From the Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970
Received for publication, October 2, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Myosin heavy chain kinase (MHCK) A phosphorylates
mapped sites at the C-terminal tail of Dictyostelium myosin
II heavy chain, driving disassembly of myosin filaments both in
vitro and in vivo. MHCK A is organized into three
functional domains that include an N-terminal coiled-coil region, a
central kinase catalytic domain unrelated to conventional protein
kinases, and a WD repeat domain at the C terminus. MHCK B is a
homologue of MHCK A that possesses structurally related catalytic and
WD repeat domains. In the current study, we explored the role of the WD
repeat domains in defining the activities of both MHCK A and MHCK B
using recombinant bacterially expressed truncations of these kinases
either with or without their WD repeat domains. We demonstrate that
substrate targeting is a conserved function of the WD repeat domains of
both MHCK A and MHCK B and that this targeting is specific for
Dictyostelium myosin II filaments. We also show that the
mechanism of targeting involves direct binding of the WD repeat domains
to the myosin substrate. To our knowledge, this is the first report of
WD repeat domains physically targeting attached kinase domains to their substrates. The examples presented here may serve as a paradigm for
enzyme targeting in other systems.
Complex cellular processes such as cytokinesis, cell migration,
and receptor capping depend on the ability of myosin II (conventional myosin) to bring about contraction at specific locations within the
cell (1). In Dictyostelium, myosin II undergoes a dynamic assembly/disassembly process that permits its specific spatial and
temporal localization within the cell (2). In response to extracellular
chemoattractants such as cAMP or folic acid, myosin II is recruited
from the cytoplasm to the cell cortex in a process that involves myosin
II bipolar filament assembly (3-5).
MHC1 phosphorylation is the
major route by which myosin filament assembly is controlled in
Dictyostelium. Phosphorylation of mapped sites at threonine
residues 1823, 1833, and 2029 of MHC leads to disassembly of filaments
both in vitro and in vivo (6-9). The importance
of these target sites in controlling myosin filament assembly has been
demonstrated from studies showing that conversion of these threonines
to aspartic acid residues creates a "pseudophosphorylated" myosin
that is unable to assemble into the cytoskeleton. Conversely, myosin
overassembles into the cytoskeleton if these regulatory sites are
either removed by truncation of MHC (10) or converted to
nonphosphorylatable alanine residues (9).
MHC phosphorylation appears to be regulated in Dictyostelium
through the combined activities of at least one MHC phosphatase (11)
and a group of MHC kinases (2, 12). Dictyostelium MHC kinase
(MHCK) A was the first of the heavy chain kinases to be purified.
Subsequent characterization of purified MHCK A revealed that its
catalytic activity can drive myosin filament disassembly in
vitro by phosphorylating MHC (13). Cloning and sequence analysis of the MHCK A gene has revealed that the catalytic domain of MHCK A is
unrelated to any of the conventional protein kinases. As a result, MHCK
A has become the prototype defining a new family of protein kinases
(14, 15). A structurally related kinase, MHCK B, has been identified
and cloned from Dictyostelium via sequence homology to the
catalytic domain of MHCK A. Although native MHCK B has not yet been
purified, a bacterially expressed truncation of MHCK B phosphorylates
MHC preferentially at the mapped regulatory sites discussed above (16).
Another Dictyostelium homologue of MHCK A that has been
identified by cDNA and genomic sequencing has been named MHCK C,
although it has not yet been characterized at the protein level
(GenBankTM accession number AF079447).
The MHCK A group of kinases in Dictyostelium share a
conserved domain structure consisting of the novel kinase catalytic
domain and a C-terminal domain containing seven tandem copies of the WD
repeat motif. The WD repeat is a conserved amino acid motif that recurs
four to sixteen times and is found in a large and heterogeneous family
of proteins. The general makeup of the repeating unit, although not
absolutely conserved among WD repeats, begins with a pair of GH
residues followed by a variable core of about 36-46 amino acids and
ends with the WD residues (17). The crystal structure of the
Functionally, the WD repeat class of proteins has been implicated in
the regulation of a number of cellular functions such as signal
transduction, RNA processing, gene regulation, vesicular traffic, and
cytoskeletal assembly; however, no common function or interaction has
been attributed to the WD repeat domain specifically (17). Insight on
the function of the WD repeat domain of MHCK A has been gained from a
previous study showing that a Dictyostelium-expressed truncation of MHCK A lacking its WD repeat domain (MHCK A- In the current report, we describe results from experiments exploring
the structure/function relationship between the conserved WD repeat
domains of MHCK A and MHCK B and the kinase activity of each enzyme.
For these studies, the catalytic domains of MHCK A and MHCK B, either
with or without their WD repeat domains, were expressed in bacteria as
recombinant glutathione S-transferase (GST) fusion proteins
and then assayed for kinase activity toward several different
substrates, including Dictyostelium myosin II. We found that
the WD repeat domains of both MHCK A and MHCK B stimulate kinase
activity toward myosin II substrate but not toward the other substrates
tested. These results provide strong support for the hypothesis that
MHCK B, like MHCK A, functions physiologically as an MHC kinase.
Binding experiments presented here reveal that the mechanism by which
the WD repeats promote kinase activity toward myosin substrate is
likely to involve a direct interaction with myosin II. To our
knowledge, this represents the first time a WD repeat domain has been
shown to physically target an attached kinase domain to its substrate.
