From the
The substrate binding properties of skeletal muscle myosin light
chain kinase were investigated with a synthetic peptide containing the
photoreactive amino acid p-benzoylphenylalanine (Bpa)
incorporated amino-terminal of the phosphoacceptor serine
(BpaKKRAARATSNVFA). When photolyzed at 350 nm, the peptide was
cross-linked stoichiometrically to myosin light chain kinase in a
Ca
The
cross-linked photoadduct was digested, labeled peptides were purified
by high performance liquid chromatography, and sites of covalent
modification were determined by amino acid sequencing and analysis. The
covalent modification in the catalytic core occurred on Ile-373 (66%)
and in a peptide containing residues Asn-422 to Met-437 (14%),
respectively. Lys-572 in the autoinhibitory region accounted for 20% of
the incorporated label. The coincident covalent modification of the
autoinhibitory domain suggests that it is located near the catalytic
site. Based upon a model of the catalytic core, the substrate peptide
is predicted to bind in the cleft between the two lobes of the kinase.
The orientation of the substrate peptide on myosin light chain kinase
is similar to the orientation of the substrate recognition fragment,
but not the high affinity binding fragment, of inhibitor peptide of
cAMP-dependent protein kinase in the catalytic subunit of the
cAMP-dependent protein kinase.
Myosin light chain kinase catalyzes the
Ca
In contrast to the cAMP-dependent protein
kinase, myosin light chain kinases are dedicated protein kinases that
only phosphorylate the regulatory light chains of myosin
physiologically (Stull et al., 1986). Studies with peptide
substrates showed the consensus phosphorylation sequence for the
regulatory light chain from smooth muscle to include
KKR XXR XXS XVF (Kemp and Pearson, 1991). The
specific spatial arrangement of basic residues amino-terminal and
hydrophobic residues carboxyl-terminal of the phosphoacceptor serine
are important in peptide substrates. This unique sequence contributes
in part to the high specificity of myosin light chain kinase, although
there are recognition determinants in other subdomains of the light
chain (Zhi et al., 1994).
In cAMP-dependent protein kinase,
the structure of an inhibitor peptide (PKI)
In this investigation we characterized the conditions for the
covalent labeling of rabbit skeletal muscle myosin light chain kinase
with a synthetic peptide substrate containing a photoreactive amino
acid, Bpa, at the P-9 position that is immediately amino-terminal of
the three basic residues that are substrate recognition determinants
(BpaKKRAARATSNVFA). The structure of this peptide is based upon the
sequence of the smooth muscle myosin light chain, which is a good
substrate for the skeletal muscle myosin light chain kinase (Michnoff
et al., 1986). The locations of the covalently modified sites
in the catalytic core of the kinase were determined following digestion
of the peptide-kinase complex and purification of reacted peptides by
HPLC.
Truncated
myosin light chain kinase was constructed by deleting residues
1-256 by oligonucleotide mutagenesis of the cDNA for the
full-length kinase (Zoller and Smith, 1987). Deletion of the DNA was
confirmed by DNA sequencing. The cDNA for the truncated myosin light
chain kinase was subcloned into baculovirus transfer vector pVL 1392
and cotransfected into Sf9 insect cells with BaculoGold
Myosin
light chain kinase was also purified from rabbit skeletal muscle
(Herring et al., 1990a). Myosin regulatory light chain was
prepared from fresh rabbit skeletal muscle according to Blumenthal and
Stull (1980). Calmodulin was purified from bovine testis (Bowman et
al., 1992).
The photolysis
mixture containing 10 mg of myosin light chain kinase cross-linked to
peptide I was applied to a DEAE-Sephacel column (1.4
Peptide I
was also phosphorylated by myosin light chain kinase in the presence of
[
The lyophilized digest was
dissolved in 1 ml of 0.1% trifluoroacetic acid (eluent A) and applied
to reverse phase HPLC. A Vydac C8 column (4.6
Amino acid analysis was
performed with a Pico-Tag system from Waters Corp. (Milford, MA)
following precolumn derivatization of residues with phenyl
isothiocyanate according to manufacturer's recommendations.
Hydrolysis was performed for 22 h at 110 °C in the presence of
vapor-phase, constant-boiling HCl containing 1:200 (v/v) phenol as a
scavenger.
Peak III was applied to a Vydac C18 column
(4.6
Peak IV was first rechromatographed with the
same conditions used for peak I and subsequently purified with a linear
gradient of 0-100% eluent B (eluent A, 0.12% trifluoroacetic
acid; eluent B, 70% acetonitrile, 0.1% trifluoroacetic acid) in 100 min
at 40 °C. Sequencing of the radioactive peak showed that the sample
contained two peptides, both containing one cysteine. The sample was
therefore reduced, carboxymethylated, and purified with a linear
gradient of 0-100% eluent B (eluent A, 0.2% 6 N HCl;
eluent B, 0.2% 6 N HCl, 70% acetonitrile) for 100 min
(Fig. 8 D).
The purified peptides derived from peaks
I, II, III, and IV were subjected to amino-terminal sequence analysis
(). Residues from peptide I itself were not identified in
the sequence analysis because the amino terminus was blocked by the
previous
Peak III contained an atypical V8 cleavage product on
the carboxyl-terminal side of Ser-567, which precedes the purified
peptide containing residues Gln-568 to Met-576. Following CNBr
digestion, methionine may form mixtures of homoserine, homoserine
lactone, and methylthiocyanate and would not be detected under
conditions used for sequencing. Lys-572 at cycle 5 was not observed,
suggesting that this residue was covalently cross-linked to peptide I
and remained on the filter. Values for amino acid residues after cycle
5 were similar to the initial values.
The peptide purified from peak
IV was derived from the carboxyl-terminal cleavage of myosin light
chain kinase by V8 protease and contained the sequence Asn-422 to
Asp-437. Because there was no significant decrease in the yield of the
residue values during sequencing, it was not possible to identify the
specific labeled side chain. This result probably reflects
decomposition of this specific photoadduct during the sequencing
process.
p-Benzoylphenylalanine has been introduced as a
photoreactive amino acid into synthetic peptides that bind calmodulin
(O'Neil et al., 1989). It has also been substituted for
the phosphorylatable serine residue of a synthetic peptide substrate
for the cAMP-dependent protein kinase (Miller and Kaiser, 1988). When
photolyzed at 350 nm, the appropriate target protein was cross-linked
in a time- and concentration-dependent manner with stoichiometric
incorporation of peptide. In the present work, the incorporation of Bpa
into the amino terminus of a synthetic peptide substrate for myosin
light chain kinase had no significant effect on its phosphorylation
properties, indicating that the binding of peptide I to the catalytic
site had not been perturbed. The stoichiometric cross-linking of Bpa to
the kinase with the proportional loss of enzyme activity indicates that
the amino terminus of the peptide is in close proximity to the
catalytic core. Incorporation of Bpa into selective positions of other
synthetic peptide substrates may provide useful reagents for examining
the biochemical properties of other protein kinases.
