From the
Ca
The
pseudosubstrate model for autoinhibition proposes that basic residues
within the autoinhibitory region mimic basic residues in the substrate
and bind to defined acidic residues within the catalytic core. Charge
reversal mutations of these specific acidic residues, however, had
little or no effect on the K
Activation of smooth/nonmuscle myosin light chain kinase by
Ca
Recent mutational analyses have identified
several acidic residues in the catalytic core that may bind the
inhibitory sequence but not the light chain. When the charge of these
residues was reversed by mutation, the respective K
A three-dimensional
model has been proposed for the catalytic core of smooth muscle myosin
light chain kinase, and a portion of the bound autoinhibitory region
that includes the expanded pseudosubstrate sequence (Knighton et
al., 1992). This model is based on the known crystal
structure of the catalytic subunit of cAMP-dependent protein kinase in
complex with its inhibitory peptide, PKI. A substrate binding groove
has been defined utilizing the position of the PKI peptide in the
crystal structure of this complex (Kemp et al., 1994). It is
predicted that specific acidic residues lining this substrate-binding
groove of the myosin light chain kinase catalytic core form salt
bridges with basic residues in both the autoinhibitory sequence and the
regulatory light chain substrate. Since the role of these acidic
residues was not examined in a previous study (Gallagher et
al., 1993), we have experimentally tested the predictions of this
pseudosubstrate model using selected site mutagenesis to identify
interacting residues. A modified molecular mechanism for autoinhibition
is proposed.
To examine the calmodulin
activation properties of mutant myosin light chain kinases in COS cell
lysates, Ca
The
Ca
Individual substitution of basic residues Lys
The resulting mutant kinases were characterized with
respect to catalytic and activation properties. One of the charge
reversal mutants, E858K, was catalytically inactive ()
even though it was expressed at high levels and comigrated with the
152-kDa wild-type recombinant myosin light chain kinase (data not
shown). A more conservative mutation, E858Q, resulted in the expression
of an active kinase with V
All of the other catalytic core
mutants exhibited no significant changes in
[Ca
Minor yet statistically significant
increases in the K
As a result of previous mutational and deletion analysis of
the myosin light chain kinase regulatory domain, some residues
immediately amino-terminal to the calmodulin-binding sequence were
implicated in autoinhibition (Shoemaker et al., 1990;
Ito et al., 1991; Fitzsimons et al., 1992; Yano et al., 1993). Our data indicate
that basic residues even further amino-terminal to the originally
proposed pseudosubstrate sequence and close to the carboxyl terminus of
the catalytic core could be involved in maintaining myosin light chain
kinase in an inhibited state (Fig. 1). In each case, both single
charge reversal and alanine substitution mutations of residues
Lys
Many acidic
residues in the catalytic core appear to have specific interactions
with the autoinhibitory sequence, but not light chain. The conservative
neutral mutation, E858Q, created a kinase with wild-type catalytic
properties but a 7-fold lower K
The
recently reported crystal structure of a related kinase, twitchin, may
provide further insights into the structural basis for intrasteric
inhibition of myosin light chain kinase (Hu et al., 1994). The portion of the kinase carboxyl-terminal to the
catalytic core extends across the surface of the large lobe of the
catalytic core similar to the distinct pathway proposed for the
autoinhibitory sequence of myosin light chain kinase (Fig. 3). It
then extends through the active site between the large and small lobes.
Acidic residues in the twitchin kinase, Glu
Recombinant and mutant myosin light chain kinases were
expressed in COS cells as described under ``Experimental
Procedures.'' Values are means ± S.E. for at least three
experiments with lysates from transfections representing at least two
independent mutations. [Ca
Mutagenesis,
expression, and measurements of kinetic properties of myosin light
chain kinases were performed as described under ``Experimental
Procedures.'' Values represent means ± S.E. for at least
three experiments with lysates from transfections representing at least
two independent mutants. The relative K
We thank Phyllis Foley for the preparation of this
manuscript, Patricia J. Gallagher for antibodies to myosin light chain
kinase, and James M. Mottonen for technical assistance with the
MOLSCRIPT software.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/calmodulin activates myosin light chain
kinase by reversal of an autoinhibited state. The effects of
substitution mutations on calmodulin activation properties implicate 4
of the 8 basic residues between the catalytic core and the
calmodulin-binding domain in maintaining autoinhibition. These residues
are further amino-terminal to the basic residues comprising the
previously proposed pseudosubstrate sequence and suggest involvement of
the connecting region in intrasteric autoinhibition.