Plasmid Constructs--
All DNA manipulations were carried out
using standard methods. For the MHCK A constructs, an intermediate
plasmid (pMEX) containing the entire MHCK A DNA coding region was used
to generate the inserts for the GST fusion vector. The numbers
corresponding to the restriction sites are derived from the MHCK A DNA
sequence (GenBankTM accession number P42527). For the
pGST-A-CAT construct, pMEX was digested with HindIII (base
pair 1493) and BstXI (base pair 2529), and the resultant
1036 base pair fragment was treated with Klenow polymerase (New England
Biolabs) to yield blunt ends. The pGST-A-CAT construct was completed by
ligating the 1036-base pair fragment into pGEX-2T (Amersham Pharmacia
Biotech) and then digesting with SmaI to generate an in
phase fusion with GST. The pGST-A-CATWD construct was made in a similar
manner, but the fragment was released from pMEX by digesting only with
HindIII and then treating the fragment with Klenow
polymerase to yield blunt ends. The upstream end is contained within a
bacterial polylinker downstream of the native MHCK A termination codon.
The 2.0-kilobase insert fragment was then ligated into pGEX-2T cut with
SmaI.
MHCK B constructs were made from an intermediate plasmid,
pMM3.1, which contains the full-length cDNA-coding region of MHCK B
(GenBankTM accession number P90648). pMM3.1 contains the
entire downstream end of MHCK B and has an EcoRI linker
inserted next to codon 13 at the upstream end of the MHCK B coding
region. For the pGST-B-CAT construct, a 1.4-kilobase insert fragment
was isolated from pMM3.1 using an internal EcoRV site
(nt1391) that lies 3' of the catalytic domain of MHCK B and an
XbaI site in the upstream polylinker of pMM3.1. The
XbaI site was made blunt by treatment with Klenow polymerase
and then ligated into pGEX-2T vector that had been digested with
EcoRI and made blunt. This ligation recreated the upstream
vector EcoRI site (junction of blunt EcoRI
ligated with blunt XbaI). This plasmid insert also contains
the EcoRI site that lies adjacent to codon 13 of the MHCK B
coding region. Digestion of this intermediate with EcoRI was
performed to cut at the upstream vector EcoRI site and at
the internal EcoRI site. Subsequent recircularization yielded pGST-B-CAT, which has codon 13 of MHCK B fused directly to the
upstream GST domain encoded by the vector and an artificial stop codon
following residue 459 of MHCK B. This stop codon lies at a position
~110 amino acids downstream of the conserved catalytic domain portion
of the native protein. The pGST-B-CATWD construct was prepared in a
similar fashion. pMM3.1 was digested with EcoRI, releasing a
2.2-kilobase restriction fragment that has the entire downstream end of
the MHCK B coding region and an upstream EcoRI site adjacent
to codon 13 of MHCK B. This fragment was ligated into pGEX-2T to yield
pGST-B-CATWD.
A GST fusion protein containing a 15-kDa segment of the
Dictyostelium myosin II heavy chain was expressed in
Escherichia coli from the plasmid pGST2029. Construction of
pGST2029 involved the subcloning of a segment of myosin II tail
centered on the MHCK A target site mapped to position 2029 (7) into the
pGEX-3X (Amersham Pharmacia Biotech) expression plasmid. An 80-amino
acid region of the myosin II gene spanning residues 1985-2064 was
amplified using primers that introduced upstream and downstream
EcoRI restriction sites. The polymerase chain reaction
product was cut with EcoRI and ligated into pGEX-3X vector
digested with EcoRI. The resulting plasmid, pGST2029, was
then electroporated into E. coli cells (strain BL21-DE3,
Novagen) for expression.
Protein Purification--
The four GST fusion
proteins were purified in a similar manner. A 100-ml culture of
E. coli (BL21-DE3 strain, Novagen) containing each plasmid
was grown overnight in a 37 °C shaker incubator. This culture was
then used to inoculate (1:50 dilution) 1-2 liters of LB (Life
Technologies, Inc.) containing ampicillin (50 µg/ml; Roche Molecular
Biochemicals), and the cultures were grown at 37 °C to log phase
(A600 of 0.6-1.0). At this time, cultures were cooled to 25 °C, and then 1 mM
isopropyl-
All subsequent purification steps were performed at 4 °C. Lysozyme
(100 µg/ml, Sigma) was added to the resuspended cells. The cell
mixture was sonicated in an ice water bath, and the resulting lysate
was spun at 150,000 × g for 15 min to remove insoluble material. The cleared lysate was incubated with glutathione-Sepharose beads (Amersham Pharmacia Biotech) for 3 to 5 h at 4 °C. The
beads were then washed several times with buffer A to remove unbound material. For samples used in autophosphorylation experiments, a
dephosphorylation step was added by incubating (12 h at 4 °C) bead-bound fusion protein in Buffer A containing 50 units of calf intestinal alkaline phosphatase (Roche Molecular Biochemicals). After
incubation, calf intestinal alkaline phosphatase was removed by washing
the beads with Buffer A. Bound fusion protein was then eluted with
buffer A containing 30 mM glutathione (Sigma), and the
eluate was dialyzed against 12.5 mM Tris, pH 7.5, in 40%
glycerol. Aliquots were frozen on dry ice and then stored at
The GST-2029 fusion protein was prepared as described above but with
the following modifications. A 1-liter culture of E. coli
(BL21-DE3) cells containing the pGST2029 plasmid was grown to log phase
at 37 °C, and then expression of the GST-2029 fusion protein was
induced with 1 mM
isopropyl-
Myosin was purified by a modification of the method of Aguado-Velasco,
et al. (20), as has been described (11). Briefly, an initial
actomyosin precipitate was subjected to several assembly/disassembly cycles followed by a final gel filtration step to remove residual actin.