Evidence was
obtained for the specific incorporation of peptide I into myosin light
chain kinase: 1) the extent of incorporation was saturable at 1 mol of
peptide I/mol of kinase; 2) the concentration of peptide I required for
half-maximal labeling was similar to the K
ATP in the presence of Mg
It has been proposed that myosin light chain kinase is
regulated by an intrasteric mechanism where the autoinhibitory domain
folds back onto the active site to inhibit light chain binding (Kemp
and Pearson, 1991; Gallagher et al., 1993). This proposal is
supported by the Ca
The major site labeled on myosin light chain kinase (peaks I and II)
is located at Ile-373, which represents 66% of the total
Peak
III contained a peptide that represents a portion of the autoinhibitory
domain from Gln-568 to Met-576. The sequence analysis suggested that
Lys-572 was covalently modified with 20% of the total incorporation.
The autoinhibitory domain may be near the peptide substrate binding
site on surface of the catalytic core of the kinase. This placement is
consistent with an intrasteric mechanism of regulation in which the
autoinhibitory domain folds back on the catalytic core and is near the
active site (Bagchi et al., 1992; Hu et al., 1994).
The general position of the autoinhibitory domain on the catalytic core
is consistent with results obtained with
Ca
Peak IV contained a peptide with residues
Asn-422 to Asp-437 representing 14% of the total radioactivity
incorporated. Unfortunately, the sequence analysis failed to identify
the covalently modified residue. This peptide is proposed to form
The cross-linking of peptide I
to three distinct sites in myosin light chain kinase is consistent with
the results on phosphorylation of peptide I bound to the kinase. The
less than stoichiometric phosphorylation (0.6 mol of
The solved crystal structures of cAMP-dependent protein
kinase and other protein serine/threonine kinases make a critical
contribution to our understanding of the backbone structure of the
catalytic cores (Taylor and Radzio-Andzelm, 1994). A common motif
includes a bilobal catalytic core with a deep cleft between the two
lobes (Fig. 9 A). The adenine portion of ATP is buried
deep within a conserved hydrophobic pocket of the small lobe with the
phosphates oriented to the opening of the cleft. Some important
structural features include the glycine-rich loop in the ATP binding
site of the small lobe and the catalytic loop with an aspartate
catalytic base in the large lobe. These structures come together at the
cleft to position the
In the
cAMP-dependent protein kinase, the five-residue consensus sequence for
substrate recognition in PKI lies along the surface of the cleft
between the two lobes near the catalytic site (Fig. 9 A).
The more distal portion of the peptide forms an amphipathic
The orientation of peptide I on myosin light chain kinase is similar
to the orientation of the substrate recognition fragment of PKI.
Although it is more extended, the binding appears to be contained
between the two lobes of the kinase. These results are not consistent
with the placement of the peptide substrate in the putative hydrophobic
substrate binding groove in a similar position as the
Although the binding properties of the peptide represent an
important part of substrate recognition, recent evidence indicates that
there are also elements of substrate recognition in the intact light
chain distal from the phosphoacceptor serine (Zhi et al.,
1994). Additional investigations will be needed to establish the
complete determinants of substrate recognition in the protein
substrate.
Rates of phosphorylation were measured at different peptide
concentrations as described under ``Experimental
Procedures.'' Kinetic constants were determined from
Lineweaver-Burke plots and mean values with S.E. are shown for at least
three measurements.
We thank Faming Zhang for assistance in preparing
figures of the modeled kinases. We also thank William F.DeGrado for the
generous gift of Bpa used in the chemical synthesis of the peptide and
Phyllis Foley for assistance in the preparation of this manuscript.
Coordinates for the cAMP-dependent protein kinase catalytic subunit and
myosin light chain kinase were kindly provided by Janusz Sowadski.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/calmodulin-dependent manner. Peptide incorporation
into kinase inhibited light chain phosphorylation, and the loss of
kinase activity was proportional to the extent of peptide incorporated.
After peptide I was incorporated into myosin light chain kinase, it was
partially phosphorylated in the absence of
Ca
/calmodulin. The extent of phosphorylation
increased in the presence of Ca
/calmodulin.
/calmodulin-dependent phosphorylation of the
regulatory light chain of myosin. Light chain phosphorylation is
thought to be responsible for smooth muscle contraction, cell shape
changes, receptor capping, and potentiation of skeletal muscle
contraction (Kamm and Stull, 1985; Stull et al., 1986, 1991;
Sweeney and Stull, 1990; Sweeney et al., 1993). Myosin light
chain kinases contain a central catalytic core that is homologous to
other protein kinases, including the cAMP-dependent protein kinase
(Hanks et al., 1988). Our knowledge of the binding and
phosphorylation of peptide substrates to the catalytic core of the
cAMP-dependent protein kinase is based upon extensive biochemical
studies complementing the crystal structure of the enzyme (Taylor
et al., 1993).
(
)
containing a pseudosubstrate sequence for the consensus
phosphorylation sequence has been determined (Knighton et al.,
1991a, 1991b). Two basic residues important for recognition as a
substrate are close to the phosphoacceptor site at the P-2 and P-3
positions and bind in a plane between the two lobes of the kinase.
Mutagenesis studies of the rabbit skeletal and smooth muscle myosin
light chain kinases suggest that the arginine at the P-3 position in
the smooth muscle regulatory light chain binds to homologous residues
in both kinases (Herring et al., 1992; Gallagher et
al., 1993). However, there also are more distal basic residues at
the P-6 to P-8 positions that are specificity determinants in peptide
substrates. It has been proposed that three basic residues in a
putative pseudosubstrate sequence found in an internal autoinhibitory
domain bind to three specific acidic residues preceding near or in
-helix G (Knighton et al., 1992). This proposed model was
based upon the structure of the catalytic core of the cAMP-dependent
protein kinase and its bound pseudosubstrate inhibitor peptide, PKI.