value for
regulatory light chain. From a total of 20 acidic residues on the
surface of the substrate binding lobe of the catalytic core, 7 are
implicated in binding directly or indirectly to the autoinhibitory
domain but not to the light chain. Only 2 acidic residues near the
catalytic site may bind to the autoinhibitory domain and the arginine
at P-3 in the light chain. Exposure of these 2 residues upon
calmodulin binding may be necessary and sufficient for light chain
phosphorylation.
/calmodulin results in phosphorylation of myosin
regulatory light chain that plays important roles in initiation of
smooth muscle contraction, endothelial cell retraction, secretion, and
other cellular processes (Stull et al., 1995). The
smooth/nonmuscle myosin light chain kinase contains a catalytic core
homologous to that of other protein kinases and a carboxyl-terminal
regulatory domain consisting of both an inhibitory sequence and a
calmodulin-binding sequence (Kemp et al., 1994; Stull et
al., 1995). Initially, inspection of the linear sequence within
the regulatory domain revealed a similar number and sequential
arrangement of 4 basic residues with those shown to be important
substrate determinants in a synthetic peptide containing residues
11-23 of the myosin regulatory light chain (see Fig. 1).
Thus, Kemp et al.(1987) proposed that the regulatory domain
contained a pseudosubstrate inhibitory sequence whereby 4 specific
basic residues in myosin light chain kinase mimic the basic substrate
determinants in the light chain peptide substrate. Binding of the
pseudosubstrate sequence to the active site inhibited activity.
Intrasteric inhibition involves an autoinhibitory sequence that folds
back on the catalytic site to inhibit kinase activity as opposed to an
allosteric mechanism whereby a conformational change induced at a site
distinct from the active site would be responsible for regulation of
enzyme activity (Kemp and Pearson, 1991). The sequence comprising the
pseudosubstrate region was later expanded to include overlap with the
complete amino terminus of the light chain (Faux et al.,
1993). However, these additional
residues(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) are not
important for substrate binding and thus are not part of the consensus
phosphorylation sequence (Kemp and Pearson, 1990).
Figure 1:
Sequence comparison of the
regulatory domain of rabbit myosin light chain kinase with myosin light
chain. Boxedresidues in the myosin light chain
kinase sequence are those basic residues that were previously noted to
be similar in sequential arrangement to the basic residues in the light
chain (boxed) that form part of the consensus phosphorylation
sequence (Kemp et al., 1987). The proposed
pseudosubstrate sequence was later extended to include all residues
(Ser-Val
) that overlap the amino
terminus of the light chain (Knighton et al., 1992). Circledresidues identify basic residues within the
regulatory domain implicated in this study to bind to the catalytic
core. The catalytic core ends with
Leu
.
Proteolysis
studies have supported the hypothesis that myosin light chain kinase
contains an autoinhibitory sequence (Walsh et al., 1982; Ikebe et al., 1987; Pearson et
al., 1988). Limited tryptic cleavage of the chicken
smooth muscle myosin light chain kinase results in a nonactivatable
form (64 kDa) that becomes constitutively active upon further digestion
(61 kDa). Because of differences in the reported cleavage sites, there
is disagreement on whether the inhibited form contains the
pseudosubstrate sequence (Pearson et al., 1988; Ikebe et al., 1989).
(
)values decreased with
no significant effect on the K
or V
values for the regulatory light chain
(Gallagher et al., 1993). It was proposed that a
lowered K
value reflected a weakened binding of
the inhibitory region to the catalytic core. In this series of
mutations, only 2 acidic residues, located near the catalytic site in
the cleft between the two lobes of the kinase, were identified as
binding to both the inhibitory region and the arginine residue at the
P-3 position in the light chain substrate.