Phosphorylation Assays--
All kinase assays were performed at
25 °C in 10 mM TES, pH 7.0, 2 mM
MgCl2, 0.5 mM dithiothreitol, and 0.5 mM [
Unless indicated otherwise, each kinase was allowed to
auto-phosphorylate for 10 min at 25 °C before starting the kinase
reaction with the addition of substrate. Kinase reactions containing
MH-1 peptide substrate were terminated by blotting a fraction of the reaction mixture onto P-81 filter paper (Whatman) and then processing the filter for scintillation counting (22). For reactions in which the
substrate was either GST-2029 or myosin II, the reaction was stopped by
adding an equal volume of 2× SDS-PAGE sample buffer at each
time point, boiling for 2 min, and then performing SDS-PAGE on the
samples. The resulting gels were stained with Coomassie Blue, and
protein bands of interest were excised for scintillation counting to
quantify phosphate incorporation. Analysis of fusion protein
autophosphorylation was performed by incubating the kinase constructs
(200 nM) in the kinase reaction described above and then
blotting a fraction of the reaction mix to P-81 filter paper as
described for the peptide assay.
Protein Concentration Determination--
Purified
kinase constructs were subjected to SDS-PAGE with a series of known
amounts of BSA on the same gel. After Coomassie Blue staining, the
protein bands were quantified by scanning densitometry, and the
concentration of each purified fusion protein was determined by
comparing the densitometry values for the purified kinases with those
of the BSA standards in the same gel.
Myosin II Cosedimentation Assays--
Centrifuge tubes used
throughout these experiments were pretreated with Sigmacote (Sigma) and
3% BSA in 25 mM Tris, pH 7.5. Kinase fusion proteins used
in cosedimentation assays were precleared by centrifugation at
95,000 × g for 10 min in a Beckman Ti Tabletop Ultracentrifuge in a TLA 100.3 rotor. The resulting supernatants were
transferred to clean centrifuge tubes and then mixed with the other
binding assay components to give 40-µl reaction mixes containing 0.3 µM kinase fusion protein, 1.0 µM
Dictyostelium myosin II, 0.1% BSA, 2.5 mM TES,
pH 7.0, 40 mM KCl, 2 mM MgCl2, and
5% sucrose. Under these conditions, greater than 80% of the myosin II
is in filaments (25). After incubation for 10 min at 4 °C, the
binding reactions were centrifuged for 10 min at 95,000 × g, and equal volumes of pellet and supernatant fractions were resolved by SDS-PAGE. The myosin II and BSA were visualized by
Coomassie Blue staining of gels. The distribution of kinase fusion
proteins in pellets and supernatants was determined from Western blots
probed with anti-GST antibody (Upstate Biotechnology, NY). The relative
amounts of kinase fusion protein in the pellet and supernatant
fractions were quantified by densitometric analysis of the developed
Western blots.
Construction of MHCK A and MHCK B Truncations Expressed
and Purified as GST Fusion Proteins--
In earlier studies, domain
truncations of MHCK A were generated for expression in
Dictyostelium cells. Biochemical analysis of the purified
constructs revealed that the WD repeat domain of MHCK A was necessary
for efficient phosphorylation of intact myosin II but not for
phosphorylation of a myosin-based peptide (MH-1) substrate (19). In the
current study, we explored further the role of WD repeat domains in
defining the kinase activities of both MHCK A and its recently
identified homologue, MHCK B. For this study, truncated forms of both
kinases were constructed, expressed, and purified as GST fusion
proteins either lacking or containing their WD repeat domains (Fig.
1). The GST-A-CAT fusion protein
construct contains the minimal region of MHCK A that is necessary for
basal kinase catalytic activity (23). The GST-A-CATWD fusion protein
encompasses this segment and the C-terminal WD repeat domain of MHCK A. In contrast to MHCK A, the catalytic properties of MHCK B have not been
studied thoroughly, and nothing is known of the WD repeat domain
contribution to MHCK B kinase activity. Although a fine level mapping
of the catalytic domain of MHCK B has not been performed, comparison
with the MHCK A sequence suggests that the region of MHCK B necessary
for basal kinase catalytic activity is probably contained within
residues 100-340 (16). The GST-B-CAT fusion protein used in this study encompasses this region, and the GST-B-CATWD construct includes this
region as well as the more distal WD repeat domain of native MHCK B. All of the constructs described above were expressed in E. coli and purified to near homogeneity (Fig. 1).
The Role of the WD Repeat Domain in Defining MHCK A Catalytic
Activity toward Various Substrates--
The GST-A-CAT and GST-A-CATWD
constructs were assayed for kinase activity toward intact myosin II.
Initial experiments determining the time course of MHC phosphorylation
by each of the GST fusion proteins revealed that only the GST-A-CATWD
construct phosphorylated myosin efficiently (Fig.
2A). The GST-A-CAT construct
exhibited kinase activity that was barely detectable above background
levels. Similar results were obtained when GST-A-CAT and GST-A-CATWD
activities were analyzed over a range of myosin II concentrations (Fig.