Chemicals and Reagents
V8 protease from
Staphylococcus aureus was obtained from Boehringer Mannheim,
DEAE-Sephacel was from Pharmacia, protein grade Triton X-100
(hydrogenated) was from Calbiochem, and enzyme grade urea (ultrapure)
was from Life Technologies Inc. Acetonitrile (UV) was from Burdick and
Jackson Co. Sequence grade guanidine-hydrochloride, trifluoroacetic
acid, HFBA, and HCl were from Pierce. All other reagents were of
analytical grade.
Peptide Synthesis
A photoreactive amino acid, Bpa,
was incorporated as the amino-terminal residue of the peptide
BpaKKRAARATSNVFA (peptide 1). Bpa is a derivative of benzophenone and
is stable under the conditions of peptide synthesis. The diarylketone
in Bpa can preferentially react with C-H bonds rather than water
via a biradical mechanism with relatively low energy ultraviolet
radiation (Kauer et al., 1986; O'Neil et al.,
1989). Peptide synthesis was conducted with a model 430A automated
peptide synthesizer from the Applied Biosystems Division of Perkin
Elmer (Foster City, CA). Standard manufacturer's programming and
chemicals were employed. t-Butoxycarbonyl amino acids were
coupled as the hydroxybenzotriazole active esters, and the activation
and coupling reactions were performed in N-methylpyrrolidone.
The t-butoxycarbonyl derivative of Bpa was synthesized from
DL-Bpa (Molecular Probes Inc., Eugene, OR) using the
di- t-butyl dicarbonate method described by Stewart and Young
(1984). The peptide was made radioactive (0.046 µCi/nmol) by solid
phase reaction of the side chain protected peptide with
[H]acetic anhydride (Amersham Corp.) in
N-methylpyrrolidone to acetylate the N-
-amino
group of the amino-terminal residue. The structure of the peptide was
verified by fast atom bombardment mass spectrometry, which provided a
value for the molecular mass of 1713 u (expected value, 1712.98 u).
Recombinant Myosin Light Chain Kinases
A 1930-base
pair fragment of the rabbit skeletal muscle myosin light chain kinase
cDNA, including the entire coding region (nucleotides 210-2140)
(Herring et al., 1990a, 1990b) was subcloned into baculovirus
transfer vector pVL 1393 and cotransfected into Sf9 insect cells with
wild-type baculovirus DNA. The recombinant virus was isolated and
purified by screening for expression of myosin light chain kinase in
infected cells by immunoblotting and measurements of kinase activity
(Herring et al., 1990a, 1990b). Experiments were performed
with both tissue-purified and recombinant myosin light chain kinase.
The catalytic and activation properties are essentially identical, and
no differences were noted in cross-linking experiments.
linearized baculovirus DNA (Pharmingen). The latter DNA contains
a lethal deletion so that the transfected virus DNA cannot make viral
particles in insect cells unless the deletion is complemented by a
co-transfected polyhedron-based baculovirus transfer vector. The
purification of recombinant virus, assay of plaques, infection of
insect cells with recombinant virus, and collection of cells were
performed as described by Pharmingen. The expression of the truncated
kinase was verified by Western blotting,
Ca
/calmodulin-dependent kinase activity, and amino
acid analysis as described previously (Herring et al., 1990a,
1990b).
Protein Purification
Myosin light chain kinase was
expressed in the Sf9 cells infected by recombinant virus. The cells
were incubated at 27 °C for 4 days and harvested by centrifugation.
The cell pellets were lysed on ice for 20-30 min in 1% Nonidet
P-40, 20 mM MOPS (pH 7.0), 1 mM dithiothreitol, 0.5
mM EGTA, 10% glycerol, 2 mM MgCl, 0.2
mM phenylmethylsulfonyl fluoride, 0.04 mM leupeptin,
0.6 µM aprotenin, 0.015 mM N
- p-tosyl-L-lysine
chloromethyl ketone, 0.015 mM pepstatin A. After
centrifugation, the supernatant fraction was applied to a DEAE-Sephacel
column previously equilibrated with 20 mM MOPS (pH 7.0), 0.5
mM EDTA, 1 mM dithiothreitol, and 0.2 mM
phenylmethylsulfonyl fluoride buffer. The kinase was eluted with
0-0.3 M NaCl gradient for the full-length kinase and
0-0.2 M NaCl for the truncated kinase. The fractions
containing kinase were identified by enzyme activity assay and
SDS-PAGE. Myosin light chain kinases were further purified to
homogeneity by calmodulin-Sepharose affinity chromatography as
described previously (Herring et al., 1990a, 1990b).
Photoaffinity Labeling
Photolysis mixtures
typically contained 50 mM MOPS (pH 7.0), 1 mM
dithiothreitol, 0.2 mM CaCl, 5 µM
calmodulin, 4 µM myosin light chain kinase, and 30
µM peptide I, unless otherwise indicated. Photolysis was
performed in borosilicate glass tubes at 25 °C for 10 min with a
light source (four Rayonet RPR-3500 A lamps, Southern N.E. Ultraviolet
Co.) held at a distance of 1.5 cm from the samples. After photolysis,
the protein was separated by SDS-PAGE (7.5% polyacrylamide) according
to Laemmli (1970), except all buffers contained 1 mM EGTA.
Following electrophoresis, gels were stained, destained, and washed
with water for 1 h. Myosin light chain kinase bands (13 µg of
myosin light chain kinase/band) were excised from the gel and cut into
1-mm slices. A same sized gel slice without myosin light chain kinase
was excised to determine the radioactive background. One ml of 50
mM ammonium bicarbonate (pH 8.4) and 65 µg of
trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone
(Worthington) in 1 mM HCl were added to each sample and
incubated at 37 °C for 24 h on a shaker. Aliquots of each sample
were added to 15 ml of CytoScint (ICN), and the radioactivity was
quantitated by liquid scintillation spectrometry.
2.3 cm)
previously equilibrated with 10 mM MOPS (pH 7.0), 1
mM dithiothreitol, and 0.5 mM EGTA. The column was
sequentially eluted with linear gradients of 200 ml of 0-70
mM NaCl and 140 ml of 70-300 mM NaCl in the
buffer. Aliquots of selected fractions were applied for SDS-PAGE
(Laemmli, 1970) to identify the kinase fractions. The fractions
containing kinase were pooled, and cold acetone was added to a final
concentration of 80% (v/v) to precipitate the kinase. The kinase was
collected by centrifugation and dried by air.