Oligonucleotide-directed Mutagenesis
A
1,660-base pair 5`-BamHI-XbaI-3` cDNA fragment,
representing the carboxyl-terminal half of rabbit smooth/nonmuscle
myosin light chain kinase (Gallagher et al., 1991),
was subcloned into M13 bacteriophage. Mutagenesis was performed using
the oligonucleotide-directed in vitro mutagenesis system
(Amersham Corp.) and oligonucleotides designed to produce mutant cDNAs
having the desired substitutions. For each mutant cDNA, the desired
nucleotide substitutions were verified by DNA sequencing (Sanger et
al., 1977).
Expression of Wild-type and Mutant Smooth Muscle Myosin
Light Chain Kinases
All wild-type and mutant myosin light chain
kinases were expressed in COS cells. In each case, two clones of each
mutant were subcloned into a pCMV expression vector as
described previously (Gallagher et al., 1991).
Subsequent expression was accomplished by transfection into COS cells
using DEAE-dextran and chloroquine (Herring et al.,1990).
Myosin Light Chain Kinase Assays
COS cell lysates,
prepared as described by Gallagher et al.(1991), were used to
determine recombinant wild-type and mutant myosin light chain kinase
activity (Gallagher et al., 1991). The quantity of
wild-type and mutant smooth muscle myosin light chain kinases present
in COS cell lysates was determined by immunoblotting with purified
smooth muscle myosin light chain kinase as a standard and probing with
antiserum raised against the carboxyl-terminal telokin portion of
rabbit smooth muscle myosin light chain kinase (Gallagher and Herring,
1991). The Ca/calmodulin-dependent activity of
wild-type and mutant smooth muscle myosin light chain kinases was
measured by
P incorporation into myosin regulatory light
chain purified from chicken gizzards (Blumenthal and Stull, 1980).
Ca
/calmodulin-independent background activity of the
extracts was measured as radioactivity incorporated in the presence of
3 mM EGTA. V
and K
values were determined from Lineweaver-Burke
double-reciprocal plots after performing kinase assays under varying
regulatory light chain concentrations.
activation assays were performed as
described by Herring(1991). The relative K
values of the mutant kinases within the COS cell extracts were
measured at 1 µM calmodulin with varying Ca
concentrations from 75 nM to 100 µM with a
Ca
/EGTA buffer system (Potter and Gergely, 1975). The
added calmodulin is in great excess of the endogenous calmodulin
contributed by the COS cell lysate and therefore provides controlled
conditions to establish the relative concentrations of
Ca
/calmodulin required for kinase activation. The
free Ca
concentration was the determinant of the
actual Ca
/calmodulin concentration. To assess the
quantitative changes in the calmodulin activation properties (K
) of mutant myosin light chain kinases, the
ratio of activities at Ca
concentrations that
resulted in less than maximal activity to the maximal activity measured
at 100 µM Ca
was determined as described
previously (Miller et al., 1983; Fitzsimons et al., 1992). The ratio of activities at a specific Ca
concentration that is less than that required for maximal
activity increases quantitatively as the K
value
for a mutant myosin light chain decreases relative to the wild-type
enzyme. Although the ratio of activities does not allow determination
of the absolute value of K
, it may be used to
calculate the -fold change in K
relative to
wild-type myosin light chain kinase that has an average K
value of 1 nM (Miller et
al., 1983; Stull et al., 1990; Fitzsimons et
al., 1992). It is proposed that a decrease in the K
value (<1 nM) reflects a more
easily activated kinase due to a weakening of the binding between the
inhibitory region and the catalytic core.
Properties of Myosin Light Chain Kinase Mutated in the
Connection Region
To further define the boundaries of myosin
light chain kinase inhibitory sequence, we created charge reversal and
alanine substitution point mutations at basic residues amino-terminal
to the proposed pseudosubstrate region of myosin light chain kinase (Fig. 1) (Kemp et al., 1987). Full-length cDNA
was constructed in the vector pCMV for transient expression
in COS cells. Immunoblot analysis of COS cell lysates demonstrated that
all of the mutant myosin light chain kinases were full-length and
expressed at levels (5-20 µg/ml) similar to previously
obtained results (Fitzsimons et al.,1992; Gallagher et al., 1993). All active mutant myosin light chain
kinases were dependent on Ca
for activity.
