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219
Protein kinase inhibition: natural and synthetic variations on a
theme
Susan S Taylor* and Elzbieta Radzio-Andzelm
How a protein
physiological
solved
kinase
is turned
function
crystal
subfamilies
structures
reveals
are effective.
inhibitors
Although
target
is due to the extended
inhibitors
occupy.
mechanisms
Although
or natural
site, specifically
high degree
targeting
is proving
to be very successful,
designing
inhibitors
that target
kinase
site
can be achieved
that these
of the ATP binding
is also wide
other surfaces
site
latitude
for
of the kinases.
Addresses
Department of Chemistry and Biochemistry, University of California at
San Diego, La Jolla, California 92093-0654, USA
*e-mail: [email protected]
Current Opinion
in Chemical
Biology
1997, 1:219-226
http://biomednet.com/elecref/1367593100100219
0
kinases, which are activated
through complex
but tightly
controlled
kinase cascades
in response
to a number
of
factors, including
stress and cytokines,
represent
only one
set of targets where selective
inhibitors
are actively being
sought.
as well
product
of the protein
there
by
the ATP binding
of specificity
surface
of
conformations.
other than the active
most synthetic
the active
site, a remarkably
which
mimetic
to surfaces
protein
that are utilized
into inactive
and adenine
as complementarity
Examination
different
of strategies
kinases
for its
activity.
representing
a variety
nature to lock protein
Pseudosubstrate
off is as critical
as is its catalytic
Current Biology Ltd ISSN 1367-5931
Abbreviations
catalytic
C
cdk
cyclin-dependent kinase
MAP
mitogen-activated protein
myosin light chain kinase
MLCK
PKA
CAMP-dependent protein kinase
PKG
cGMP-dependent protein kinase
PKI
protein kinase inhibitor
PRS2
peripheral recognition site 2
R
regulatory
Introduction
Fischer
and Krebs [l] were the first to demonstrate,
in
1955, that the reversible
equilibrium
between
active and
inactive
conformations
of a protein
could be regulated
by the post-translational
addition
of a phosphate
moiety.
Not only are there
thousands
of proteins
that utilize
this regulatory
mechanism,
but the number
of protein
kinases that catalyze
these phosphoryl
transfer
reactions
also number
in the hundreds
(reviewed
in [Z]). It is
predicted
that the human
genome
alone encodes
over
2000 protein
kinases.
Since
these
enzymes
regulate
almost every process
in the eukaryotic
cell by serving
as on/off
switches,
it is critical
that every
kinase
be
tightly regulated
physiologically.
They are turned off and
on by very precise
signals;
thus if one could develop
strategies
for selectively
regulating
a specific
kinase by
therapeutic
intervention,
one could
control
an entire
signaling
pathway. The mitogen-activated
protein (MAP)
Although
the catalytic
core of protein
kinases
has been
evolutionarily
conserved
in all eukaryotic
protein kinases
that phosphorylate
serine,
threonine,
and tyrosine
(reviewed in [Z]), the mechanisms
by which the inhibition
of each kinase is achieved
vary considerably.
Although
the details differ, a continuing
theme
is the utilization
of modular domains for multiple
functions.
As the crystal
structures
of more protein kinases have been solved, not
only of the catalytic
cores but also of the cores plus
their regulatory
domains,
a variety of motifs for regulation
have been identified.
These examples,
summarized
below,
not only provide a foundation
for thinking
more broadly
about the molecular
features that regulate each kinase, but
also provide wide latitude for developing
inhibitors
which
extend
beyond
those obtainable
using the conventional
approach of designing
molecules
that bind to the enzyme’s
active site. The purpose
of this review is to summarize
some of the mechanisms
used physiologically
to achieve
protein kinase inhibition
and compare
them to the mechanism that is used by most known synthetic
and natural
product inhibitors.
Three specific inhibition
mechanisms
will be discussed.
Twitchin
and CAMP-dependent
protein
kinase (PKA) are examples
of protein
kinases
that are
regulated
by a pseudosubstrate
mechanism;
cdk2 and the
inactive
insulin receptor
provide
examples
of inhibition
that is achieved
by an adenine
mimetic
mechanism;
Hck
and Src provide examples
of inhibition
that is achieved
by
locking the enzyme
into an inactive
conformation
using
surfaces other than the active site. Inhibition
mechanisms
are discussed
in the context
of the active enzyme
and
the requirements
for activity at the active site cleft. The
inhibitor
domains
can
be part
of the
same
polypeptide
chain, as in twitchin and Hck, or part of a separate subunit,
as in PKA and cdk2. In many cases these
inhibitory
domains
serve dual functions
its inactive state and binding
conformation.
