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J Biol Chem, Vol. 273, Issue 51, 34049-34056, December 18, 1998


Binding of 9-Anthroylcholine Monitors the Interactions of Adenosine Cyclic 3',5'-Phosphate-dependent Protein Kinase with MgATP, Substrates, and Regulatory Subunits*

Dean A. Malencik and Sonia R. AndersonDagger

From the Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7305

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The isolated catalytic subunit of cAMP-dependent protein kinase and smooth muscle myosin light chain kinase undergo interactions with the fluorescent dye 9-anthroylcholine (9AC) that are responsive to the two enzymes' associations with substrates and effectors. Additionally, the binding of 9AC is highly sensitive to subtle structural or functional differences among closely related protein kinases. Skeletal muscle myosin light chain kinase and the catalytically active chymotryptic fragment of the gamma -subunit of phosphorylase kinase do not associate with 9AC. The 1:1 fluorescent complex of the isolated catalytic subunit of cAMP-dependent protein kinase with 9AC exhibits a dissociation constant of 21 µM. The association of the catalytic subunit with either of the regulatory subunits, RI and RII, results in decreases in the observed 9AC fluorescence that are reversed upon the addition of cAMP. The effects of MgATP and of polypeptide substrates (Kemptide, troponin I, protamine) on the 9AC-catalytic subunit complex are consistent with a general noncompetitive model in which the interactions of 9AC and the other ligands with the enzyme are mutually antagonistic but not purely competitive.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Phosphorylation and dephosphorylation are ubiquitously occurring protein modifications that function in the control of diverse cellular processes. Protein phosphorylation was discovered in conjunction with studies on the enzymology of glycogen degradation. Phosphorylase kinase, which catalyzes the phosphorylation of glycogen phosphorylase (1-3), and cAMP-dependent protein kinase, whose many functions include the phosphorylation and activation of phosphorylase kinase (4-7), were the first protein kinases to be identified. The two enzymes belong to a large family of eukaryotic protein kinases which share a common "kinase catalytic core" but exhibit varying regulatory mechanisms and specificities (8-10). Regulation of the individual enzymes often involves specific association with another protein. The holoenzyme of cAMP-dependent protein kinase contains two catalytic subunits and two regulatory subunits, each of which binds two molecules of cAMP. Association with cAMP causes the enzyme tetramer to dissociate into two catalytically active 40.8-kDa subunits (C) and a dimeric regulatory subunit-cAMP complex (5).
<UP>R<SUB>2</SUB>C<SUB>2,inactive</SUB></UP>+4<UP>cAMP</UP> ⇌ 2 <UP>C<SUB>active</SUB></UP>+<UP>R</UP><SUB>2</SUB> · (<UP>cAMP</UP>)<SUB>4</SUB> (Eq. 1)
This reversible reaction is a major component in cAMP-mediated signal transduction. There are two principal types of regulatory subunit, RI (43 kDa) and RII (45 kDa), which differ in both their tissue distributions and functional properties (11-14).

The x-ray crystallographic analyses that were conducted with the isolated catalytic subunit of cAMP-dependent protein kinase demonstrated the binding of a peptide substrate analogue at a site near the entrance to a bilobal cleft, which contains the ATP-binding region. One of the two lobes functions principally in nucleotide binding while the other larger lobe participates in peptide binding and catalysis (15-17). Crystal studies with an overexpressed truncated form of the catalytically active gamma  subunit of phosphorylase kinase, which possesses 33% sequence identity with the catalytic core (residues 42-297) of cAMP-dependent protein kinase, revealed a similar three-dimensional structure (18). The "catalytic cores" of still other protein kinases have been modeled from the coordinates established by Knighton et al. (15). These enzymes include smooth muscle myosin light chain kinase, which exhibits 30% sequence identity with the aligned 261-residue sequence of cAMP-dependent protein kinase; skeletal muscle myosin light chain kinase, which displays 46% identity with the aligned sequence of the smooth muscle enzyme (19); and protein kinase C (20).