Purification of Phosphorylated Peptide
Peptide I
was phosphorylated in the dark in 0.4 ml of 50 mM MOPS (pH
7.0), 10 mM magnesium acetate, 1 mM dithiothreitol,
0.2 µM calmodulin, 0.2 mM CaCl, 2
nM myosin light chain kinase, 50 µM peptide I,
and 1 mM ATP at 30 °C for 2 h. The reaction was terminated
by the addition of 30% acetic acid. The sample was applied to a column
containing 1.6 ml of Dowex 1-X8 (Bio-Rad) preequilibrated in 30% acetic
acid to remove nucleotides. The column was washed with 30% acetic acid.
An aliquot of each fraction was added to CytoScint to measure the
H radioactivity by liquid scintillation spectrometry.
Nearly 96% of the applied
H radioactivity was recovered in
the flow-through. The sample was lyophilized, redissolved in 0.1%
trifluoroacetic acid, and applied to a Spice Cartridge C18 column
(Analtech, Inc.). After washing the column in 13 ml of 0.1%
trifluoroacetic acid, the peptide was eluted with 0.1% trifluoroacetic
acid in acetonitrile. About 70% of the original peptide was recovered
with 0.9 mol of phosphate incorporated per mol of peptide. Peptide I
was also exposed to the same procedures, except ATP was omitted in
phosphorylation mixture. This provided a control sample for comparing
cross-linking efficiency of nonphosphorylated and phosphorylated
peptide I to myosin light chain kinase.
Two-dimensional Phosphopeptide Mapping
Myosin
light chain kinase (13 µg) was photolabeled with peptide I in the
presence of Ca/calmodulin as described above. A
control experiment was performed by replacing the photoreactive peptide
I with the unlabeled peptide. Then, 10 mM magnesium acetate
and 1 mM [
-
P]ATP were added in the
presence or absence of 4 mM EGTA. The reaction mixture was
incubated for 2 min or 50 min at 30 °C. The reaction was terminated
by the addition of SDS-PAGE sample buffer and immediately placing in
boiling water for 4 min. The samples were subjected to SDS-PAGE (7.5%
polyacrylamide). After electrophoresis, the gel was briefly stained
(about 5 min), destained, and soaked in water. The
P
radioactivity associated with myosin light chain kinase was measured by
a radioanalytic imaging system (AMBIS Systems, Inc.). The kinase bands
were excised and digested with trypsin (25 µg, Boehringer Mannheim)
in 0.7 ml of 50 mM ammonium bicarbonate (pH 8.4) at 37 °C
overnight. The gel slices were removed, and the solutions were
lyophilized. At least 87% of the initial radioactivity was recovered.
The samples were resuspended in electrophoresis buffer containing
acetic acid/formic acid/water (8.7:2.5:88.8 (v/v/v)) and spotted onto
Kodak cellulose thin-layer plates (160 µm thick, 20
20 cm).
Pyronin G (0.5%) was spotted near the sample. Peptides were
electrophoresed at 1000 V at 4 °C until the tracking dye migrated 7
cm. After drying, the plate was subjected to thin-layer chromatography
in 1-butanol/pyridine/acetic acid/water (60:100:20:80 (v/v/v/v)) in the
second dimension. Autoradiography on either Kodak X-Omat or Kodak Blue
Brand films gave a map showing the locations of
[
P]phosphopeptides. The radioactivity on the
plate was quantified by a radioanalytic imaging system.
-
P]ATP without cross-linking. Following
separation on Dowex 1-X8, the eluate containing
[
P]peptide I was lyophilized and digested in 0.5
ml of 50 mM ammonium bicarbonate (pH 8.4) containing 3 µg
of trypsin at 37 °C overnight. The digest was lyophilized and
subjected to two-dimensional phosphopeptide mapping as described above.
Myosin Light Chain Kinase Hydrolysis and Peptide
Purification
The dried kinase was dissolved in 2 ml of 70%
formic acid. CNBr (300 mg) was added to the sample at a molar ratio of
methionine to CNBr of 1:1000. Following incubation at room temperature
overnight in the dark, the digestion was terminated by the addition of
HO followed by lyophilization. The dried CNBr digest was
dissolved in 1 ml of 50 mM NH
HCO
, 1
mM dithiothreitol, 4 M urea, and 20 mM
methylamine. The pH was adjusted to 8.2. V8 protease (150 µg) was
added to the sample and incubated at room temperature overnight.
Another 100 µg of V8 protease was added and incubated for 5 h. The
final V8/protein ratio was 1:30 (w/w). After centrifugation, the
supernatant fraction was lyophilized.
250 mm, 5 µm,
330 A) was used with a Waters HPLC apparatus (Waters, Millipore Corp.).
The flow rate was 0.8 ml/min, and the gradient was composed of the
following linear segments at room temperature: 0-15% eluent B
(90% acetonitrile, 0.1% trifluoroacetic acid), for 20 min; 15-40%
eluent B, for 130 min; 40-60% eluent B, for 10 min followed by
100% eluent B for over 20 min. Fractions were collected at 1-min
intervals with absorbance measured at 214 nm. Aliquots of 40 µl
were added to CytoScint, and
H radioactivity was measured
by liquid scintillation spectrometry. The fractions containing
radioactivity were further purified at 40 °C on an Aquapore RP-300
column (100
2.1 mm, 7 µm; Brownlee Labs) with an Applied
Biosystems 103A Separation System (ABI). Specific details are presented
under ``Results'' and in the figure legends.
Carboxylmethylation
Peptide samples were
lyophilized and reduced in 80 µl of 5 M
guanidine-hydrochloride, 0.5 M Tris-HCl (pH 8.5), 2
mM EDTA, and 10 mM 2-mercaptoethanol at 37 °C for
1 h. Iodoacetic acid was added to 15 mM and incubated at 37
°C for 15 min. The reaction was terminated by the addition of
another 20 mM 2-mercaptoethanol and incubating at 37 °C
for 20 min.
Amino Acid Sequencing and Analysis
Automated
degradation was conducted using a model 475A amino acid sequencer from
the Applied Biosystems Division of Perkin Elmer (Foster City, CA),
fitted with an on-line model 130A PTH-derivative analyzer for
identification of residues. Manufacturer's programming and
chemicals were employed throughout.