2B). Although kinetic parameters for MHC phosphorylation
could not be determined because of the insolubility of myosin II
substrate at higher concentrations (
The recombinant GST-A-CAT and GST-A-CATWD fusion proteins were tested
next for kinase activity toward the MH-1 peptide. MH-1 is a previously
described (21) 16-residue peptide corresponding to the mapped MHCK A
target site at residue 2029 of MHC. Both GST-A-CAT and GST-A-CATWD
phosphorylated MH-1 substrate efficiently (Fig. 2C and Table
I) and exhibited specific activities
within an order of magnitude to those reported previously for both
native MHCK A (13) and recombinant MHCK A catalytic domain expressed as
a His6-tagged fusion protein (Table I) (23). Our results showing a lack of difference between the activities of GST-A-CAT and
GST-A-CATWD toward MH-1 demonstrate that the presence or absence of the
WD repeat domain does not affect the basic catalytic activity of MHCK
A. Moreover, these results, together with the myosin II phosphorylation
results described above, demonstrate unequivocally that the recombinant
WD repeat domain is necessary and sufficient to stimulate MHCK A
catalytic activity toward Dictyostelium myosin II.
The lack of WD repeat targeting to the MH-1 peptide suggests that
a larger structural target, perhaps filamentous myosin II, is needed
for the WD repeat domain stimulatory effect. To characterize further
the specificity and possible structural requirements for the WD repeat
domain effect on kinase activity, the GST-A-CAT and GST-A-CATWD fusion
proteins were tested for kinase activity toward a novel substrate,
GST-2029. This substrate contains a GST domain fused to a 15-kDa
segment of the Dictyostelium myosin II tail that is centered
on the mapped MHCK A phosphorylation site at residue 2029 of MHC.
GST-2029 lacks regions of the myosin tail necessary for bipolar
filament assembly (24); however, previous studies of
Dictyostelium myosin II, along with structure prediction
analysis of the GST-2029 sequence, indicate that this construct, unlike
the MH-1 peptide, is likely to form a coiled-coil dimer. Assays using
the GST-A-CAT and GST-A-CATWD constructs revealed that both fusion
proteins phosphorylated GST-2029 equally well with no significant
enhancement of activity by the WD repeat domain (Fig. 2D).
As with myosin II substrate, kinetic values could not be determined for
GST-2029 because this substrate was insoluble at concentrations
required to reach VMAX conditions. Our results showing
kinase activity toward MH-1 peptide and GST-2029 is unaffected by the
presence of the WD repeat domain indicate that the enhancement of
kinase activity by the WD repeat domain is specific to myosin II.
Characterization of MHCK B Catalytic Activity and Identification of
the Role of the WD Repeat Domain--
To assess whether the WD repeat
domain of MHCK B confers similar targeting properties toward myosin II,
the recombinant GST-B-CAT and GST-B-CATWD fusion proteins were assayed
for kinase activity toward myosin II filaments, MH-1 peptide, and
GST-2029. As was observed for the MHCK A-derived fusion proteins, the
GST-B-CAT and GST-B-CATWD constructs displayed nearly equivalent levels of activity toward MH-1 (Figs.
3C and Table I) and GST-2029
(Fig. 3D) substrates. However, when myosin II was used as
the substrate, the WD repeat containing construct (GST-B-CATWD)
displayed significantly higher activity toward native myosin II
filaments, relative to the GST-B-CAT fusion protein (Fig. 3,
A and B). Although the stimulatory effect of the
WD repeat domain from MHCK B was not as dramatic as with MHCK A,
several independent preparations of the MHCK B constructs have
consistently shown WD repeat-dependent stimulation of
kinase activity toward myosin II. The lower level of stimulation by the
WD repeat domain of GST-B-CATWD may be due to improper folding of this
domain when expressed in E. coli or may reflect a
fundamentally lower stimulatory effect of this domain relative to the
corresponding domain of MHCKA. Further studies with purified native
MHCK B may be needed to resolve this point.
Comparison with the activities of the MHCK A constructs reveals that
the MHCK B-derived fusion proteins exhibited lower rates of kinase
activity toward GST-2029 and myosin II. However, when a
nonphysiological substrate such as myelin basic protein was included in
the reaction, the MHCK B fusion proteins showed equivalent or higher
rates of substrate phosphorylation (Table
II), indicating that the inherent
catalytic activities of these constructs is not lower than those of the
MHCK A fusion proteins. Moreover, although neither the MHCK A nor the
MHCK B fusion proteins phosphorylated nonphysiological myosins from
skeletal or smooth muscle sources very well, the MHCK B fusion proteins
displayed significantly higher activity toward these substrates than
did the MHCK A fusion proteins (Table II).
Role of the WD Repeat Domain in the Autophosphorylation of MHCK A
and MHCK B--
The fusion proteins of both MHCK A and MHCK B were
also assessed for the ability to autophosphorylate. Previous studies
have shown that autophosphorylation is a major mechanism regulating native MHCK A, resulting in a 50-fold increase in kinase activity (21).
In the current study, fusion protein constructs derived from MHCK A
consistently autophosphorylated to levels of 1-2 mol Pi/mol kinase (Fig.
4A). The level of
autophosphorylation does not change if the WD repeat domain of MHCK A
is absent. In comparison, the MHCK B-derived fusion proteins
autophosphorylated to a level of 15-20 mol Pi/mol kinase
(Fig. 4B), with some preparation-to-preparation variability
in autophosphorylation stoichiometry. For both the MHCK A and MHCK
B-derived fusion proteins, the presence or absence of the
WD repeat domain does not significantly affect autophosphorylation rate or stoichiometry.