Noncovalently bound fluorescent ligands have facilitated dynamic solution studies of the interactions of numerous enzymes with their substrates and effectors (21, 22). For example, our laboratory employed the fluorescent dyes 8-anilino-1-naphthalenesulfonate in the monitoring and characterization of the glycogen phosphorylase b to a conversion (23, 24) and 9-anthroylcholine in analyses of the binding equilibria of smooth muscle myosin light chain kinase (25, 26). Nevertheless, the fluorescent probe approach has been used infrequently with protein kinases. 5,5'-Bis[8-(phenylamino)-1-naphthalenesulfonate] (bis(ANS)),1 a fluorescent dye that first was found to compete with NADH in binding by lactate dehydrogenase (27, 28), also interacts with the regulatory subunits of cAMP-dependent protein kinase. Association of the regulatory subunits with either cAMP or the catalytic subunit leads to the dissociation of a major portion of the previously bound bis(ANS) molecules. However, neither bis(ANS) nor the parent molecule 8-anilino-1-naphthalene-sulfonate affects nucleotide binding by the isolated catalytic subunit (29).

1,N6-Ethenoadenosine cyclic 3',5'-phosphate and lin-benzoadenosine 5'-phosphate are fluorescent nucleotide derivatives that have been applied to competitive displacement experiments with cAMP and the regulatory subunits (30, 31) and with ATP and the isolated catalytic subunit (32), respectively. However, the emission spectrum and quantum yield of linbenzoadenosine 5'-phosphate do not change appreciably on complex formation facts which mean that fluorescence polarization measurements are required for the detection of binding. The highly fluorescent 1,N6-ethenoadenosine 5'-triphosphate is simply not bound by the catalytic subunit (32). Overall, there is no noncovalent fluorescent probe that is known to be applicable to fluorescence intensity measurements with the catalytic subunit of cAMP-dependent protein kinase.

The identification and application of a fluorescent probe that binds reversibly to either the "kinase catalytic core" or to a region that is functionally connected to the core are the objectives of the experiments described in this report. The studies are based on the premise that such a probe would be sensitive to subtle structural variations that exist among closely related protein kinases and to the binding of ligands and effectors by the enzymes that interact with the probe. Experiments with the isolated catalytic subunit of cAMP-dependent protein kinase, skeletal muscle myosin light chain kinase, and the catalytically active 33-kDa chymotryptic fragment derived from the gamma -subunit of phosphorylase kinase show that 9-anthroylcholine undergoes selective binding by individual protein kinases. The investigation also considers the relationships between 9-anthroylcholine binding and the association of the isolated catalytic subunit of cAMP-dependent protein kinase with substrates and effectors.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- The synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), salmine, ATP, ADP, AMP-PNP, cAMP, and dithiothreitol were purchased from Sigma. Sephacryl S-200 superfine gel filtration medium was obtained from Pharmacia, Piscataway, NJ. Histone H2A was supplied by Calbiochem, Inc. 9-Anthroylcholine was supplied by Molecular Probes, Inc. All other reagents were of the highest grade commercially available. Buffers were prepared from distilled water that had undergone a final purification in a Milli-Q reagent water system. Most of the experiments were performed in a buffer containing 5.0 mM MOPS, 0.10 M KCl, 0.5 mM EGTA, 5 mM Mg(CH3CO2)2 (in the nucleotide binding determinations), 1 mM dithiothreitol, pH 7.3, 25 °C.

Protein Preparations-- The catalytic subunit of type II cAMP-dependent protein kinase was prepared from bovine heart as described by Peters et al. (33), with the modifications outlined in our earlier report (30). Final enzyme fractions were concentrated by ultrafiltration with an immersible ultrafiltration unit (Millipore Immersible CX NMWL 10,000 daltons), dialyzed against 1 volume of glycerol, and stored at -80 °C. The purified enzyme has a specific activity of 23 µmol of 32P/min/mg in the catalytic assay employing Malantide as phosphate acceptor (34). RII was prepared from bovine heart essentially as described by Dills et al. (35). Regulatory subunit free of cAMP was prepared according to Builder et al. (36). cAMP-free RI from bovine skeletal muscle was a generous gift from Dr. Janice Bohnert. As detailed by Bohnert et al. (30), both RI and RII were fractionated on Sephacryl S-200 in order to remove aggregates.