Miscellaneous
The activities of myosin light chain
kinase were measured by P incorporation into rabbit
skeletal muscle myosin light chain as described previously (Blumenthal
and Stull, 1980). Myosin light chain kinase concentrations were
determined by the dye-binding method of Bradford (1976) with bovine
-globulin as the standard. Calmodulin concentrations were
determined by UV absorbance at 277 nm using an extinction coefficient
of 3300 M
cm
(Klee,
1977). The concentrations of phosphorylatable light chain and peptides
were determined by stoichiometric
P incorporation
(Blumenthal and Stull, 1982).
Phosphorylation Properties for Synthetic Peptide
Substrates
The phosphorylation properties of an unlabeled
synthetic peptide, KKRAARATSNVFA, and peptide I containing Bpa
(BpaKKRAARATSNVFA) were compared (). The
Kvalues obtained with the two peptides
for myosin light chain kinase were similar. There was a slight decrease
in the V
value obtained with the labeled peptide
compared with the unlabeled peptide. These values are similar to those
previously reported for the unlabeled peptide and myosin light chains
from rabbit skeletal or chicken smooth muscles (Michnoff et
al., 1986). These results suggest that the introduction of Bpa
into the synthetic peptide did not significantly alter its properties
as a substrate for rabbit skeletal muscle myosin light chain kinase.
Effect of Ca
The maximal extent of photoaffinity labeling of
myosin light chain kinase with peptide I was 0.92 mol of peptide I/mol
of kinase in the presence of Ca/Calmodulin and ATP on
the Covalent Incorporation of Peptide I into Myosin Light Chain
Kinase
/calmodulin, whereas
the extent of cross-linking in the presence of EGTA was less than 0.15
mol of peptide/mol of kinase (Fig. 1 A). MgATP did not
affect the extent of cross-linking in the presence of EGTA; however, it
inhibited the extent of peptide incorporation in the presence of
Ca
/calmodulin (Fig. 1 A). ATP in the
absence of Mg
had no effect on the
Ca
/calmodulin stimulated cross-linking of peptide I
to myosin light chain kinase.
Figure 1:
Conditions for the covalent
incorporation of peptide I into myosin light chain kinase. A,
effect of Ca/calmodulin and MgATP. The cross-linking
reactions were irradiated for 10 min in 50 mM MOPS (pH 7.0)
and 1 mM dithiothreitol in the presence of 0.2 mM
CaCl
plus 5 µM calmodulin ( Ca/CaM) or
3 mM EGTA. Additional conditions included 10 mM
magnesium acetate plus 0.2 mM ATP ( MgATP) or 0.2
mM ATP alone. B, effect of prephosphorylation on the
extent of peptide I incorporation into myosin light chain kinase.
Peptide I was phosphorylated by myosin light chain kinase and purified
as described under ``Experimental Procedures.''
Nonphosphorylated peptide I was treated similarly but without MgATP in
the phosphorylation reaction mixture. The cross-linking reactions were
irradiated for 10 min in the presence of
Ca
/calmodulin under standard conditions at the
indicated peptide concentrations. Solidbars are
phosphorylated peptide I, and the openbars are
nonphosphorylated peptide I. The values represent means ± S.E.
for at least three determinations.
The possibility was considered that
the inhibitory effect of MgATP on peptide I incorporation into kinase
was related to rapid phosphorylation and subsequent poor binding of the
phosphorylated peptide. To address this possibility, peptide I was
phosphorylated by myosin light chain kinase and purified.
Nonphosphorylated peptide I was treated identically, except ATP was
eliminated from the phosphorylation reaction mixture. The extent of
cross-linking of the phosphorylated peptide I was substantially less
than the nonphosphorylated form at 2 different peptide concentrations
(Fig. 1 B). These results collectively indicate that the
cross-linking of peptide I is
Ca/calmodulin-dependent, and phosphorylation of the
substrate peptide significantly decreases its affinity for the kinase.
Effect of Peptide I Concentration on the Covalent
Incorporation into Myosin Light Chain Kinase
When the
full-length myosin light chain kinase was incubated with different
concentrations of peptide I and irradiated for 10 min, the extent of
incorporation was affected (Fig. 2). A maximal extent of
incorporation of 0.92 mol of peptide I/mol of kinase was obtained at 30
µM peptide I. The concentration required for half-maximal
incorporation was 8 µM, similar to the
Kvalue ().
Figure 2:
Effect of peptide I concentration on the
covalent incorporation into myosin light chain kinase. The reaction was
performed in the presence of Ca/calmodulin as
described in the legend to Fig. 1 with various concentrations of
peptide I. Two forms of myosin light chain kinase were used for the
cross-linking, including kinase purified from rabbit skeletal muscle
(
) and the recombinant truncated form lacking residues
1-256 (
). The values represent means ± S.E. for at
least three determinations.
Myosin light
chain kinase truncated at the amino terminus (residues 1-256
deleted) has catalytic and activation properties similar to the
full-length kinase. It has Kand
V
values of 6.5 µM and 2000 pmol of
P incorporated/min/pmol of kinase compared with 4
µM and 2400 pmol of
P incorporated/min/pmol
of full-length kinase (data not shown). Furthermore, the
K
value (concentration of
Ca
/calmodulin required for half-maximal activation)
for the truncated kinase is 2 nM, which is similar to the 1
nM value obtained with the full-length enzyme (Blumenthal and
Stull, 1980). As shown in Fig. 2, the concentration dependence
for cross-linking peptide I to the truncated kinase was similar to the
full-length myosin light chain kinase.
Specificity of Peptide I Incorporation into Myosin Light
Chain Kinase
To provide additional information about the
specificity of peptide I incorporation into the substrate binding site
of myosin light chain kinase, competition experiments were performed.
The extent of incorporation at 10 µM peptide I was
measured in the presence of Ca/calmodulin and myosin
light chain or the nonlabeled peptide (Fig. 3). Increasing
concentrations of these 2 substrates inhibited peptide I incorporation,
whereas bovine serum albumin had no effect. Concentrations required for
half-maximal inhibition were about 20 µM. These results
are consistent with a specific interaction between peptide I and the
active site of myosin light chain kinase.
Figure 3:
Effect of substrates on the extent of
peptide I incorporation into myosin light chain kinase. The
cross-linking reactions were performed in the presence of 10
µM peptide I and Ca/calmodulin as
described in the legend to Fig. 1. Different concentrations of bovine
serum albumin (
), rabbit skeletal muscle myosin light chain
(
), and the nonlabeled peptide substrate described in Table I
(
), respectively, were added to the reaction mixtures before
irradiation for 2.5 min. The values represent means ± S.E. for
at least three determinations.