Structural features
by inhibiting
the kinase in
to other ligands in the active
of the protein kinase core
All known eukaryotic
protein
kinases
share a conserved
catalytic
core, in spite of considerable
diversity
in size,
mechanism
of regulation
and activation,
and subcellular
localization.
Since the structural
features
of this core in
its active conformation
are best understood
in functional
terms for PKA, one of the simplest
protein
kinases,
we
shall use it as a prototype
for the entire
family. The
220
Next generation therapeutics
Modes of inhibition
Pseudosubstrate
inhibition
The mechanism
of inhibition
whereby
the active site
of the kinase is occupied
by an inhibitor
peptide
that
resembles
a substrate was first suggested
as the mechanism
for inhibition
of the catalytic
subunit
of PKA by its
regulatory
subunits
(discussed
in [14]). This strategy
as
a general inhibition
mechanism
was more fully explored
by Kemp and co-workers
[15] with myosin light chain
kinase (hILCK).
The crystal structures
of PKA [4] and
twitchin
[16**] support this mechanism
of inhibition.
The
crystal structure
of the C subunit of PKA w;1s first solved
as a binary complex with a peptide
derived from the heat
stable protein kinase inhibitor
(PKI) (residues
5-24). The
PKI sequence
resembles
the consensus
sequence
for PKA
substrates.
As predicted
by the binding of peptide
analogs
to PKA [17], this pseudosubstrate
occupies
the active site
cleft [18]. In addition
to this consensus
sequence,
which
alone is not sufficient
to convey high affinity binding, PKI
has an essential amphipathic
helix that lies amino-terminal
to the consensus
site. This helix binds to a site peripheral
to the active site cleft. This peptide,
PKI (residues
5-2-l),
binds with high affinity (Ki =0.2 nhl) in the presence
of
ATP [17,19].
The regulatory
(R) subunits
of PKA contain
a similar
which
is thought
to occupy
the
consensus
sequence,
active site cleft in a similar
manner.
To achieve
high
affinity
binding,
however,
the R subunit
interacts
with
the surface on the C subunit
that lies carboxy-terminal
to the consensus
site. This region includes
the essential
phosphorylation
site, Thr197, and the basic residues
that
surround
this phosphate
[ZO]. This site is designated
as
peripheral
recognition
site 2 (PRSZ), to distinguish
it from
the surface that is required
by PKI [21]. Binding of these
inhibitors
is thus bipartite.
One segment
that resembles
a substrate
binds to the active site cleft. while a second
segment.
contiguous
in PKI but not contiguous
in R, binds
to an additional
site peripheral
to the active site cleft.
Both PKI and R are competitive
inhibitors
with respect
to peptide
substrates.
The structure
of twitchin
(Figure
2) provides
probably
the most dramatic
example
to date of pseudosubstrate
inhibition
[ 16”]. In this case, the conserved
kinase core is
followed by an extended
pseudosubstrate
sequence,
which
occupies the active site cleft as was predicted
earlier. This
is followed by an IgG domain. The structure
of the kinase
domain
and the autoinhibitor
segment
alone define the
core and confirmed
the pseudosubstrate
mechanism
of
kinase inhibition
[ZZ], while the subsequent
structure
of
the kinase core plus the IgG domain
shows the active
site completely
occluded
by the adjacent
domain
[ 16”].
The pseudosubstrate
is docked
into the active site cleft
while
the IgG domain
securely
anchors
itself on the
Protein
Figure
kinase
inhibition:
natural
and synthetic
variations
on a theme
Taylor
and Radzio-Andzelm
221
1
(b)
.oop
Active
conformation
of the conserved
core of the catalytic
subunit
of PKA. (a) In the center
is the conserved
c 1997 Current Opmvx
I” Chemical Biology
core in its closed
conformation
wit h
the two lobes (residues 40-l 27 and 126-300)
shaded differently. The conserved residues (G50, G52, G55, K72, E91, Dl66,
N171, D164,
E206, D220 and R260, in single letter code for amino acids) are shown as balls. ATP is bound at the base of the active site cleft between the
two lobes while the inhibitor peptide PKI (residues 5-241, shown in white, binds on the surface of the large lobe. (b) An expanded view of the
small lobe where the central role of the glycine-rich
loop is emphasized. The glycine-rich
loop (residues 49-57)
and the linker that joins the two
lobes (residues 120-l 27) are shown in black. The structures
were generated using PDB file 1 ATP.
surface that lies carboxy-terminal
to the active site cleft.
