3-D
reconstruction were therefore necessary.
3-D reconstructions of reconstituted thin filaments
Electron micrographs of two different sets of F-actin–
MLCK-147 were analyzed independently by the first and
last authors. Density maps of reconstituted filaments were
calculated from the averages of the Fourier transform layer
line data (not shown). All maps obtained showed typical
two-domain actin monomers that could be further divided
into identifiable subdomains (Figs. 2 and 3). When com-
pared with maps generated from pure F-actin controls, each
separately calculated reconstruction showed obvious extra
density lying between the inner domain of one actin and the
outer domain of the next monomer along the left-handed
genetic helix of F-actin (Figs. 2 and 3, arrows). Since the two
data sets were so similar, they were combined for further
analysis. The extra mass, attributable to the MLCK peptide,
and its location became especially apparent when difference
densities computed between maps of F-actin–MLCK-147
and F-actin were superimposed on the F-actin reconstruc-
tions (filled black region in Figs. 2 and 3, respectively). The
computed difference densities were statistically significant at
99.95%. The statistical analysis con-
firmed that the only significant difference between the con-
trol and MLCK maps was the mass bridging successive
monomers. Inspection of the surface views of reconstruc-
tions (Fig. 3) showed that part of the MLCK density is
connected to a relatively broad part of the back of actin sub-
domain-1. The opposite side of MLCK approaches a protu-
berance on the bottom of subdomain-4 of the next actin
monomer along the F-actin genetic helix (subdomains noted
on four successive actin monomers). The MLCK density
therefore appears to link respective monomers together.
the position of the MLCK-147 peptide on F-actin. (a) F-actin control;
actin subdomains are noted on four of the successive actin monomers
(subscripts distinguish the particular monomer labeled; monomers
with odd-numbered subscripts present face-on views of actin, and
ones with even-numbered subscripts show reverse-side views).
(b) MLCK-147–decorated F-actin; note the extra mass (arrows) not
present in control filaments that link the bottom of subdomain-4 of
each actin monomer to the back of subdomain-1 of the adjacent
one. (c) To further define the relative position of MLCK-147, differ-
ence densities (filled black region) were calculated as described
above and then superimposed on the F-actin reconstruction. Again,
note the position of the difference density bridging between neigh-
boring monomers and then extending downward toward the barbed
end of the filament. Connectivity was not noted between axially
adjacent MLCK-147 densities, and therefore the results did not allow
us to determine if the MLCK peptide is arranged longitudinally
along F-actin as suggested by binding studies (Smith and Stull,
2000). However, any possible linkage, predicted by Smith and Stull
(2000), between successive binding domains may have been too
thin or too disordered to be detected by the methods applied.
Surface views of thin filament reconstructions showing
614 The Journal of Cell Biology
Volume 154, 2001
Fitting MLCK on the atomic model of F-actin
To further define the MLCK-147 binding site on F-actin, the
MLCK difference densities were localized on the refined
atomic model of F-actin (Lorenz et al., 1993) by fitting the
atomic model within the envelope formed by the actin compo-
nent of our reconstructions (compare with Vibert et al., 1997).
The fitting (Fig. 4) indicated that one side of MLCK-147 (red)
attached to COOH-terminal residues on subdomain-1 of one
actin monomer and that the opposite side of the peptide
closely approached an
-helix (representing actin residues
228–232) protruding from subdomain-4 of the neighboring
monomer. The MLCK binding site is distinct from consensus
actin binding sites previously observed (McGough, 1998).
Molecular model of MLCK–actin binding
Circular dichroism studies indicate that MLCK-147 is
largely unstructured without
(unpublished data). Despite this, our observations here indi-
cate that, once associated with F-actin, MLCK-147 assumed
a fairly compact shape, as if a structured conformation was
induced by binding to actin. Further inspection of the resi-
dues involved in the docking site of MLCK on actin re-
vealed a stretch of charged and hydrophobic residues at the
COOH-terminal region of F-actin complementary to those
in the MLCK-147 binding motif, suggesting that binding
might occur as illustrated in the model in Fig. 5, in which
the DFRXXL residues of MLCK-147 associate with the
Arg374, Val372, Glu366, and Tyr364 of actin, respectively.