Studies Exploring the Mechanism of WD Repeat-mediated
Enhancement of Myosin Heavy Chain Kinase Activities--
The results
presented thus far demonstrate that both MHCK A and MHCK B require
their WD repeat domains to phosphorylate myosin II efficiently but do
not depend on the presence of their WD repeat domains for basic
catalytic activity toward other substrates. However, it is not clear
whether the WD repeat domain interacts with myosin II to physically
target the kinase catalytic domain to its substrate or if an
interaction between the WD repeat domain and myosin II results in
stimulation of the inherent catalytic activity of the kinase. As a
means of testing the latter possibility, the GST-A-CAT and GST-A-CATWD
constructs were assayed for kinase activity toward MH-1 peptide in the
presence and absence of myosin II. Under these conditions, neither
kinase construct was stimulated to phosphorylate MH-1 peptide above
normal levels, thus indicating that the kinase catalytic activity is
not affected by an interaction between the WD repeat and myosin II
(Fig. 5). These findings further support
a role for the WD repeat domain in physically targeting the kinase
specifically to myosin substrate.
To explore the possibility that WD repeat targeting of MHCK A and MHCK
B involves a direct interaction with myosin, we assayed all four of the
fusion proteins for cosedimentation with myosin filaments. Results from
these assays revealed that up to 40% of the WD repeat containing
constructs of both MHCK A and MHCK B bound directly to myosin II
filaments under the conditions used in the cosedimentation assay (Fig.
6, A-C). In contrast, the
GST-A-CAT and GST-B-CAT fusion proteins, both of which lack WD repeat
domains, exhibited significantly less cosedimentation with myosin II
filaments. Sedimentation of GST alone was not detectable in the
presence or absence of myosin (data not shown). A GST fusion protein
containing only the WD repeat domain of MHCK A was also tested, but
high basal sedimentation caused by aggregation made this construct unsuitable for these assays (data not shown). To determine whether the
catalytic domain contributes to myosin II binding, we assayed GST-A-CATWD and GST-B-CATWD for cosedimentation with myosin II filaments in the presence of MH-1 peptide. Under these conditions, an
interaction between the catalytic domain and myosin II should be
inhibited by peptide binding to the substrate-binding site. Our results
demonstrate that the presence of MH-1 peptide at either 100 and 400 µM has no detectable effect on MHCK A or MHCK B binding to myosin II filaments and thus suggest that the interaction of the
kinases with myosin II is mediated mainly through their WD repeat
domains with little contribution from the catalytic domains. These
results, showing that both MHCK A and MHCK B bind to myosin filaments
in a manner that requires the presence of a WD repeat domain, indicate
that the mechanism by which the WD repeat domain promotes myosin
phosphorylation involves physically targeting the kinases to myosin
substrate.
In the experiments presented here we have shown that the
structurally related WD repeat domains of MHCK A and MHCK B share a
conserved mechanism for targeting MHC kinase catalytic activity by
binding directly to myosin substrate. This WD domain-mediated binding
facilitates the phosphorylation of MHC. For MHCK A, the functional
consequence of this phosphorylation is to promote myosin II filament
disassembly and ultimately to inhibit myosin II-mediated contraction in
cells (9, 19, 25). Further studies are needed to characterize the
physiological roles of MHCK B and to determine whether MHCK B
phosphorylation of MHC is restricted to the same or different sites
phosphorylated by MHCK A.
Using recombinant, bacterially expressed GST fusion proteins of MHCK A
and its homologue MHCK B, we have shown that both kinases are targeted
by their WD repeat domains to phosphorylate myosin II substrate
specifically. Assays of these constructs revealed that full kinase
activity toward native myosin substrate is achieved only with MHCK A
and MHCK B fusion protein constructs containing their corresponding WD
repeat domains. MHCK A and MHCK B fusion proteins lacking their WD
repeat domains exhibit reduced ability to phosphorylate myosin II. In
contrast, activities of the kinases toward soluble substrates such as
the MH-1 peptide or GST-2029 are not affected by the presence or
absence of their WD repeat domains. This demonstrates that the basal
kinase catalytic activities of both MHCK A and MHCK B are independent
of their WD repeat domains. It is noteworthy that target recognition
appears to involve an extended structural component because
phosphorylation of GST-2029, a myosin-based substrate that is predicted
to fold into a coiled-coil conformation but that lacks the region
necessary for filament formation, is not stimulated by the presence of
the WD repeat domain.
The mechanism by which the WD repeat domain targets the
kinase to myosin II does not involve stimulation of the inherent
activity of the catalytic domain because the ability of GST-A-CATWD to phosphorylate MH-1 peptide is not enhanced in the presence of myosin II
(Fig. 5). Kinetic analysis of kinase activity over a range of myosin
concentrations from 0.25 to 1.5 µM revealed no saturation
of the reaction rate (Figs. 2B and 3B).
Solubility limitations preclude assays at fully physiological myosin II
concentrations (~5 µM; Ref. 19); however, the lack of
saturation or of an apparent Vmax in these tests
suggests that the WD repeat domains confer an elevated affinity of the
kinases for myosin but do not lead to formation of a rigid
substrate-enzyme complex. Further studies will be needed to determine
whether there are cellular mechanisms that may dynamically regulate
this affinity in vivo and whether substrate binding requires
a fully assembled bipolar myosin filament or simply an extended region
of the monomeric myosin tail.
Using myosin II cosedimentation assays we have determined that the
conserved WD repeat domains of both MHCK A and MHCK B target MHC kinase
catalytic activity by binding directly to myosin II substrate. To our
knowledge, this represents the first report of a WD repeat domain
physically targeting an attached kinase domain to its substrate.