Myosin light chain kinase was prepared from both smooth muscle (turkey gizzard) and striated muscle (rabbit skeletal) as described, respectively, by Sobieszek and Barlko (37) and Blumenthal and Stull (38). The two purified enzymes exhibited the expected apparent molecular weights on sodium dodecyl sulfate polyacrylamide gel electrophoresis---90,000 (skeletal) and 135,000 (smooth). No lower molecular weight components were detected. Catalytic assays showed nearly absolute calmodulin dependence, with specific activities of 12.5 µmol of 32P/mg for the smooth muscle enzyme (see Ref. 25) and 7.1 µmol of 32P/min/mg for the skeletal muscle enzyme (38). The concentrations of the myosin light chain kinases were verified in stoichiometric fluorescence titrations of dansylcalmodulin and in titrations performed in the presence of 9-anthroylcholine in the case of the smooth muscle enzyme (25, 26). The 33-kDa catalytically active chymotryptic fragment of rabbit muscle phosphorylase kinase (30 µmol of 32P/min/mg) was prepared and assayed according to the method of Malencik et al. (39); telokin was isolated from turkey gizzard following the protocol of Ito et al.(40); calmodulin was purified from bovine brain as described by Schreiber et al. (41); and rabbit skeletal muscle troponin I was prepared according to Kerrick et al. (42). All proteins were 95% or more homogeneous in sodium dodecyl sulfate-gel electrophoresis.

Protein Concentration-- The concentration was routinely determined by UV absorption using values of A280 nm0.1%, 1 cm = 1.49 for C (33), 0.80 for RI (36), 0.60 for RII (43), 0.20 for calmodulin (44), 0.90 for the catalytic fragment of phosphorylase kinase (39), and 1.1 for myosin light chain kinase (45). The following molecular masses were used to calculate molarity from protein concentration: C = 40.8 kDa, RI = 43 kDa, RII = 45 kDa (see review by Taylor et al. (10)), CaM = 16.7 kDa (44); phosphorylase kinase fragment = 33 kDa (46); smooth muscle MLCK = 108 kDa (47, 48); and skeletal muscle MLCK = 65 kDa (49).

Fluorescence Measurements-- Measurements of fluorescence anisotropy (r = (Iparallel  - Iperp )/(Iparallel  + 2Iperp ) and total fluorescence intensity (Iparallel  + 2Iperp ) were obtained by using the SLM 4000 fluorescence polarization spectrophotometer. The excitation wavelength was fixed at 365 nm, which is close to an absorption maximum for 9-anthroylcholine. For maximum sensitivity in the polarization measurements, the fluorescence was observed through a Schott-KV filter with either a 418- or 389-nm cutoff limit. Quartz fluorescence cuvettes (previously treated with 1% dichlorodimethylsilane in chloroform) of 2-, 5-, or 10-mm path length were used throughout the experiments. By using differing path lengths, we obtained a linear relationship between fluorescence intensity and the concentration of free 9-anthroylcholine up to concentrations of ~50 µM.

The performance of the binding experiments and the data analyses are the same as those that we described previously for smooth muscle myosin light chain kinase (25, 26). Side by side comparisons were made of the fluorescence intensities of the enzyme/9AC mixtures (F) with the fluorescence of the standard containing the equivalent concentrations of 9AC (F0). Computerized analyses for the determination of dissociation constants and maximum fluorescence changes employed Graph Pad Prism version 2.01 software. Fluorescence emission spectra, corrected for the wavelength dependence of grating transmission and detector response, were determined with the Perkin-Elmer LS-50 fluorescence spectrophotometer. Both fluorometers were connected to circulating constant temperature baths (±0.1 °C).

Ultracentrifugation-- Direct determinations of the quantities of 9-anthroylcholine bound by the isolated catalytic subunit were obtained with the Beckman XL-A Optima analytical ultracentrifuge (50, 51). The sedimentation of the solutions was carried out at 40,000 rpm and monitored at 362 nm (an absorption maximum of 9AC) and at either 280 or 295 nm, depending on protein concentration. The samples analyzed included the three different concentrations of C-subunit (0.9, 1.8, and 3.7 mg/ml), sedimented in both the presence and absence of 100 µM 9AC, and a reference containing only 100 µM 9AC, which does not sediment. The reference sectors of the cells contained the buffer blank (0.10 M KCl, 10 mM MOPS, 0.5 mM EGTA, 2 mM dithiothreitol, pH 7.3). The sedimentation monitored at any of the three wavelengths demonstrated a single component with s20,w = 3.3 S. Measurement of the absorbance at 362 nm of the supernatant solution and of the plateau region, performed after the boundaries were well separated from the meniscus and with radial dilution correction applied, yielded the proportions of free and bound dye.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Survey of Four Different Protein Kinases for the Binding of 9-Anthroylcholine-- The emission spectra shown in the inset to Fig. 1 demonstrate the large changes in fluorescence that occur when the catalytic subunit (C) of cAMP-dependent protein kinase (2.5 µM) is added to a solution containing 5.0 µM 9-anthroylcholine (9AC). Since these effects resemble those reported for smooth muscle MLCK, we followed the protocol established with that enzyme in order to determine a dissociation constant (K2 = [9AC][C]/[C-9AC]) for the C subunit·9AC complex (25, 26). Fig. 1 shows the relative fluorescence intensities (F) found in titrations of a fixed concentration (2.0 µM) of enzyme with varying quantities of 9AC versus those obtained in titrations of buffer alone. The smooth curve through the data points was calculated for a simple equilibrium PX right-left-harpoons  P + X with K2 = 21.9 ± 0.7 µM (chi 2 = 0.999) and for a difference between the fluorescence intensities of totally free and bound 9AC at saturation of 7.0. The relative values of K2 and of the concentration of the C-subunit are consistent with the assumption made in the computations that, within experimental error, most of the 9AC is unbound. Corrections for binding based on the premise that the C-subunit has one 9AC site reduce K2 to 20.9 µM. The dissociation constant of the C-subunit·9AC complex is close to that reported for smooth muscle MLCK (17.1 µM) (26).