Relationship between Peptide I Incorporation and
Inactivation of Myosin Light Chain Kinase Activity
Peptide I was
photolyzed with myosin light chain kinase for various times, and
aliquots were withdrawn for measurements of the extent of peptide I
incorporation and kinase activity toward myosin light chain. The extent
of incorporation was dependent upon the time of irradiation with a
maximal incorporation of 0.91 mol of peptide I incorporated/mol of
kinase at 10 min (Fig. 4 A). The apparent half-time of
the photoincorporation was 2 min. There was no significant
photoinactivation in the absence of the photoreactive peptide I
(Fig. 4 A). However, in the presence of peptide I, there
was a time-dependent inactivation of catalytic activity toward myosin
light chain with a half-time similar to that obtained for
incorporation. The loss of kinase activity was proportional to the
extent of peptide I incorporation with extrapolated maximal
inactivation obtained at 1 mol peptide I incorporated/mol of kinase
(Fig. 4 B).
Figure 4:
Effect of peptide I incorporation on
myosin light chain kinase activity. Photolabeling was performed under
conditions described in legend to Fig. 1. After irradiation the extent
of peptide I incorporation and kinase activity was assayed as described
under ``Experimental Procedures.'' A, effects of
time on photolabeling () and photoinactivation (
) of myosin
light chain kinase by peptide I.
, myosin light chain kinase
activity in the absence of peptide I but in the presence of the same
concentration of nonlabeled peptides. B, relationship between
extent of peptide I incorporation and photoinactivation of myosin light
chain kinase. The values represent means ± S.E. for at least
three determinations. Symbols without S.E. bars are shown where the
S.E. values are smaller than the symbol.
Phosphorylation of Peptide I Cross-linked to Myosin Light
Chain Kinase
The possibility was considered that peptide I could
be phosphorylated when it was cross-linked to myosin light chain
kinase. Peptide I not cross-linked to myosin light chain kinase was
phosphorylated, followed by tryptic digestion for a control in the
two-dimensional phosphopeptide mapping. The migration of the
phosphopeptides from peptide I and autophosphorylated myosin light
chain kinase was distinct (Fig. 5, upperpanel,
insert). After the incorporation of 0.92 mol of peptide I/mol
of kinase, the complex was incubated at 30 °C in standard reaction
mixture containing [-
P]ATP with or without
EGTA. At 2 and 50 min, aliquots of the reaction mixture were applied to
SDS-PAGE. The bands containing kinase alone or kinase plus peptide I
were excised, digested with trypsin, and subjected to two-dimensional
phosphopeptide mapping. As described previously (Gao et al.,
1992), incubation of myosin light chain kinase alone with
[
-
P]ATP resulted in a time-dependent
P incorporation stimulated by
Ca
/calmodulin (Fig. 5, upperpanel). This intramolecular autophosphorylation of myosin
light chain kinase does not effect catalytic activity or calmodulin
activation. A similar pattern of incorporation was found with the
myosin light chain kinase-peptide I complex (Fig. 5, upperpanel). However, the extent of incorporation in the
presence of Ca
/calmodulin was not as large as that
obtained with myosin light chain kinase alone. Measurements of the
distribution of
P in the myosin light chain kinase-peptide
I complex showed that both were phosphorylated (Fig. 5, upperpanel). Most of the radioactivity was incorporated into
the peptide I with a maximum of 0.63 mol of
P
incorporated/mol of peptide I in the presence of
Ca
/calmodulin at 50 min. The extent of
phosphorylation of peptide I in the presence of EGTA was 0.36 mol of
P incorporated/mol of peptide I. A similar pattern of
P incorporation into myosin light chain kinase in the
kinase-peptide complex was similar to that obtained with kinase alone,
although the extent of autophosphorylation was less than the extent of
phosphorylation of peptide I in the complex (Fig. 5, upperpanel). Thus, although the kinase peptide complex does
not phosphorylate light chain (Fig. 4 B), it can
phosphorylate itself and the peptide bound to it. Furthermore,
Ca
/calmodulin stimulates the phosphorylation of both
myosin light chain kinase and peptide in the photoreacted complex.
However, the covalent incorporation of peptide I into kinase inhibited
the extent of autophosphorylation in myosin light chain kinase.
Figure 5:
Phosphorylation of peptide I cross-linked
to myosin light chain kinases. Upper panel, peptide I was
cross-linked to the full-length myosin light chain kinase and then
phosphorylated in the presence of CaCl or EGTA as described
under ``Experimental Procedures.'' After SDS-PAGE, the kinase
bands were excised and digested with trypsin. The digestions were
subjected to two-dimensional phosphopeptide mapping. Measurements of
the extent of myosin light chain kinase autophosphorylation without
peptide I incorporation were made after performing the cross-linking
reaction with the unlabeled substrate peptide described in Table I
( openbars). The extent of phosphorylation
of the peptide I-myosin light chain kinase complex was also measured
( solidbars). Phosphorylation of cross-linked peptide
I and myosin light chain kinase (autophosphorylation) was distinguished
by two-dimensional phosphopeptide mapping ( inset) that
separates peptides from peptide I ( I) and myosin light chain
kinase ( M and M), respectively. The radioactivity in
cross-linked peptide I ( hatchedbar) and cross-linked
myosin light chain kinase ( cross-hatched bar) were measured as
described under ``Experimental Procedures.'' Lower panel, peptide I was cross-linked to truncated myosin light chain kinase
lacking autophosphorylation sites (residues 1-256 deleted) and
then phosphorylated. After SDS-PAGE,
P in kinase bands was
quantified by liquid scintillation spectrometry. Phosphorylation was
measured in the absence (
,
) and presence (
,
)
of Ca
/calmodulin with (
,
) or without
(
,
) peptide I cross-linked to the
kinase.
The
truncated myosin light chain kinase without the amino-terminal
autophosphorylation sites was also used to examine the phosphorylation
of peptide I cross-linked to the kinase (Fig. 5, lowerpanel). The truncated kinase alone showed no
autophosphorylation in the presence of EGTA or
Ca/calmodulin. However, after cross-linking 0.9 mol
of peptide I/mol of kinase incubation of the complex with
[
-
P]ATP and magnesium acetate resulted in a
time-dependent incorporation of
P into the complex
(Fig. 5, lowerpanel). The maximal extent of
phosphorylation at 60 min was 0.51 and 0.38 mol of
P
incorporated per mol of kinase in the presence of
Ca
/calmodulin and EGTA, respectively. These results
are similar to those obtained with the full-length kinase.