In hlLCK.
a related family member,
the pseudosubstrate
peptide
domain overlaps
with a calmodulin
binding
site
(discussed
in [23]). When Car+ is bound to calmodulin,
the CaZ+/calmodulin
complex competes
favorably with the
inhibitory
domain,
thus exposing
the active site of the
enzyme.
Twitchin
is activated
in a similar fashion,
by
binding to another protein complex,
CaZ+/SlOO [El].
PKA and twitchin
are assembled
as fully active modules
that are maintained
in an inactive
state by formation
of
a stable complex
with an inhibitor.
In the case of PKA,
the inhibitor
is a separate
protein, whereas
with twitchin
(and IZILCK) the inhibitor
is a contiguous
domain of the
kinase itself. In both cases, a secondary
messenger
causes
the release of the inhibitor
segment
[14,23].
cyclin (see Figure 3). The carboxyl
terminus
of p27Q~l
specifically
folds over onto the small lobe of the kinase
core. The glycine-rich
loop, a key component
of the
nucleotide
positioning
motif, becomes
a distorted
and
discontinuous
part of the p sheet.
The Tyr88 residue
of p27Kipl folds into the adenine
binding
pocket
and
hydrogen
bonds with the backbone
carbonyl
of Glu81
and the backbone
amide
of Leu83,
both in the short
linker
strand
that joins the two lobes.
A comparison
of the active site filled by ATP and the corresponding
adenine binding pocket of cdkZ-cyclin-p27Kil’l
filled with
the Tyr88 of p27Kipl is also shown
in Figure
3. How
widely this mechanism
is utilized by other protein kinases
must await crystal structures
of other inhibited
enzymes.
However,
given the critical importance
of protein-protein
interactions
for kinase signaling
pathways.
this is likely to
be used
Adenine
mimetic
repeatedly.
inhibition
While pseudosubstrate
inhibition
is very attractive
and
logical, it is not the only mechanism
by which kinase
inhibition
is achieved.
Another
mechanism
that has onlv
been recognized
more recently utilizes a peptide
segment
that mimics
the adenine
ring of ATP. This
pepcide
functions
as an adenine
mimetic
and fills the shielded
pocket
that is typically
occupied
by the nucleotide
base in the active kinase.
To date, the best example
of this mechanism
is represented
by the structure
of
p27Kllll bound to the cdk2-cyclin
complex
[ZS”]. In this
structure
p27KiPl spans the surface
of both cdk2 and
The
structure
of a truncated
form
of the insulin
receptor
provides another example in which a peptide sequence
can
fold into the active site cleft and mimic the adenine ring of
ATP, thus inhibiting
enzymatic
activity [26]. In this case,
it is Tyrl158,
part of the activation
loop, that folds into
the adenine binding pocket. The tyrosine hydroxyl makes
contacts similar to those seen in the cdk2-cyclin-p27W~~
complex.
This crystal
structure
suggests
an intriguing
mechanism
whereby
the dephosphorylated
tyrosines
in
the activation
loop stabilize
the inactive
conformation
of the enzyme.
The full physiological
relevance
of this
222
Next
Figure
2
generation
therapeutics
Autoregulation
of the twitchin and Hck kinases [16”,26”].
Autoregulation
(inhibition of kinase activity) is achieved using two different
mechanisms
that result in the active site being maintained in an inhibited state by domains that flank the core. (a) The structure of the twitchin
kinase core joined to the autoinhibitor
segment and the IgG domain. The autoinhibitor
segment (residues 6199-6255)
is locked firmly into
place at the active site cleft by numerous contacts and by the positioning
of the IgG domain. (b) The structure of the Hck kinase core with its
amino-terminal
&c-homology
flanking SH2 and SH3 domains. In this case the SH2 and SH3 domains bind to Tyr526 in the carboxy-terminal
tail and to the linker strand that joins the SH2 domain to the core, respectively.