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  		Fig. 1.   Fluorescence titrations of protein kinases with 9-anthroylcholine. The traces show the relative fluorescence intensities (F) obtained upon the addition of varying concentration of 9AC to: open circle , 2.0 µM 33-kDa fragment of the gamma -subunit of phosphorylase kinase; , 2.0 µM rabbit skeletal muscle myosin light chain kinase; Delta , 2.0 µM isolated catalytic subunit of cAMP-dependent protein kinase; bullet , to buffer alone (F0). The smooth curve was calculated for a dissociation constant of 21.9 µM (Delta ) and for a difference of 7.0 in the relative fluorescence intensities of the bound and totally free 9AC at saturation of the isolated catalytic subunit. See text for details. Excitation: 365 nm; emission, Schott KV 389 filter. The inset shows the fluorescence emission spectra of 5.0 µM 9-anthroylcholine recorded in the absence (2) and presence (1) of the isolated catalytic subunit of cAMP-dependent protein kinase (2.5 µM). Conditions: 0.10 M KCl, 5 mM MOPS, 0.5 mM EGTA, 1.0 mM dithiothreitol, pH 7.3, 25 °C.

We also performed analytical ultracentrifugation experiments in order to compare the directly determined concentration of bound 9-anthroylcholine to the quantity that is predicted for a single binding site with Kd = 21 µM. To ensure that a significant fraction of dye was bound, the measurements were made at protein concentrations that equal or exceed the value of Kd (0.9 to 3.7 mg/ml). The comparisons given in Table I show that the calculated and observed values of, the average number of moles of 9AC bound per mole isolated catalytic subunit are similar.

                              
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Table I
9-Anthroylcholine binding by the isolated catalytic subunit of cAMP-dependent protein kinase
The following conditions were used: 0.10 M KCl, 10 mM MOPS, 0.5 mM EGTA, 2.0 mM dithiothreitol, 100 µM 9-anthroylcholine, pH 7.3 (12 °C).

Fluorescence titrations with skeletal muscle MLCK and the 33-kDa catalytically active fragment of the gamma -subunit of phosphorylase kinase2 show little detectable interaction with 9AC (Fig. 1). Since the fluorescence intensity of 9AC does not necessarily change on binding, we also determined the fluorescence anisotropies of the samples. The complex of smooth muscle MLCK with 9AC has an anisotropy under these conditions of 0.147 while free 9AC has a value of 0.067 (25). The anisotropies obtained with skeletal MLCK and the 32-kDa phosphorylase kinase fragment are close to the latter value (not shown), verifying that little association with 9AC takes place. Telokin, an independently expressed protein containing the last 154 amino acid residues of smooth muscle MLCK (40), also fails to bind 9AC.