/calmodulin activation properties of the wild-type
and mutant kinases were determined by performing assays at a high
calmodulin concentration with a Ca
/EGTA buffer used
to vary the free Ca
concentration and hence the
Ca
/calmodulin concentration (Miller et al.,
1983). Lys
is 21 residues amino-terminal to basic
residues (Arg
-Lys
) in the calmodulin
binding domain that form the core of the proposed pseudosubstrate
structure (Fig. 1). Both K953D and K953A mutants required
significantly less Ca
for half-maximal activation
(0.26 and 0.42 µM, respectively) relative to wild-type
kinase (0.65 µM) with corresponding decreases in K
(0.16 and 0.53 nM from 1.0
nM) ( and Fig. 2A). These mutations
that produced a 6.3- and 1.9-fold decrease in K
values were not accompanied by any significant changes in
catalytic properties (). In contrast to these results,
mutant K956E had no significant effects on Ca
activation or catalytic properties ().
Figure 2:
Calcium activation curves for mutant
myosin light chain kinases. Myosin light chain kinase mutants were
expressed in COS cells, and activity was measured in cell lysates at 1
µM calmodulin at different Ca concentrations. The data were normalized to the percent maximal
activity. Symbols represent a mean value of at least three independent
assays, each performed in duplicate. Data are presented without error
bars for clarity. A, mutations within the regulatory domain of
myosin light chain kinase are as follows:
, wild-type;
,
K953D;
, K956E;
, K961E;
, K962E;
, R967E. B, mutations within the catalytic core of myosin light chain
kinase are as follows:
, wild-type;
, E858Q;
,
D896K;
, E900R;
, D911K/D914K.
[Ca
]
values are 0.65 ±
0.02, 0.13 ± 0.02, 0.57 ± 0.05, 0.57 ± 0.03, and
0.78 ± 0.09 nM, respectively.
The K961E
and K961A mutants required significantly less Ca for
half-maximal activation (0.30 and 0.31 µM) with 5- and
3.2-fold decreases in K
values, respectively (). The charge reversal mutant K961E selectively changed
the activation properties of the kinase with no change in V
or K
values (). In contrast, the charge reversal and alanine
substitution mutations of Lys
had little effect on
activation and catalytic properties. The V
and K
values were not changed for the charge
reversal mutation. The K
value for light
chain was not measured for the alanine substitution, but the specific
activity of the kinase was not different for wild-type enzyme. The
charge reversal mutation K962E resulted in a modest decrease in
[Ca
]
from 0.65 to 0.47 mM with only a 1.6-fold decrease in K
. By
comparing these relative K
values with that
previously reported (Fitzsimons et al., 1992) for the double
mutant KK961/962EE (0.20 nM), it is evident that of these 2
residues, Lys
is more sensitive to substitution.
and
Arg
with oppositely charged acidic residues showed a
marked decrease in K
value (0.03 and 0.09
nM, respectively) relative to the 1 nM value for
wild-type kinase (). These values can be compared with the
previously reported values (Fitzsimons et al., 1992)
for the individual alanine mutants K965A and R967A (0.11 and 0.25
nM) as well as the double mutants K965A/R967A and K965E/R967D
(0.04 nM and inactive, respectively). None of the mutations
caused significant changes in V
or K
values except for K965E in which the
33-fold decrease in K
was associated with lower V
and K
values () (Fitzsimons et al., 1992).
Properties of Myosin Light Chain Kinase Mutated in the
Catalytic Core
Acidic residues Glu,
Asp
, Glu
, and Asp
(corresponding residues for myosin light chain kinase from
chicken smooth muscle are Glu
, Asp
,
Glu
, and Asp
, respectively) within the
myosin light chain kinase catalytic core are predicted by the proposed
pseudosubstrate model to be involved in binding both the inhibitory
sequence in the kinase and the regulatory light chain (Knighton et
al.,1992). This hypothesis was tested by charge reversal
mutations of these residues. Three neighboring acidic residues,
Asp
, Asp
, and Asp
, were
chosen as controls for the charge reversal mutation studies. These
latter residues are found in the vicinity of those proposed to bind to
both the autoinhibitory sequence and the regulatory light chain but are
not predicted to bind to either light chain or the autoinhibitory
sequence.