Comparison of binding of MLCK and other
The amount of MLCK in smooth muscle and nonmuscle
cells is substantially less than the amount of actin-binding
sites (Hartshorne, 1987; Stull et al., 1998), and it is therefore
likely that MLCK will be targeted to actin filaments partici-
pating in myosin II–based motility (compare with North et
al., 1994a; Kamm and Stull, 2001). These filaments contain
several actin-binding proteins involved in regulating thin fila-
ment function that could potentially compete with MLCK
for actin-binding sites. Since the locations of all major
smooth muscle and key nonmuscle thin filament-associated
proteins are known, their binding and that of MLCK could
be compared structurally. Inspection of Fig. 6, a composite
of difference density maps localizing the myosin binding site
and positions of tropomyosin, caldesmon, calponin, and
of F-actin. The atomic model of F-actin (Lorenz et al., 1993) was fit-
ted to the EM reconstructions as previously described (Vibert et al.,
1997), and the location of the MLCK difference density on the atomic
model was established. Two neighboring actin monomers are dis-
played as ?-carbon chains: one in green (face-on view of actin) and
the other in yellow (reverse-side view; note that these two monomers
correspond to actin monomers 1 and 2 in Fig. 3). The fitted MLCK
density (red) approaches an ?-helix projecting from actin subdomain-4
on one monomer (residues 228–232 highlighted in magenta) and
comes into close contact with the COOH-terminal ?-helix of actin
subdomain-1 on the other (residues 364–375 highlighted in white).
Without an atomic model of MLCK-147 available as a reference for
molecular fitting, an exact description of the volume, shape, orienta-
tion, and contact points of the MLCK density on F-actin is precluded
by the resolution and positional accuracy of the data.
Location of MLCK-147 densities on the atomic structure
interaction with the MLCK-binding motif. The amino acid sequence
and 3-D structure of chicken aortic actin were evaluated in light of
both the molecular fitting depicted in Fig. 4 and data on the
DFRXXL binding motif. The space-filling model shows that the
COOH-terminal actin residues Arg374 (green), Val372 (yellow),
Glu366 (magenta), and Tyr364 (off-white) on the actin surface
could make alternating electrostatic and hydrophobic interactions
with DFRXXL (drawn schematically). If this were true, then the
apparent binding to residues 228–232 of a laterally adjacent actin
monomer (as in Fig. 4) would occur with flanking amino acids of
the MLCK-147 construct.
Space-filling model of actin showing possible sites of
Myosin light chain kinase binding to F-actin |
Hatch et al. 615
MLCK-147 on F-actin, demonstrates that the MLCK dock-
ing site is distinct from that of the others. The absence of any
steric clash between the binding of MLCK and the other ac-
tin-binding proteins would favor the stability of the MLCK
interaction. Moreover, the unique binding of MLCK would
not interfere with myosin docking and cycling on actin. It is
noteworthy that caldesmon and calponin densities do over-
lap structurally with each other (Fig. 6), consistent with sig-
nificant binding competition of caldesmon and calponin ob-
served in vitro and histological segregation in vivo (Fürst et
al., 1986; Small et al., 1986; Lehman, 1991; Makuch et al.,
1991; North et al., 1994a,b); in contrast, MLCK binding
to native smooth muscle thin filaments is not blocked by
the presence of tropomyosin, caldesmon, and/or calponin
(Smith et al., 1999). Together, studies on the structure of
smooth muscle thin filaments demonstrate that actin-bind-
ing proteins adopt characteristic positions on the thin fila-
ment consistent with distinct functions. In the case of the
short MLCK in smooth muscle, its NH
domain is anchored to a site where no other actin-binding
protein to date has been localized (compare with
1998). The long MLCK present in nonmuscle cells has two
additional DFRXXL actin-binding motifs, and thus, it is pre-
dicted that its location will be similar except for a maximal
stoichiometry of one long MLCK per five actin monomers in
the thin filament. In both the short and long MLCKs, the in-
tervening PEVK, Ig-like, and fibronectin modules would ex-
tend the catalytic core to myosin thick filaments, thereby
allowing regulatory light chain phosphorylation upon activa-
tion by Ca
–calmodulin. The design of the MLCK mole-
cule is therefore well adapted for its part in the cascade of
events leading to myosin II–based cellular motility.