In this structure of the inhibited kinase, the active site cleft is
fully exposed but locked into a conformation
that cannot support catalysis. The structure of twitchin was generated using PDB file 1 KOA. The
coordinates
for Hck were kindly provided
by John Kuriyan
(The Rockefeller
mechanism
for the insulin
receptor
awaits a structure
solution of the full-length
protein,
since both membrane
localization
and the carboxy-terminal
tail may contribute
to
the conformation
of the core. The physiological
relevance
of this mode of inhibition
is also unclear since in the cell,
where ATP concentrations
are high, ATP may displace
Tyr1158
and lead to structural
instability.
It is clear,
however,
that the adenine
binding
pocket
can be an
important
potential
target for physiological
inhibition.
Other
mechanisms
inactive
for locking
the kinase
core in an
conformation
The recently solved structures
of two nonreceptor
tyrosine
kinases, Src and Hck, illustrate
another novel mechanism
for locking the catalytic
core into an inactive
conformation [27”,28**].
These
structures
(of the core plus the
contiguous
Src-homology
domains,
SH2 and SH3,) also
emphasize
the importance
of having a full length protein
structure
as opposed
to structures
of isolated domains.
It
is very easy to overinterpret
the significance
of crystal
packing in isolated domains
when the natural partner, in
this case the kinase core, is missing.
In the case of Src
and Hck, the finely tuned inhibition
mechanism
was only
revealed when the structures
of the proteins containing
all
three domains were solved [27**,28”].
The orientation
of the Hck kinase core relative
to the
SH2 and SH3 domains
is shown
in Figure
2b. This
conformation
is stabilized
by two specific
interactions:
the phosphorylated
Tyr at the carboxy-terminus,
Tyr527,
University,
New York).
is hydrogen
bound
to the adjacent
SH2 domain,
and
the proline-rich
segment
(which links the SH2 domain
to the kinase core) serves as an intramolecular
docking
site for the SH3 domain.
Consequently,
the kinase core
is physically
constrained
in a conformation
that cannot
support catalysis.
In addition
to restricting
the conformational flexibility
of the core, two specific regions of the
kinase core are either incorrectly
aligned
or disordered.
The
activation
loop is disordered
because
it is not
phosphorylated
[B”].
Activation
of Src correlates
directly
with the phosphorylation
of Tyr416 in its activation
loop
[29]. In addition, the C helix in the small lobe is twisted so
that the conserved
Glu310 is facing away from Lys295, its
partner
to the
in the active enzyme.
Instead
Glu310 is exposed
solvent
and faces the activation
loop, where
it
actually
forms an ion pair with the highly
conserved
Arg385, which immediately
precedes
the catalytic
loop.
Following
phosphorylation
of Tyr416, this same arginine
helps to assemble
the activation
loop into its correct
and active form (in the inhibited
conformation
the same
residue
helps to stabilize
the inactive
enzyme).
Correct
phosphorylation
of this loop is essential
for full activity of
most protein kinases (reviewed
in [30’]). In PKA, lack of
this essential
phosphate
(on Thr197) results in a decrease
in the efficiency
of phosphoryl
transfer
and an increase
in the Km for ATP [31]. In spite of its distance
from the
active site cleft, this phosphate
is linked by an extensive
network of interactions
to most of the residues involved in
recognition
of both ATP and peptide
substrates,
as well as
those involved
in catalysis.
Protein
kinase
inhibition:
natural
and synthetic
variations
on a theme
Taylor and Radzio-Andzelm
223
Figure 3
fb)
Beta 3
Linker
p27(84-93)
Linker
’ ATP
Structural views of the cdk2 kinase bound to an inhibitor, p2i’W
[25”1. (a) This structure shows cdk2-cyclin
A bound to an inhibitor peptide,
p2Wpt,
and reveals a novel mechanism of inhibition. p27W
flanks the surface of both cdk2 and cyclin A with the p27W
carboxy-terminal tail
binding to the adenine binding pocket of cdk2. (b) A view of the adenine pocket with Tyr88 hydrogen bonding to the linker strand and Lys33 of
the kinase core. In this conformation the glycine loop is very distorted. (c) The corresponding view of this region when ATP is bound, rather than
p27W.
to the cdk2-Cyclin
A complex. Amino acid residues are designated using single letter code in the figure. The coordinates for cdk were
kindly provided by Nikola Paveltich (Memorial Sloan-Kettering Cancer Center, New York).