Identification of the Labeling Sites on
Kinase
Myosin light chain kinase cross-linked with peptide I was
separated from free peptide and calmodulin using DEAE-Sephacel
chromatography. The free positively charged peptide I appeared in the
flow-through fraction. The kinase-peptide I complex was eluted with
sequential salt gradients (Fig. 6) and shown to be free of
calmodulin by SDS-PAGE. The purified kinase-peptide I complex was
sequentially hydrolyzed by CNBr and V8 protease as described under
``Experimental Procedures,'' and the products were purified
by C8 reverse phase HPLC. Four major radioactivity peaks (peak I, II,
III, and IV) were isolated (Fig. 7). The respective radioactivity
peaks were pooled, concentrated, made to 1% Triton X-100, and applied
to reverse phase rechromatography.
Figure 6:
Isolation of myosin light chain kinase
( MLCK) cross-linked to peptide I by DEAE-Sephacel
chromatography. The column (1.4 2.5 cm) was equilibrated with
the buffer A (10 mm MOPS (pH 7.0), 1 mM dithiothreitol, and
0.5 mM EGTA) and sequentially eluted with two linear NaCl
gradients (200 ml of 0-70 mM NaCl and 140 ml of
70-300 mM NaCl; dashedline) in buffer
A at a flow rate of 1 ml/min. Absorbance was monitored at 280 nm
( solidline). Fractions (3.8 ml) were collected and
analyzed by SDS-PAGE. The content of the fractions are marked above the
absorbance peaks. Kinase containing fractions indicated by arrows were pooled.
Figure 7:
HPLC separation of the CNBr and V8 digest
of myosin light chain kinase cross-linked to peptide I. The digestion
product was separated on a Vydac C8 R-P column (4.6 250 mm, 5
µm) and eluted with a flow rate of 0.8 ml/min at room temperature.
Fractions were collected at 1-min intervals, and
H
radioactivity was measured in 40 µl to identify peptide I
containing fragments ( dottedline). Absorbance was
monitored at 214 nm ( solidline). The gradient is
illustrated in the dashedline corresponding to
percentage of eluent B. Eluent A, 0.1% trifluoroacetic acid. Eluent B,
90% acetonitrile, 0.1% trifluoroacetic acid. Major radioactive peaks
I-IV were marked.
Aliquots from peak I were
chromatographed at 40 °C on an Aquapore RP-300 (100 2.1 mm,
7 µm) column connected with ABI HPLC system. A linear gradient from
0-100% eluent B (0.1% HFBA, 55% acetonitrile) and eluent A (0.13%
HFBA) was started at 30 min and continued for another 100 min. All
peaks were collected individually, and radioactivity was measured in
aliquots (Fig. 8 A). Radioactivity was associated with a
single absorbance peak.
Figure 8:
Final HPLC purification of radioactive
peaks I-IV. Dried samples were dissolved in eluent A with 1% Triton
X-100 and applied to an Aquapore RP-300 column (100 2.1 mm, 7
µm) and eluted with a flow rate of 50 µl/min at 40 °C. The
linear gradient started at 30 min from 0-100% eluent B over 100
min. Absorbance was monitored at 214 nm ( solidlines). Background absorbance from solvent was marked
with dashedlines. All fractions were manually
collected, and 5-µl aliquots of each fraction were used for
H radioactivity measurements. The bars represent
the radioactivity of fractions. A, peak I (eluent A, 0.13%
HFBA; eluent B, 55% acetonitrile, 0.1% HFBA); B, peak II
(eluent A, 0.12% trifluoroacetic acid; eluent B, 50% acetonitrile, 0.1%
trifluoroacetic acid); C, peak III (eluent A, 0.13% HFBA;
eluent B, 80% acetonitrile, 0.1% HFBA); D, peak IV (eluent A,
0.2% 6 N HCl; eluent B, 70% acetonitrile, 0.2% 6 N
HCl).
Peak II was first rechromatographed under
conditions similar to those used for peak I, except that eluent B
contained 80% acetonitrile. The eluted radioactive fraction was
subsequently purified by rechromatography with a linear gradient of
0-100% eluent B (0.1% trifluoroacetic acid, 50% acetonitrile) for
100 min (Fig. 8 B). Radioactivity was associated with a
single absorbance peak.
250 mm, 5 µm) with a Waters HPLC System. The same
eluent system described in the legend to Fig. 7was used, and a
shallow linear gradient was developed from 21-26% eluent B for
100 min at room temperature. The isolated radioactive peak was
subsequently purified under the same conditions used for peak I
purification, except that eluent B contained 80% acetonitrile. The
profile of the final purification of peak III is shown in
Fig. 8C.
H-acetylation. Peaks I and II yielded the same
CNBr fragment of myosin light chain kinase. Only the first two residues
were identified as glutamate and tyrosine, but as this sequence only
occurs once in this myosin light chain kinase, it is likely to
represent the sequence starting at Glu-371. The labeling of Ile-373 by
Bpa apparently blocked the Edman degradation. The differences in the
retention times for the two peptides with identical sequences from
peaks I and II suggest that two isomers were formed during photolysis
arising from different atomic positions of insertion of the Bpa moiety
into the Ile side chain. Amino acid analysis of peak I provided results
consistent with the identification of peak I based on sequence analysis
(I). The results showed that the composition of peak I
consisted of residues predicted for the Glu-371 to Glu-374 fragment
obtained after CNBr and V8 protease digestions in addition to residues
in peptide I.
value; 3) the loss of kinase activity was proportional to the
extent of incorporation; 4) the incorporation was inhibited by peptide
or protein substrates; and 5) incorporation was
Ca
/calmodulin-dependent. Similar conclusions were
reached regarding the incorporation of a Bpa-peptide substrate for the
cAMP-dependent protein kinase where stoichiometric incorporation and
protection with peptide substrate suggested specificity of
photoaffinity labeling (Miller and Kaiser, 1988). However, an important
difference is that Bpa replaced the phosphoacceptor serine at the P
position in the peptide labeling of cAMP-dependent protein kinase,
whereas Bpa is at the P-9 position in relation to the phosphoacceptor
serine in peptide I.
inhibited the incorporation of peptide I into myosin light chain
kinase. This inhibition is probably indirect and related to the
rapid-equilibrium random Bi-Bi catalytic mechanism (Geuss et
al., 1985). Phosphorylated peptide has a lower affinity for the
kinase compared to the nonphosphorylated peptide, and, in the presence
of MgATP, the rate of phosphorylation is much more rapid than the rate
of cross-linking. Inhibition of cross-linking does not occur in the
presence of ATP alone since Mg
is required for
catalysis.