Materials and methods
Construction and expression of MLCK-147: the cDNA fragment of the
-terminal 1–147 amino acids of rabbit smooth muscle MLCK was am-
plified by PCR and subcloned into TOPO cloning vector pCR 2.1 (Invitro-
gen). After sequencing, the cDNA was then subcloned into the expression
vector pET 28a (Novagen) with a His-tag sequence at the NH
MLCK-147. The expression constructs were transformed into bacteria
strain BL21 (DE3), and protein expression was induced by IPTG. After cen-
trifugation, the cells were resuspended in 20 mM Tris/HCl, pH 7.0, 1 mM
EDTA, and 0.1% NP-40, sonicated, and centrifuged again. The soluble
MLCK-147 peptide was purified by His-Bind Quick column as described
by the manufacturer (Novagen). The purified MLCK-147 binds to F-actin
value of 0.26
M and a stoichiometry of one MLCK-147
to three actin molecules. MLCK-147 contains the three DFRXXL motifs
present in the 75–amino acid NH
-terminal fragment studied by Smith and
Rabbit skeletal muscle F-actin was purified by standard methods (Spu-
dich and Watt, 1971). The F-actin used was stable and did not aggregate,
an advantage for the structural studies performed. Different actin isoforms
are highly conserved (we used skeletal muscle
form variability, in fact, occurs mainly at the acidic NH
amino acids of actin (Vanderkerckhove and Weber, 1978; Lehman et al.,
1996) whose position is distal from the site of MLCK-147 binding (see Re-
sults and discussion). There were no significant differences in binding af-
finity of NH
-terminal MLCK fragments to skeletal muscle F-actin and de-
tergent washed native smooth muscle myofilaments; the stoichiometry of
binding was the same (Smith and Stull, 2000; unpublished data).
-actin in our studies); iso-
Electron microscopy and helical reconstruction
M) was decorated with excess MLCK-147 (2
50 mM KCl, 1 mM EGTA, 1 mM MgCl
imidazole buffer (pH 7.2) at room temperature (
ments were immediately applied to carbon-coated electron microscope
grids and negatively stained as previously described (Moody et al., 1990).
Prolonged decoration was avoided, since F-actin and MLCK-147 incubated
together for as short as 5 min formed very large and unusable filament ag-
gregates. Electron micrograph images of decorated filaments were recorded
on a Philips CM120 electron microscope at 60,000
low-dose conditions (
12e /Å). Micrographs were digitized using a ZEISS
SCAI scanner at a pixel size corresponding to 0.7 nm in the filaments. Well-
stained regions of filaments were selected and straightened as previously
described (Egelman, 1986; Hodgkinson et al., 1997a). Helical reconstruc-
tion was carried out by standard methods (DeRosier and Moore, 1970;
Amos and Klug, 1975; Owen et al., 1996) as previously described (Vibert et
al., 1993, 1997). Two independently analyzed sets of filaments totaling 28
filaments were chosen for averaging, based on their relative phase agree-
ment. The reconstruction had a resolution (Owen and DeRosier, 1993) of
2.5–3.0 nm; the positional accuracy of the method is
al., 1990). Difference density analysis to define MLCK position on actin was
carried out as previously described (Xu et al., 1999), and differences be-
tween maps were evaluated statistically using a Student’s
and Flicker, 1987; Trachtenberg and DeRosier, 1987). Fitting of reconstruc-
tions to the atomic resolution structure of F-actin (Lorenz et al., 1993) was
carried out as previously described (McGough and Way, 1995; Vibert et al.,
1997; Xu et al., 1999) using the program O (Jones et al., 1991).
M) in solutions of
, 0.2 mM ATP, 1 mM DTT, 10 mM
25°C). Reconstituted fila-
0.5 nm (Milligan et
t test (Milligan
This work was supported by National Institutes of Health grants HL-36153
(to W. Lehman), HL-26043 (to J.T. Stull), and HL-62468 (to R. Craig) and
by Shared Instrumentation grant RR-08426 (to R. Craig).
Submitted: 15 May 2001
Revised: 13 June 2001
Accepted: 3 July 2001
Amos, L.A., and A. Klug. 1975. Three-dimensional image reconstruction of the
contractile tail of T4 bacteriophage.
Chalovich, J.M., A. Sen, A. Resetar, B. Leinweber, R.S. Fredricksen, F. Lu, and
Y.-D. Chen. 1998. Caldesmon: binding to actin and myosin and effects on
the elementary steps in the ATPase cycle.