, K
, and V
/K
ratio values
similar to those of the wild-type kinase. E858Q did, however, require
significantly less Ca
(0.13 µM) for
half-maximal activation relative to the wild-type kinase (0.65
µM) with a 7-fold decrease in the K
value (, Fig. 2B). These results are
consistent with the binding of this residue to or near the inhibitory
domain but not to the light chain.
]
and K
values from that of the wild-type kinase (, Fig. 2B). These results are consistent with the
interpretation that these acidic residues do not bind to basic residues
in the autoinhibitory domain.
values for regulatory
light chain were observed for D896K, D898K, E900R, D911K/D914K, D911R,
D913K, and D914K mutants (19.8, 19.3, 11.6, 33.7, 10.2, 9.6, and 16.0
µM, respectively, versus 4.7 µM for
wild-type kinase). In several cases (E900R, D911R, D913K, and D914K)
the changes were only about 2-fold and could result from nonspecific
electrorepulsion rather than perturbation of specific salt bridge bonds
between the respective residues in the catalytic core and the light
chain. The V
values were similar to wild-type
kinase except for D913K, which was lower (8.7 µmol of
P incorporated per min/mg). The changes in the V
/K
ratios for the
charge reversal mutants were also modest (). The largest
change was noted in the double mutant D911K/D914K (5.8-fold). However,
single mutations in these residues resulted in only 1.7- and 2.4-fold
changes.
, Lys
, Lys
, and
Lys
resulted in kinases that were more easily activated
by Ca
/calmodulin (decreased
[Ca
]
and K
values relative to wild-type). The results with Lys
are most interesting since this residue is close to the junction
between the catalytic core and the connecting region. These findings
support the hypothesis that an intrasteric, autoinhibitory mechanism
for myosin light chain kinase involves structures that extend beyond
the proposed pseudosubstrate sequence. They also support the idea that
the connecting region between the catalytic core and calmodulin binding
domain may be bound to the surface of the catalytic core similar to the
binding of the carboxyl-terminal tail of twitchin kinase to its
catalytic core (Hu et al., 1994). The multiple intramolecular
contacts made by this connecting region may be involved in
autoinhibition and, because of the extensive contacts, single mutations
would not be expected to produce profound effects on
Ca
/calmodulin activation properties. However,
Shoemaker et al.(1990) showed that multiple charge reversal
mutations in a portion of this autoinhibitory sequence could result in
a constitutively active myosin light chain kinase.
value. This
result is consistent with the suggestion that Glu
is
involved in maintenance of autoinhibition but not in binding to the
regulatory light chain. As predicted, D898K, D913K, and D914K each
displayed similar [Ca
]
and K
values as wild-type myosin light chain kinase,
suggesting that these acidic residues do not bind to or near the
autoinhibitory sequence. However, evidence was not obtained that other
predicted acidic residues bind to the autoinhibitory sequence
(Asp
, Glu
, and Asp
). Thus,
the acidic residues on the surface of the catalytic core, which have
been implicated from this and previous mutational analyses (Gallagher et al., 1993) in binding to or near the
autoinhibitory sequence, are predicted to be mainly in the D
-helix (Fig. 3). The placement of these residues defines a
binding pathway distinct from that of PKI binding to the G
-helix
of cAMP-dependent protein kinase. By inference then, this pathway is
also distinct from the purported substrate-binding groove that predicts
pseudosubstrate as well as substrate binding contacts with residues in
the G
-helix (Asp
, Glu
, and
Asp
) (Knighton et al., 1992; Kemp et al., 1994). In fact, none of these acidic residues nor any
of those in the vicinity of the G
-helix (Asp
,
Asp
and Asp
) can be implicated by our
mutational analyses in binding to or near the autoinhibitory domain.
Additional proposed contacts include Glu
, which is found
in a loop spatially situated between the D
-helix and the G
-helix, and Glu
, which is found in the active site
cleft on the D
-helix (Knighton et al., 1992).
