©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Photoaffinity Labeling of a Peptide Substrate to Myosin Light Chain Kinase (*)

Zhong-Hua Gao (§) , Gang Zhi , B. Paul Herring (¶) , Carolyn Moomaw (1), Lynn Deogny (1), Clive A. Slaughter (1), James T. Stull (**)

From the (1) Department of Physiology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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/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.

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.


INTRODUCTION

Myosin light chain kinase catalyzes the Ca/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).

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)() 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.

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.


EXPERIMENTAL PROCEDURES

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.

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 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).

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).

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.

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 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.

Peptide I was also phosphorylated by myosin light chain kinase in the presence of [-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 NHHCO, 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.

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 (4.6 250 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.

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.

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).


RESULTS

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/Calmodulin and ATP on the Covalent Incorporation of Peptide I into Myosin Light Chain Kinase

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, 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.

Peak III was applied to a Vydac C18 column (4.6 250 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.

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 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.

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.


DISCUSSION

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 Kvalue; 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.

ATP in the presence of Mg 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.

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/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.

The major site labeled on myosin light chain kinase (peaks I and II) is located at Ile-373, which represents 66% of the total 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.

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/calmodulin-dependent protein kinase II (Brickey et al., 1994).

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 -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.

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 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.

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 -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).

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 -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.

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 -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).

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.

  
Table: Phosphorylation of synthetic peptide substrates

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.


  
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



FOOTNOTES

*
This work was supported in part by grants from the National Institutes of Health (HL06296) and the Bashour Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Laboratory of Biochemistry, NCI, NIH, Bethesda, MD 20892.

Present address: Krannert Inst. of Cardiology, Indianapolis, IN 46202-4800.

**
To whom correspondence should be addressed: Dept. of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235-9040. Tel.: 214-648-6849; Fax: 214-648-2974.

The abbreviations used are: PKI, inhibitor peptide of cAMP-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; MOPS, 3-( N-morpholino)propanesulfonic acid; Bpa, p-benzoylphenylalanine; HFBA, heptafluorobutyric acid; HPLC, high performance liquid chromatography; PTH, phenylthiohydantoin.


ACKNOWLEDGEMENTS

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.


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