Cohen, P. 2000. The regulation of multisite phosphorylation—a 25 year update.
Trends Biochem. Sci. 25:596–601.
Dabrowska, R., S. Hinkins, M.P. Walsh, and D.J. Hartshorne. 1982. The binding
J. Mol. Biol. 99:51–73.
Acta Physiol. Scand. 164:427–435.
muscle actin-binding proteins. Binding locations of MLCK-147 (red),
smooth muscle tropomyosin (TM, green; data from Lehman et al.,
2000), calponin (CP, magenta; data from Hodgkinson et al., 1997a),
and caldesmon (CD, yellow; derived from selected data in
Hodgkinson et al., 1997b) were plotted on a transverse section of
F-actin. The myosin-binding site on actin (from data of S-1–decorated
thin filaments in Milligan et al., 1990; Vibert et al., 1997) is high-
lighted (MY, ochre). Note that the MLCK-binding site is far from
each of the other actin-binding proteins.
Composite map displaying the relative positions of smooth
616 The Journal of Cell Biology
Volume 154, 2001
of smooth muscle myosin light chain kinase to actin.
DeRosier, D.J., and P.B. Moore. 1970. Reconstruction of three-dimensional im-
ages from electron micrographs of structures with helical symmetry.
Egelman, E.H. 1986. An algorithm for straightening images of curved filamentous
Erdödi, F., M. Ito, and D.J. Hartshorne. 1996. Myosin light chain phosphatase.
Biochemistry of Smooth Muscle Contraction, M. Bárány, editor. Academic
Press, San Diego. 131–141.
Fürst, D.O., R.A. Cross, J. DeMey, and J.V. Small. 1986. Caldesmon is an elon-
gated flexible molecule localized in the actomyosin domains of smooth mus-
EMBO J. 5:251–257.
Gallagher, P.J., and J.T. Stull. 1997. Localization of an actin binding domain in
smooth muscle myosin light chain kinase.
Guerriero, V., D.R. Rowley, and A.R. Means. 1981. Production and characteriza-
tion of an antibody to myosin light chain kinase and intracellular localiza-
tion of the enzyme.
Hartshorne, D.J. 1987. Biochemistry of the contractile process in smooth muscle.
In Physiology of the Gastrointestinal Tract, L.R. Johnson, editor. Raven
Press, New York. 423–482.
Herring, B.P., S. Dixon, and P.J. Gallagher. 2000. Smooth muscle myosin light
chain kinase expression in cardiac and skeletal muscle.
Hodgkinson, J.L., M. EL-Mezgueldi, R. Craig, P. Vibert, S.B. Marston, and W.
Lehman. 1997a. 3-D image reconstruction of reconstituted smooth muscle
thin filaments containing calponin: visualization of interactions between
F-actin and calponin.
J. Mol. Biol. 273:150–159.
Hodgkinson, J.L., S.B. Marston, R. Craig, P. Vibert, and W. Lehman. 1997b.
Three-dimensional image reconstruction of reconstituted smooth muscle
thin filaments: effects of caldesmon.
Jones, T.A., J.Y. Zou, S.W. Cowan, and M. Kjeldgaard. 1991. Improved methods
for the building of protein models in electron density maps and the location
of errors in these models.
Kamm, K., and J.T. Stull. 1985. The function of myosin and myosin light chain
kinase phosphorylation in smooth muscle.
Kamm, K.E., and J.T. Stull. 2001. Dedicated myosin light chain kinases with di-
verse cellular functions.
J. Biol. Chem
Kanoh, S., M. Ito, E. Niwa, Y. Kawano, and D.J. Hartshorne. 1993. Actin-binding
peptide from smooth muscle myosin light chain kinase.
Lehman, W. 1991. Calponin and the composition of smooth muscle thin fila-
ments. J. Muscle Res. Cell Motility. 12:221–224.
Lehman, W., P. Vibert, R. Craig, and M. Bárány. 1996. Actin and the structure of
thin filaments. In Biochemistry of Smooth Muscle Contraction. M. Bárány,
editor. Academic Press, San Diego. 47–62.
Lehman, W., P. Vibert, and R. Craig. 1997. Visualization of caldesmon on smooth
muscle thin filaments. J. Mol. Biol. 274:310–317.
Lehman, W., V. Hatch, V. Korman, M. Rosol, L. Thomas, R. Maytum, M.A.