Although further examples are needed, the fact that the WD repeat
domains of both MHCK A and MHCK B are substrate targeting domains may
indicate that MHCK A, B, and C have a conserved domain structure that includes a
catalytic domain unrelated to conventional protein kinases, with a WD
repeat domain that lies C-terminal to the catalytic domain. Members of
the emerging family of eEF-2 kinases, present throughout the animal
kingdom (15, 28, 29), contain a catalytic domain highly related to that
of MHCK A, B, and C but do not have detectable WD repeat motifs.
However, these enzymes all contain a C-terminal domain of roughly
similar size to the WD repeat domains observed in MHCK A, B, and C. Recent work from Pavur and colleagues (30) has revealed that the
C-terminal segment of eEF-2 kinases also acts as a targeting domain
toward the eEF-2 substrate. Moreover, these researchers identified a
degenerate repeat motif present in the eEF-2 kinase targeting domain.
This 36-amino acid repeat is intriguingly similar in size to the WD
repeat motif (~40 amino acids) but is not detectably related to WD
repeats at the primary sequence level.
The actin-fragmin kinase of Physarum may also display a
related mode of targeting. This enzyme contains a highly divergent kinase catalytic domain with a fold related to conventional protein kinases (31). C-terminal to the catalytic domain, this enzyme contains
a domain with a predicted Results from our studies of WD repeat domain targeting of MHC kinase
activity provide strong evidence that MHCK B is a physiologically relevant myosin heavy chain kinase. Although the isolated catalytic domain of MHCK B has been shown to preferentially phosphorylate the
mapped MHCK A target sites on MHC (16), we have found that the initial
rate of activity of the MHCK B constructs toward myosin II and
myosin-like substrates (MH-1 and GST-2029) is significantly lower than
that observed for the MHCK A fusion proteins (Table I). This does not
appear to be due to inherently lower kinase activity because the MHCK B
fusion proteins phosphorylate myelin basic protein, a nonphysiological
substrate, at a higher rate than do the MHCK A constructs. In
comparison with MHCK A, the MHCK B fusion protein shows greater
substrate promiscuity, exhibiting relatively higher initial rates of
kinase activity toward skeletal muscle and smooth muscle myosin
substrates. Although further studies of the activity and substrate
specificity of native MHCK B will be important, the results presented
here raise the possibility that myosin II may not be the only cellular
substrate for MHCK B.
In summary, we have shown that the catalytic activities of MHCK A and
MHCK B are targeted specifically to myosin II by a mechanism that
involves direct binding via their associated WD repeat domains. We
suggest that this targeting mechanism may be conserved among an array
of identified protein kinases that have
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of the heterotrimeric G-protein complex (G
)
has revealed that its seven WD repeats fold into a ring-like
-propeller structure consisting of seven "blades" with each
blade folded into a four-stranded anti-parallel
-sheet structure
(18). Although no structural information is available for the WD repeat domains of MHCK A, B, and C, the conservation of their seven repeats with those of the G
subunit indicates that the
seven-bladed
-propeller structure is also likely to occur in these
Dictyostelium proteins.
WD) was
unable to phosphorylate myosin II in vitro and in
vivo. In contrast, MHCK A-
WD phosphorylated a soluble
myosin-based peptide (MH-1) just as well as full-length MHCK A (19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactoside was added to induce fusion
protein synthesis. Induced cultures were incubated for 15-20 h at
25 °C on a rotating platform. Cells were then harvested by
centrifugation at 6,000 × g for 5 min, and the resulting cell pellet was washed in 25 mM Tris buffer, pH
7.5, and then centrifuged again. The cell pellet was resuspended (5 ml/g of pellet weight) with Buffer A (25 mM Tris, pH 7.5, 150 mM KCl, 5 mM dithiothreitol, and 1 mM EDTA) containing 175 µg/ml phenylmethylsulfonyl
fluoride and 2× concentration of the protease inhibitor mixtures,
PIC-I and PIC-II. PIC-I was added from a 1,000 × stock containing
2 mg/ml antipain, 10 KIU/ml aprotinin, 10 mg/ml benzamidine, 1 mg/ml
leupeptin, 5 mg/ml Pefabloc, and 10 mg/ml L-1-chloro-3[4-tosylamido]-7-amino-2-heptanone-HCl
N-
-tosyl-L-lysine chloromethyl ketone in
ddH2O. PIC-II (1,000×) contains 1 mg/ml chymostatin, 1 mg/ml pepstatin, and 5 mg/ml
L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone tosyl-l-phenylalanine chloromethyl ketone in ethanol.
80 °C
until used in assays.
-D-thiogalactoside. The culture was grown for
3 h at 37 °C, and then cells were harvested by centrifugation.
The cell pellet was resuspended in Tris-buffered saline containing 175 µg/ml phenylmethylsulfonyl fluoride and 1 mg/ml pepstatin. The cell
slurry was frozen in a dry ice/methanol bath and stored overnight in a
80 °C freezer. The following components were added after thawing
the cells for purification of GST-2029: 1 mM
dithiothreitol, 1 mM benzamidine, 100 µg/ml
L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone tosyl-l-phenylalanine chloromethyl ketone, 50 µg/ml
L-1-chloro-3[4-tosylamido]-7-amino-2-heptanone-HCl N-
-tosyl-L-lysine chloromethyl ketone, 1 mM EDTA, and 100 µg/ml lysozyme. After a 10-min
incubation on ice, Sarkosyl was added to 1.5%, and the cells were
sonicated until fully lysed. Triton X-100 (2%) was added prior to
centrifuging the sonicate and the cleared supernatant was incubated
with glutathione-Sepharose beads for 45 min at 4 °C. Unbound
material was removed by washing the beads with buffer containing 20 mM Tris, pH 7.5, 50 mM NaCl, and 1 mM EDTA. Fusion protein was eluted from the washed beads
using 10 mM glutathione in wash buffer. The peak of eluted
protein was determined by Bradford assay (Pierce), and pooled fractions
were treated with aquacide (CalBiochem) to concentrate. The resulting concentrate was dialyzed into 10 mM TES, pH 7.0, 1 mM EDTA, and 1 mM dithiothreitol and then
stored in aliquots at
80 °C until used.