However, Glu
also does not appear to bind the light
chain. These results suggest that the autoinhibitory sequence may bind
on the surface that comprises the substrate binding pocket immediately
surrounding the catalytic site, but it is likely to have more contacts
with the D
-helix than with the G
-helix.
Figure 3:
Ribbon diagram of a model of the
catalytic core of smooth/nonmuscle muscle myosin light chain kinase.
The ribbon was drawn using the program MOLSCRIPT (Kraulis, 1991). The
model is derived from the chicken smooth muscle myosin light chain
kinase catalytic core model (Knighton et al., 1992).
The letters displayed correspond to the respective
-helices as defined for the cAMP-dependent protein kinase
structure (Knighton et al., 1991). The D and G
-helices are highlighted in red and yellow, respectively, for clarification. Some amino acid
residues are displayed in ball-and-stick.
The 2 acidic residues, Glu
and Glu
(highlighted in blue), are proposed to bind to basic
residues in both the autoinhibitory domain and the arginine at
P-3 in the regulatory light chain. The 7 acidic residues,
Glu
, Glu
, Glu
,
Glu
, Asp
, Glu
, and
Glu
(highlighted in red), that display a
distinct pathway projecting from the catalytic site to the end of the
catalytic core have been implicated, from this and previous mutational
analyses (Gallagher et al., 1993), in binding to or near the
inhibitory domain, but not to the light chain. Charge reversal
mutations of several other acidic residues (highlighted in yellow), including Asp
, Glu
, and
Asp
, which had been predicted by the pseudosubstrate
theory to bind to both the autoinhibitory domain and the regulatory
light chain, had little or no effect on the catalytic or activation
properties of the kinase. These and other residues (Asp
and Asp
) are predicted not to play important roles
in binding either the regulatory light chain or the autoinhibitory
domain.
These results
also raise a question regarding the identification of acidic residues
in the catalytic core that bind to the 3 basic residues in the
consensus phosphorylation sequence of the light chain at P-6 to
P-8 positions (Fig. 1). The V/K
ratios
obtained with the intact light chain substrate for the respective
charge reversal mutations at Glu
, Asp
,
Asp
, Asp
, and Asp
are only
slightly decreased (2-5-fold) relative to the V
/K
ratio of the
wild-type kinase and are minor compared with the 50-fold effects of
alanine substitutions for basic residues in synthetic peptide
substrates (Kemp et al., 1983; Kemp and Pearson, 1985). The
decreases in V
/K
ratios for regulatory light chain in the charge reversal
mutant kinases are still much smaller in magnitude compared with the
340-fold decrease in the V
/K
ratio reported for the charge reversal mutation of arginine
at the P-3 position in the regulatory light chain (Zhi et
al., 1994). Even with an alanine substitution at P-3 in the
light chain, the V
/K
ratio decreased 35-fold. The decreases in the V
/K
ratio values
were modest with individual alanine substitutions of the basic residues
in the consensus phosphorylation sequence at P-6 to P-8 in
the intact light chain. It is unlikely then that acidic residues
Glu
, Asp
, Asp
,
Asp
, and Asp
are important in substrate
binding and phosphorylation. This conclusion is consistent with the
proposal that it is other interactions, involving the arginine at
P-3 and the hydrophobic residues at P+1 to P+3, that
are most important for substrate recognition in addition to subdomains
I and II of the regulatory light chain (Zhi et al., 1994).
Thus, only a portion (Arg
at P-3 and hydrophobic
residues at P+2 and P+3) of the previously described
consensus phosphorylation sequence is important for substrate
recognition of the intact light chain. As the importance of the basic
residues found amino-terminal to the phosphorylatable serine at
P-6 to P-8 positions is diminished in the protein substrate
(Zhi et al., 1994), the definition of the pseudosubstrate must
also be modified. This hypothesis may explain why only 2 of the 20
acidic residues predicted to be on the substrate-binding surface of the
catalytic core appear to be important in substrate recognition. These 2
residues, Glu
and Glu
, are located near the
catalytic site in the cleft between the two lobes of the kinase and are
implicated in binding to arginine at P-3 in the regulatory light
chain. When these 2 residues are mutated, the V
/K
ratio
decreases 138- and 72-fold, respectively (Herring et al.,
1992). These 2 residues also appear to bind to or near the
autoinhibitory domain (Gallagher et al., 1993).