Geeves, J.E. Van Eyk, L.S. Tobacman, and R. Craig. 2000. Tropomyosin
isoforms modulate the localization of tropomyosin strands on actin fila-
ments. J. Mol. Biol. 302:593–606.
Lin, P.-J., K. Luby-Phelps, and J.T. Stull. 1997. Binding of myosin light chain ki-
nase to cellular actin-myosin filaments. J. Biol. Chem. 272:7412–7420.
Lin, P.-J., K. Luby-Phelps, and J.T. Stull. 1999. Properties of filament-bound my-
osin light chain kinase. J. Biol. Chem. 274:5987–5994.
Lorenz, M., D. Popp, and K.C. Holmes. 1993. Refinement of the F-actin model
against x-ray fiber diffraction data by use of a directed mutation algorithm. J.
Mol. Biol. 234:826–836.
MacDonald, J.A., M.A. Borman, A. Muranyi, A.V. Somlyo, D.J. Hartshorne, and
T.A. Haystead. 2001. Identification of the endogenous smooth muscle
phosphatase-associated kinase. Proc. Natl. Acad. Sci. USA. 98:2419–2424.
Makuch, R., K. Birukov, V. Shirinsky, and D. Dabrowska. 1991. Functional inter-
relationship between calponin and caldesmon. Biochem. J. 280:33–38.
Marston, S.B., and P.A.J. Huber. 1996. Caldesmon. In Biochemistry of Smooth
Muscle Contraction. M. Bárány, editor. Academic Press, San Diego, CA.
Marston, S.B., D. Burton, O. Copeland, I. Fraser, Y. Gao, J. Hodgkinson, P. Hu-
ber, B. Levine, M. El-Mezgueldi, and G. Notarianni. 1998. Structural inter-
actions between actin, tropomyosin, caldesmon and calcium binding protein
and the regulation of smooth muscle thin filaments. Acta Physiol. Scand.
Biochem. Biophys. Res.
Mol. Cell. Biochem. 173:1–57.
Am. J. Physiol. 279:
Biophys. J. 72:2398–2404.
Annu. Rev. Pharmacol. Toxicol.
McGough, A. 1998. F-actin-binding proteins. Current Opin. Struct. Biol. 8:166–
McGough, A., and M. Way. 1995. Molecular model of an actin filament capped
by a severing protein. J. Struct. Biol. 115:144–150.
Milligan, R.A., and P.F. Flicker. 1987. Structural relationships of actin, myosin,
and tropomyosin revealed by cryo-electron microscopy. J. Cell Biol. 105:29–
Milligan, R.A., M. Whittaker, and D. Safer. 1990. Molecular structure of F-actin
and the location of surface binding sites. Nature. 348:217–221.
Moody, C., W. Lehman, and R. Craig. 1990. Caldesmon and the structure of ver-
tebrate smooth muscle thin filaments: electron microscopy of isolated thin
filaments. J. Muscle Res. Cell Motility. 11:176–185.
North, A.J., M. Gimona, Z. Lando, and J.V. Small. 1994a. Actin isoform compart-
ments in chicken gizzard smooth muscle cells. J. Cell Sci. 107:445–455.
North, A.J., M. Gimona, R.A. Cross, Z. Lando, and J.V. Small. 1994b. Calponin
is localised in both the contractile apparatus and the cytoskeleton of smooth
muscle cells. J. Cell Sci. 107:437–444.
Nunnally, M., and J.T. Stull. 1984. Mammalian skeletal muscle myosin light chain
kinases: a comparison by antiserum cross-reactivity. J. Biol. Chem. 259:
Owen, C., and D.J. DeRosier. 1993. A 13-Å map of the actin–scruin filament
from the Limulus acrosomal process. J. Cell Biol. 123:337–344.
Owen, C., D.G. Morgan, and D.J. DeRosier. 1996. Image analysis of helical ob-
jects: the Brandeis helical package. J. Struct. Biol. 116:167–175.
Perry, S.V. 1983. Phosphorylation of the myofibrillar proteins and the regulation
of contractile activity in muscle. Philos. Trans. Roy. Soc. B. 302:59–71.