/calmodulin-dependence of peptide
I incorporation into myosin light chain kinase. The binding of the
autoinhibitory domain appears to inhibit specifically light chain
binding since the reactive lysine in the ATP binding site is labeled at
the same rate with 5`- p-(fluorosulfonyl)adenosine in the
presence or absence of Ca
/calmodulin (Kennelly et
al., 1991, 1992). Approximately 40% of peptide I bound to the
full-length or truncated myosin light chain kinase was phosphorylated
in the absence of Ca
/calmodulin. Thus, for this
population of cross-linked kinase, the peptide may occupy a position
that prevents the binding and hence inhibition by the autoinhibitory
domain. Ca
/calmodulin increased the maximal extent of
phosphorylation by 12 and 24% for the truncated and full-length
kinases, respectively. Thus, there appears to be a small population of
cross-linked peptide that is close to the active site but not in a
position to block the inhibitory activity of the autoinhibitory domain.
H
incorporated in peaks I-IV. The two separable radioactive peptides with
identical amino acid sequences suggest that there are multiple atomic
positions of attachment or that multiple stereoisomers are formed.
These possibilities are consistent with previous results obtained with
the chemical modification of calmodulin by a Bpa-containing peptide
(O'Neil et al., 1989). Furthermore, amino acid analysis
of peak I is consistent with the sequencing results and indicates that
the modified peptide includes only residues Glu-371 to Glu-374.
/calmodulin-dependent protein kinase II (Brickey
et al., 1994).
-strands 7 and 8 in a molecular model of myosin light chain kinase
(Knighton et al., 1992). Further discussion of the importance
of this placement is presented below.
P/mol) of peptide I bound to myosin light chain kinase
indicates that incorporation in some site(s) is not favorable for
placement of the phosphoacceptor serine into the active site. It is
possible that the covalent modification at Ile-373 allows the correct
orientation since it accounts for 66% of the peptide I incorporation
into the kinase. However, direct evidence for this possibility is not
available.
-phosphate of ATP for the chemical transfer
to the phosphoacceptor serine at the P position.
Figure 9:
Structure of cAMP-dependent protein kinase
and myosin light chain kinase. A, ribbon model of the
catalytic core of cAMP-dependent protein kinase containing bound MgATP
and peptide inhibitor PKI (Zheng et al., 1993). The small lobe
is indicated in pink; the large lobe is shown in
blue. The ATP binding loop is shown in green with the
three invariant glycine residues in balls. The catalytic loop is shown
in yellow, and MgATP is shown in white. PKI
(5-24) is shown in orange except for the five residues
comprising the consensus sequence for substrate recognition shown in
purple. B, localization and orientation of peptide I
on a ribbon model of the catalytic core of myosin light chain kinase.
The color coding is similar to that described above for cAMP-dependent
protein kinase. The model (Knighton et al., 1992) shows the
conserved features in the secondary structure of the protein kinase
family (Taylor and Radzio-Andzelm, 1994). Ile-373 is shown as a red
ball on the linker between the two lobes. The position of
-strands 7 and 8 are shown in red in the larger lobe. The
specific residue labeled by peptide I in this fragment was not
identified; however, it would appear to be close to Ile-373. The
arrow belt represents the position of the cross-linked peptide
I with the phosphoacceptor serine identified by an
S.
Smooth and skeletal
muscle myosin light chain kinases have been modeled using the crystal
structure of cAMP-dependent protein kinase as a template (Knighton
et al., 1992). According to the model, Ile-373, the major site
covalently modified in myosin light chain kinase, is located on the
polypeptide linking the two lobes together (Fig. 9 B).
Furthermore, the peak IV peptide includes -strands 7 and 8, which
are close to Ile-373. These two positions represent 80% of the covalent
modification with the remainder in the autoinhibitory domain (not shown
in the model depicted in Fig. 9 B). If the
phosphoacceptor serine of peptide I is positioned near the
-phosphate of ATP and the postulated catalytic base Asp-417
(corresponding to Asp-166 in cAMP-dependent protein kinase), the
cross-linked peptide I would lie along the cleft between the two lobes
with the orientation shown in Fig. 9 B. This model is
consistent with the identification of Glu-377 and Glu-421 as residues
that bind to the arginine at the P-3 position in the peptide (Herring
et al., 1992; Gallagher et al., 1993).
-helix, and its hydrophobic face binds to a hydrophobic groove on
the larger lobe and conveys high affinity binding. This groove has been
proposed as a substrate-binding groove for protein kinases, including
cAMP-dependent protein kinase as well as myosin light chain kinase
(Knighton et al., 1992; Kemp et al., 1994). Of
particular importance for PKI binding in the groove is Phe-10, which is
at the P-11 position relative to the pseudophosphoacceptor alanine. The
orientation of the substrate recognition fragment is almost
perpendicular to that of the
-helix in PKI. There is not a
phenylalanine or other aromatic residue in a comparable position (P-11)
in protein substrates for this kinase (Walsh et al., 1992).
Based upon a kinetic analysis of synthetic peptide substrates derived
from PKI, it was concluded that the peptide containing only a
phosphoacceptor serine in place of Ala-21 was the most effective
substrate (Mitchell et al., 1995). Furthermore, two key
residues in this peptide essential for substrate activity were Arg-18
(P-3) and Ile-22 (P+1). Most interestingly, Phe-10 (P-11) did not
affect kinetic properties positively or negatively for phosphorylation,
although its importance in the inhibitor peptide containing alanine in
place of serine was recognized (Mitchell et al., 1995). It was
proposed that the hydrophobic binding pocket that recognizes Phe-10 may
act to contribute specificity for the interaction between the catalytic
subunit of cAMP-dependent protein kinase and PKI, but not substrates.
-helix of
PKI on the catalytic subunit of cAMP-dependent protein kinase (Knighton
et al., 1992; Kemp et al., 1994). These results are
consistent with the recent suggestion that substrates bind in a
different orientation than PKI (Mitchell et al., 1995).
Table:
Phosphorylation of synthetic peptide substrates
Table:
Amino-terminal
sequence of the radioactive fragments purified from CNBr and V8 digests
of myosin light chain kinase (MLCK) cross-linked to peptide I. CM-Cys
refers to carboxylmethylated Cys.
Table:
Amino acid analysis of peak I
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.