The role of the SH3 domain
in maintaining
the kinase
core in an inactive conformation
was demonstrated
clearly
by a kinetic
analysis of the effect of Nef [32], a small,
(25-27 kDa) protein
required
for infection
by HIV. It
binds with high affinity to the SH3 domain of Hck. Nef
is a potent
activator
of the Src kinase by a mechanism
that specifically
involves displacement
of the SH3 domain
specificity.
Because the nucleotide
is deeply embedded
in
the active site cleft and extends
over such a large surface
area, there are potentially
many ways to achieve specificity.
As seen in Figure 1, the entire nucleotide-including
the
adenine,
ribose, and triphosphate
moieties -is
specifically
embraced
by the glycine-rich
loop. Crystal structures
of
several of these synthetic
inhibitors
have now been solved,
from the
regulation
and these structures
reveal at least one common
All of these inhibitors
target the adenine
binding
kinase core. This
of Src activity-a
that is quite
Synthetic
distinct
represents
strategy
from the active
a new strategy for
that targets a region
site.
and natural inhibitors
A number
of synthetic
and
natural
product
kinase
inhibitors
have now been identified,
and so far all compete
with ATP for binding.
Thus,
like p27Kip1, they have
a subsite
that mimics
the binding
of adenine.
Despite
being competitive
inhibitors
with respect to ATP, however,
and in contrast
to earlier
predictions,
a number
of
these
inhibitors
show exquisite
specificity.
Particularly
impressive
examples
are the pyridinylimidazole
inhibitors
that specifically
target ~38, a MAP kinase
[33]. Other
examples
are the pyrrolo- and pyrazoloquinazolines
that
are specific for the epidermal
growth factor (EGF) receptor
(reviewed
in [34]; [35]). Thus,
in spite
of a highly
conserved
core, a few critical differences
allow for high
feature.
pocket
whereby
one of the rings mimics the planar adenine
ring,
fills the pocket,
and also forms hydrogen
bonds to the
linker strand that joins the small and large lobes. This
hydrogen
bonding
is to the same backbone
carbonyl and
amide that are complemented
by the N6 and Nl nitrogens
of the adenine
ring of ATP.
A comparison
of the binding
of two such inhibitors
is
shown in Figure
4. H7, H8, and H89 are isoquinoline
sulfonamide
inhibitors
of PKA, PKI, and PKG. H89 shows
the highest
specificity
for PKA (Ki =O.SnM)
[36]. By
solving the crystal structures
of each of these inhibitors
in complex
with the C subunit of PKA, Engh et a/.[37-l
showed that the isoquinoline
ring mimics the adenine
ring
of ATP in the ternary complex of CATP.PKI
(5-24). The
nitrogen
in the isoquinoline
ring hydrogen
bonds to the
224
Next generation
therapeutics
linker strand, mimicking
the hydrogen
bond made by the
Nl nitrogen of the adenine ring of ATI? The higher degree
of selectivity
in H89, compared
to H7 and H8, is due to
an extended
glycine-rich
region, including
a bromine,
loop, resulting
in distortion
that interacts
of the loop.
the
The crystal structures
of cdk2 bound to three different
inhibitors
also reveal a common
mechanism
of inhibition
[38,39]. All have rings that occupy the adenine
binding
pocket,
and most also form hydrogen
bonds with the
linker strand. The orientation
of the rings differs, however.
Olomoucin,
a highly specific inhibitor,
actually contains
a purine
ring like ATP, but differs because
there is a
bulky substitution
on the N6 nitrogen.
As a consequence
of this bulky substituent,
the olomoucine
purine ring is
oriented
in a configuration
that is reversed
compared
to
the purine ring of ATP [39]. In this case, the hydrogen
bonding
involves the N3 and N9 nitrogens
in the purine
ring which replace the N6 and Nl nitrogens
in ATI?
The
indolines
are another
group
of highly
specific
ATP-based
inhibitors.
Some of these compounds
contain
an oxindole core, and are specific for the fibroblast growth
factor (FGF) receptor tyrosine kinase [40*]. The structure
of this complex reveals that the oxindole
ring binds to the
adenine
pocket of the FGF receptor and hydrogen
bonds
to the backbone
of the linker strand.
Specific inhibition
of the ~38 kinase by pyridinylimidazoles is one of the best examples
where the molecular
basis
for the specificity
of this inhibition
is understood.