and
Glu
, are equivalent to Glu
and
Glu
, respectively, in rabbit smooth/nonmuscle myosin
light chain kinase (Fig. 4). The twitchin structure shows that
these 2 acidic residues make potential contacts (3.5 Å) with
Lys
and Lys
, respectively, in
-helix
11. From sequence alignment, Lys
in the calmodulin
binding domain of myosin light chain kinase and Lys
of
twitchin are conserved. By inference, it is possible that Lys
binds to Glu
and/or Glu
. The crystal
structure of calmodulin bound to a peptide analog of the
calmodulin-binding region of chicken smooth muscle myosin light chain
kinase reveals that Lys
binds to calmodulin (Meador et al., 1992). Consistent with this finding is the observation
that charge reversal substitution (K979E) decreased
Ca
/calmodulin-binding affinity (Fitzsimons et
al., 1992). However, additional investigations will be
necessary to determine if Lys
and other residues in the
calmodulin binding domain bind both calmodulin and residues in the
catalytic core of myosin light chain kinase.
Figure 4:
Amino
acid sequence alignments for rabbit smooth/nonmuscle myosin light chain
kinase twitchin. Alignment was obtained using the GCG software BESTFIT
program (Genetics Computer Group, Inc., 1992). Bestfit allows for
optimal alignment by the insertion of gaps to maximize the number of
matches utilizing the local homology algorithm of Smith and Waterman
(1981). The dots between mismatched sequences represent
evolutionarily related amino acids (Gribskov and Burgess, 1986). Boxedsequence indicates conserved catalytic core.
The asterisk indicates Lys in myosin light chain
kinase, which is equivalent to twitchin residue Lys
. The
Lys
binds to Glu
and Glu
(Hu et al., 1994). By homology, the
indicates the
acidic residues in the catalytic core (Glu
and
Glu
) for myosin light chain kinase proposed to bind to
basic residues in the autoinhibitory domain (Lys
) and
light chain substrate (Arg
),
respectively.
Only 2 acidic residues
(Glu and Glu
) located near the catalytic
site of myosin light chain kinase have been implicated in binding to
both the regulatory light chain and the autoinhibitory sequence (herein
and Gallagher et al. (1993)). Assuming binding homology
between twitchin and myosin light chain kinase, Lys
may
bind to Glu
and Glu
. However, basic
residues Lys
, Lys
, Lys
,
Lys
, and Arg
amino-terminal to both the
calmodulin-binding domain and the proposed pseudosubstrate sequence
also appear to be involved in inhibition of myosin light chain kinase
activity. In addition, 7 acidic residues following a distinct path
across the surface of the catalytic core are implicated in binding to
or near the inhibitory sequence but not to basic residues in the
regulatory light chain. Since only 2 acidic residues (Glu
and Glu
) near the catalytic core may bind both to
the autoinhibitory domain and the arginine at P-3 in the light
chain substrate, exposure of these 2 residues upon calmodulin binding
may be necessary and sufficient for light chain phosphorylation. We
conclude that the autoinhibitory sequence operates primarily through an
intrasteric rather than a simple pseudosubstrate mechanism in
inhibiting myosin light chain kinase activity.
Table: Kinetic properties of myosin light chain kinase
containing point mutations of basic residues within the regulatory
domain
]
values represent the Ca
concentration required
for half-maximal activation at 1 µM calmodulin. The
relative K
values were calculated for at least three
Ca
concentrations. V
and K
(light chain) values were determined
from Lineweaver-Burke plots. K
LC refers to K
values for the regulatory light chain.
ND, values not determined.
Table: Kinetic properties of myosin light chain
kinases containing catalytic core point mutations
values
were calculated for at least three Ca
concentrations. V
and K
values
were determined from Lineweaver-Burke plots. K
LC refers to K
values for the regulatory light chain.
, concentration of
Ca
/calmodulin required for half-maximal activation of
myosin light chain kinase;
[Ca
]
, concentration of
Ca
required for half-maximal activation of myosin
light chain kinase; PKI, inhibitor peptide for cAMP-dependent protein
kinase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.