-32P]ATP (250-400 Ci/mol), using
recombinant kinases at 20-80 nM, as noted for each
experiment. The substrates used in this study were MH-1 peptide,
GST-2029 (38 kDa), and myosin (243 kDa). The peptide substrate MH-1
(RKKFGESEKTKTKEFL-amide) has been described previously (21)
and corresponds to the mapped MHCK A target site in the myosin II tail
at residue 2029 (underlined in peptide above). For kinetic analysis of
kinase activity, each of the substrates was used over a range of
concentrations as noted for each experiment. For determination of MHC
phosphorylation stoichiometry, myosin II was included in the reaction
mix at a concentration of 0.42 µM. For myosin substrate
specificity experiments, rabbit skeletal muscle myosin II (Sigma) and
chicken gizzard myosin II (Sigma) were dialyzed against
Dictyostelium myosin II storage buffer (5 mM
Tris pH 7.0 buffer containing 20% sucrose) prior to their addition to
kinase reaction mixes.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Recombinant MHCK fusion protein
constructs. A, schematic diagrams of full-length MHCK A
and the bacterially expressed truncations of MHCK A used in this study.
Numbering indicates positions of included amino acids from MHCK A
(GenBankTM accession number P42527). At left is
an SDS-PAGE gel (Coomassie Blue-stained) of purified GST-A-CAT and
GST-A-CATWD (2 µg each). B, schematic diagrams of
full-length MHCK B and its bacterially expressed truncations used in
this study. Numbering corresponds to included amino acids from MHCK B
(GenBankTM accession number P90648). Coomassie Blue-stained
SDS-PAGE gel at left was loaded with 2 µg of purified GST-B-CAT and
GST-B-CATWD each.
2 µM), these
results clearly show that the presence of the WD repeat domain is
required for efficient phosphorylation of myosin II by MHCK A.
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Fig. 2.
Phosphorylation of myosin II heavy chain,
MH-1 peptide, and GST-2029 using MHCK A-derived fusion proteins either
containing or lacking the WD repeat domain. A, the
stoichiometry of Dictyostelium myosin II (0.42 µM) phosphorylation by GST-A-CAT and GST-A-CATWD (both at
20 nM final concentration) was assessed over time by
subjecting kinase reactions to SDS-PAGE, Coomassie blue staining, and
then scintillation counting of the excised MHC band (see
"Experimental Procedures"). Plotted points represent the mean
values from two separate experiments. B, GST-A-CAT and
GST-A-CATWD constructs were assayed for kinase activity over a range of
myosin II concentrations using the method described for the myosin
stoichiometry experiment above. C, the MHCK A fusion
proteins were assessed for kinase activity over a range of MH-1
peptide concentrations using the filter binding method described under
"Experimental Procedures." The inset is a
Lineweaver-Burk plot of the values obtained from this assay.
D, GST-A-CAT and GST-A-CATWD activities were assessed over a
range of GST-2029 concentrations by excising GST-2029 bands from
Coomassie-stained SDS-PAGE gels followed by scintillation counting of
those bands. For all of the plots in this figure, GST-A-CAT is
represented by closed circles, and open circles
represent the activity of the GST-A-CATWD fusion protein. The GST-A-CAT
and GST-A-CATWD constructs used in all of these experiments were
preautophosphorylated for 10 min prior to their addition to the
reaction mix. For substrate concentration range experiments
(B-D), both GST-A-CAT and GST-A-CATWD were included in the
reaction mixes at a final concentration of 40 nM and
incubated with substrate for 1 min at 25 °C. For B-D,
the plotted values represent the mean activities determined from at
least four independent experiments. The vertical bars
indicate the standard error of each mean.
MH-1 kinetics constants for GST fusion proteins
View larger version (14K):
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Fig. 3.
Phosphorylation of MH-1 peptide, GST2029, and
myosin II using MHCK B-derived fusion proteins either containing or
lacking the WD repeat domain. A, the stoichiometry of
myosin II phosphorylation (0.42 µM) by GST-B-CAT and
GST-B-CATWD (20 nM each) was assessed via SDS-PAGE and
scintillation counting of the Coomassie Blue-stained MHC band.
B, the MHCK B fusion proteins (80 nM) each were
incubated (4 min at 25 °C) with a range of myosin II concentrations,
and specific activity measurements were made as described for
A. C, the activities of GST-B-CAT and GST-B-CATWD
toward MH-1 peptide were determined by incubating (2 min at 25 °C)
the fusion proteins (40 nM) with a range of peptide
substrate concentrations. The filter binding assay (see
"Experimental Procedures") was used to determine phosphate incorporation. The
inset is a Lineweaver-Burk plot of the values obtained from
this assay. D, the activities of GST-B-CAT and GST-B-CATWD
(40 nM each) were assessed over a range of GST-2029
concentrations (4 min incubation at 25 °C) by subjecting reaction
mixes to SDS-PAGE and then excising the GST-2029 band. The amount of
phosphate incorporation was determined by scintillation counting of
excised bands. Activity of the GST-B-CAT construct is represented by
the closed circles, and open circles represent
activity of the GST-B-CATWD fusion protein. The GST-B-CAT and
GST-B-CATWD constructs used in all of these experiments were
preautophosphorylated for 10 min prior to their addition to the
reaction mix. For B-D, the plotted values represent the
mean activity determined from at least four independent experiments.