These
compounds
were first identified
by screening
for
inhibitors
of the lipopolysaccharide-induced
immune
response. In a classic search for the target of these inhibitors,
Lee et al. [33] identified
~38, a member
of the MAP
kinase family. The selectivity
of these inhibitors
has been
well documented
[41]; however
the actual basis for the
selectivity
remained
unclear until crystal structures
were
solved. The crystal structure
of ~38 is similar to that of
ERK-2, another MAP kinase [42,43]. It was the structure
of ~38 bound to VK-1911, a pyridinylimidazole,
however,
that clearly
correlated
specificity
with a single
amino
acid difference
in the ATP-binding
pocket
[44*-l. The
structure-based
prediction
was confirmed
by mutagenesis
of this amino acid [44**].
Conclusions
Protein
kinases
are clearly
important
targets
for drug
design. Earlier predictions
that achieving
specificity
would
be difficult given the highly conserved
nature of the kinase
core are clearly not valid. So far most synthetic
inhibitors
target the adenine
binding
pocket at the active site cleft
and compete
with ATP for binding.
Despite
this, high
specificity
can be achieved
by this strategy [33,34,36,40*].
As the structures
of more inhibitor-kinase
complexes
are
solved, rules will hopefully
be defined
which will allow
rational
design
of inhibitors
that will target a specific
protein
kinase. We are rapidly moving in that direction.
The conserved
nature of the kinase core also makes it an
excellent
candidate
for homology
modeling,
whereby
the
Figure 4
(a)
Structure
(b)
of synthetic inhibitors bound to various protein kinases reveal a common mechanism
the active site cleft [37-l. (a) Shows ATP binding to the C subunit of PKA superimposed
with
thick line. and ATP is shown as a thin line. This structure was generated using PDB file 1YDS.
the C subunit of PKA superimposed
with ATP bound to the same pocket. In this case, H89 is
line. This structure was generated using PDB file 1 YDT.
for binding to the adenosine binding pocket at
H8 binding to the same pocket. H8 is shown as a
(b) Shows H89 binding to the active site clefi of
shown as a thin line and ATP is shown as a thtck
Protein
sequence
of the conserved
kinase
inhibition:
core of any protein
natural
kinase
and synthetic
can
be modeled
onto a three-dimensional
structural
template
[15]. Once
again, as more structures
are solved,
the
accuracy
of the modeling
also will improve.
It is also
variations
on a theme
Taylor and Radzio-Andzelm
225
subunit of CAMP-dependent protein kinase complexed with
MgATP and peptide inhibitor. Biochemistry 1993, 32:2154-2161.
11.
Hemmer W, McGlone ML, Taylor SS: The role of the glycine
triad in the ATP-binding site of CAMP-dependent protein
kinase. J B/o/ Chem 1997, 272:16946-l
6954.
clear, however,
that the design of specific inhibitors
need
not target only the active site cleft. The C helix, the
12.
Bossemeyer
D: The glycine-rich sequence of protein kinases: a
multifunctional element Trends Biochem Sci 1994, 19:201-205.
activation
loop or the segments
that flank the kinase core
are equally valid targets for drug design, as is targeting the
ligand binding sites at the active site. Although
designing
inhibitors
that bind to the active kinase is one strategy,
designing
or screening
compound
libraries for inhibitors
that prevent
kinase
activation
is an equally
plausible
strategy - one that has been explored
by nature but yet
to be exploited
by combinatorial
chemistry.
13.
Herberg F, Zimmermann B, McGlone M, Taylor SS: Importance of
the A-helix of the cat&tic
subunit of CAMP-deoendent orotein
inase for stability and f& orienting subdomain; at the ileft
interface. Protein Sci 1997, 6:569-579.
14.
Taylor SS, Buechler JA, Yonemoto W: CAMP-dependent protein
kinase: framework for a diverse family of regulatory enzymes.
Annu Rev Biochem 1990, 59:971-l 005.
15.
Kniahton DR. Pearson RB. Sowadski JM. Means AR. Ten Evck LF.
Taylor SS, Kemp BE: Structural basis for intrasteric regulation’
of myosin light chain kinase. Science 1992, 258:130-135.
16.
..
Acknowledgements
SW is supported by grants
from the National Institutes
of Health and the
American Cancer Society. ER-A was supported in part by National Institutes
of Health ‘liaining
Grant I’HS ‘1‘32 DK07233. The authors wsh to thank
N Narayana and T Diller for helpful comments
and discussions.
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