Sellers, J.R., and R.S. Adelstein. 1986. Regulation of contractile activity. In The
Enzymes. P.D. Boyer and E.G. Krebs, editors. Academic Press, Orlando,
Sellers, J.R., and M.D. Pato. 1984. The binding of smooth muscle myosin light
chain kinase and phosphatases to actin and myosin. J. Biol. Chem. 259:740–
Small, J.V., D.O. Fürst, and J. DeMey. 1986. Localization of filamin in smooth
muscle. J. Cell Biol. 102:210–220.
Smith, L., and J.T. Stull. 2000. Myosin light chain kinase binding to actin fila-
ments. FEBS Lett. 480:298–300.
Smith, L., X. Su, P.-J. Lin, and J.T. Stull. 1999. Identification of a novel actin
binding motif in smooth muscle light chain kinase. J. Biol. Chem. 274:
Somlyo, A.P., and A.V. Somlyo. 2000. Signal transduction by G-proteins, rho-
kinase and protein phosphatase to smooth muscle and non-muscle myosin
II. J. Physiol. 522:177–185.
Spudich, J.A., and S. Watt. 1971. The regulation of rabbit skeletal muscle contrac-
tion. I. Biochemical studies of the interaction of the tropomyosin-troponin
complex with actin and the proteolytic fragments of myosin. J. Biol. Chem.
Stull, J.T., J.K. Kruegger, K.E. Kamm, Z.-H. Gao, G. Zhi, and R. Padre. 1996.
Myosin light chain kinase. In Biochemistry of Smooth Muscle Contraction.
M. Bárány, editor. Academic Press, San Diego. 119–130.
Stull, J.T., K.E. Kamm, J.K. Krueger, P. Lin, K. Luby-Phelps, and G. Zhi. 1997.
Ca2?/calmodulin-dependent myosin light-chain kinases. Adv. Second. Mes-
senger. Phosphoprotein Res. 31:141–150.
Stull, J.T., P.-J. Lin, J.K. Kreuger, J. Trewhella, and G. Zhi. 1998. Myosin light
chain kinase: functional domains and structural motifs. Acta Physiol. Scand.
Tansey, M.G., K. Luby-Phelps, K.E. Kamm, and J.T. Stull. 1994. Ca2?-dependent
phosphorylation of myosin light chain kinase decreases the Ca2? sensitivity
of light chain phosphorylation within smooth muscle cells. J. Biol. Chem.
Trachtenberg, S., and D.J. DeRosier. 1987. Three-dimensional structure of the
frozen-hydrated flagellar filament: the left-handed filament of Salmonella ty-
phimurium. J. Mol. Biol. 195:581–601.
Vanderkerckhove, J., and K. Weber. 1978. At least 6 different actins are expressed
in a higher mammal. J. Mol. Biol. 126:783–802.
Vibert, P., R. Craig, and W. Lehman. 1993. Three-dimensional reconstruction of
caldesmon-containing smooth muscle thin filaments. J. Cell Biol. 123:313–
Vibert, P., R. Craig, and W. Lehman. 1997. Steric-model for activation of muscle
thin filaments. J. Mol. Biol. 266:8–14.
Volkmann, N., D. Hanein, G. Ouyang, K.M. Trybus, D.J. DeRosier, and S.
Lowey. 2000. Evidence for cleft closure in actomyosin upon ADP release.
Nat. Struct. Biol. 7:1147–1155.
Whittaker, M., E.M. Wilson-Kubalek, J.E. Smith, L. Faust, R.A. Milligan, and
Myosin light chain kinase binding to F-actin | Hatch et al. 617
H.L. Sweeney. 1995. A 35-movement of smooth muscle myosin on ADP re-
lease. Nature. 378:748– 751.
Winder, S.J., B.G. Allen, O. Clement-Chomienne, and M.P. Walsh. 1998. Regu-
lation of smooth muscle actin-myosin interaction and force by calponin.
Acta Physiol. Scand. 164:415–426.
Xu, C., R. Craig, L. Tobacman, R. Horowitz, and W. Lehman. 1999. Tropomyo-
sin positions in regulated thin filaments revealed by cryoelectron micros-
copy. Biophys. J. 77:985–992.
Ye, L.-H., K. Hayakawa, H. Kishi, M. Imamura, A. Nakamura, T. Okagaki, T.
Takagi, A. Iwata, T. Tanaka, and K. Kohama. 1997. The structure and
function of the actin-binding domain of myosin light chain kinase of
smooth muscle. J. Biol. Chem. 272:32182–32189.