The vertical bars indicate the standard error of each
mean.
Initial rates of GST-A-CATWD and GST-B-CATWD constructs towards various
substrates
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Fig. 4.
Autophosphorylation of MHCK A and MHCK
B-derived fusion proteins. Autophosphorylation reactions were
performed at 200 nM of each kinase construct, and the level
of autophosphorylation was determined with a filter binding assay as
described under "Experimental Procedures." A,
autophosphorylation of GST-A-CAT ( ) and GST-A-CATWD (
)
versus time. The inset represents the same data
plotted to a smaller scale. B, autophosphorylation of
GST-B-CAT (
) and GST-B-CATWD (
) versus time.
View larger version (13K):
[in a new window]
Fig. 5.
Phosphorylation of MH-1 peptide by GST-A-CAT
and GST-A-CATWD in the presence and absence of myosin II. The MHCK
A fusion proteins (40 nM) each were incubated (4 min at
25 °C) with 50 µM MH-1 substrate in the presence
(gray bars) or absence (black bars) of 0.5 µM myosin II. Specific activity toward MH-1 peptide was
measured using the filter binding method described under
"Experimental Procedures." Values obtained from control reactions
containing only kinase and myosin II were subtracted from experimental
values to control for MHC phosphorylation in these reactions. The
GST-A-CAT and GST-A-CATWD constructs were preautophosphorylated for 10 min prior to their addition to the reaction mix. The bars
represent the mean activity from three independent experiments with the
error bars representing the standard error of each
mean.
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Fig. 6.
Cosedimentation assays of MHCK A and MHCK
B-derived fusion proteins with myosin II filaments. Kinase fusion
proteins (0.3 µM) were incubated with myosin filaments
(1.0 µM) as described under "Experimental
Procedures." Reaction mixes were centrifuged, and equal volumes of
the resulting pellets (P) and supernatants (S)
were subjected to SDS-PAGE. Myosin and BSA were visualized by Coomassie
Blue staining of the gel, and the kinase fusion proteins were
identified by Western blotting with anti-GST antibody. A,
representative Western blot (top) and Coomassie-stained gel
(bottom) from a myosin cosedimentation assay of GST-A-CAT
and GST-A-CATWD. B, representative Western blot
(top) and Coomassie-stained gel (bottom) from a
myosin cosedimentation assay of GST-B-CAT and GST-B-CATWD.
C, bar graph of kinase construct binding to myosin II. The
values represent the means from three separate experiments performed in
duplicate, and the error bars represent the standard error
of each mean. D, bar graph of GST-A-CATWD and GST-B-CATWD
cosedimentation with myosin II in the presence and absence of MH-1
peptide. Cosedimentation results were quantified by densitometric
analysis of Western blots, and the amount of fusion protein in the
pellet fraction was divided by that in both the pellet and supernatant
to yield the values indicated by the bars.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-propeller domains present in other protein kinases
play similar roles in substrate targeting. Potential candidates for
testing this possibility include the Dictyostelium protein
MHCK C, which possesses catalytic and WD repeat domains related to
those of MHCK A, and a recently identified bacterial protein from
Thermomonospora curvata, which is predicted to contain a
conventional serine/threonine kinase domain coupled to a WD repeat
containing region (26). Recently, the WD repeat domain of a receptor
for activated protein kinase C (Rack1) has been shown to bind the
cytoplasmic region of the integrin
-subunit and has been proposed to
recruit activated protein kinase C to integrins (27). Thus, the WD
repeat domain of Rack1, although not covalently attached to protein
kinase C, may play a direct role in targeting protein kinase C activity to a substrate.
-propeller structure of the Kelch class
(32). Notably, removal of the
-propeller domain was reported to
cause a significant decrease in enzyme activity when assayed against
the native actin-fragmin substrate (other substrates were not tested).
We suggest that in this enzyme as well, the
-propeller domain may be
serving a substrate targeting role.
-propeller domains adjacent
to their catalytic domains. Although MHCK A and B fusion proteins share
the property of binding to myosin filaments via their WD repeat
domains, other aspects of their kinase activity clearly differ,
suggesting different cellular roles for these enzymes.
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ACKNOWLEDGEMENTS |
---|
We thank the Egelhoff Lab, Dr. Luke Szweda, and Dr. Graham Côté for helpful suggestions.
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FOOTNOTES |
---|
* This work was supported by research Grant GM50009 (to T. T. E.) from the National Institutes of Health and by American Cancer Society Postdoctoral Fellowship PF-99-310-01-CSM (to P. A. S.).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.
Present address: Dept. of Cell Biology and Physiology, Washington
University, St. Louis, MO 63130.
§ To whom correspondence should be addressed. Tel.: 216-368-6971; Fax: 216-368-1693; E-mail: tte@po.cwru.edu.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M008992200
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ABBREVIATIONS |
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The abbreviations used are: MHC, myosin heavy chain; BSA, bovine serum albumin; GST, glutathione S-transferase; MHCK, myosin heavy chain kinase; PIC, protease inhibitor cocktail; PAGE, polyacrylamide gel electrophoresis; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]- amino}ethanesulfonic acid.
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REFERENCES |
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