Since the binding of 9AC by smooth muscle MLCK is strongly calmodulin-dependent, with K2 decreasing from 17.1 to 3.8 µM in solutions containing calmodulin (Ca2+) and 0.10 M KCl (26), we considered whether a similar effect may occur with skeletal muscle MLCK. Fig. 2 illustrates the changes in fluorescence that are detected when calmodulin is added stepwise to solutions of 5 µM 9AC containing either skeletal or smooth muscle MLCK or buffer alone. Note that the titration of 9AC that is performed in the absence of the enzymes results in a linear increase in fluorescence that is due to the Ca2+-dependent binding of four to six 9AC molecules by calmodulin, with an average Kd congruent  440 µM (52). As we previously reported (25, 26), the initial fluorescence of the solution containing smooth muscle MLCK is enhanced (F/F0 = 6.6) and the initial rate of increase in intensity obtained upon CaM addition is 2.4-fold greater than that observed when CaM is added to 9AC alone. When skeletal muscle MLCK is present, on the other hand, the initial rate of increase in intensity observed upon CaM addition is only ~25% of that obtained in the titration of 9AC with CaM. However, the slopes approach the latter value after the end point of 1 mol of CaM/mol of either smooth or skeletal muscle MLCK is reached. Decreases in the fluorescence of CaM/9AC mixtures were found previously with troponin I, cyclic nucleotide phosphodiesterase, and melittin (26, 52), which displace 9AC molecules when they associate with CaM. Skeletal muscle MLCK is apparently similar to these proteins and does not directly interact with 9AC, either in the presence or absence of calmodulin. The addition of MgATP has no effect on the fluorescence of the skeletal muscle MLCK/9AC/CaM mixtures. Note that since the 33-kDa fragment of the gamma -subunit of phosphorylase kinase lacks a calmodulin-binding domain, it does not associate appreciably with calmodulin (46).


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  		Fig. 2.   Fluorescence titrations of 9-anthroylcholine with calmodulin performed in the presence of 1.0 µM skeletal muscle myosin light chain kinase (open circle ), of 1.0 µM turkey gizzard myosin light chain kinase (Delta ), and with no additions (bullet ). The ordinate shows the ratio of the observed fluorescence intensity (F) to the fluorescence of 9-anthroylcholine determined in the absence of protein (Fo). Conditions: 5.0 µM 9AC, 0.10 M KCl, 5.0 mM MOPS, 1.0 mM CaCl2, 1.0 mM dithiothreitol, pH 7.3, 25 °C. Path: 1 cm. Other conditions were described under Fig. 1.

Effects of Nucleotide Addition: ATP, ADP, AMP-PNP-- The addition of ATP to solutions containing the isolated catalytic subunit of cAMP-dependent protein kinase and 9-anthroylcholine leads to decreases in the average fluorescence of the dye (Fig. 3). In analyzing these data, we applied the general noncompetitive model of McClure and Edelman (53), for which the apparent dissociation constant for ATP (Kapp) is calculated from the slope of a plot of the changes in fluorescence (I0 - I) versus (I0 - I)/[ATP]. The value of Kapp is determined by the dissociation constants corresponding to the three independent equilibria shown below and by P0 and X0, the total concentrations of the enzyme and 9AC, respectively. We employed this analysis in nucleotide binding studies of smooth muscle MLCK (25, 26), for which MgATP and 9AC appeared to compete in binding (i.e. K3 >> K2).
K<SUB><UP>app</UP></SUB>=<FR><NU>(P<SUB>0</SUB>+X<SUB>0</SUB>)/K<SUB>2</SUB>+1</NU><DE>(P<SUB>0</SUB>+X<SUB>0</SUB>)/K<SUB>3</SUB>+1</DE></FR> K<SUB>1</SUB> (Eq. 2)
<UP>C</UP>−<UP>ATP</UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>K<SUB>1</SUB></LL></LIM> <UP>C</UP>+<UP>ATP</UP>
<UP>C</UP>−9<UP>AC</UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>K<SUB>2</SUB></LL></LIM> <UP>C</UP>+9<UP>AC</UP>
9<UP>AC</UP>−<UP>C</UP>−<UP>ATP</UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>K<SUB>3</SUB></LL></LIM> <UP>C</UP>−<UP>ATP</UP>+9<UP>AC</UP>
Whenever K3 >> K2, the results approach those obtained with a simple competitive model. That is,
K<SUB><UP>app</UP></SUB>=[(P<SUB>0</SUB>+X<SUB>0</SUB>)/K<SUB>2</SUB>+1]K<SUB>1</SUB> (Eq. 3)
The results of the sedimentation measurements (Table I) support the assumption that K3 makes a minor contribution to the calculated value of Kapp. The binding of 0.24 mol of 9AC/mol of C-subunit when 1 mM MgATP is present suggests3 a value for K3 of ~290 µM. With 5 µM 9AC, 0.7 µM C-subunit, and K2 = 21 µM, the calculated values of ((P0 + X0)/K2 + 1) and (P0 + X0)/K3 + 1) are 1.27 and 1.02, respectively. The smooth curves in Fig. 3, which are superimposed on the results of measurements performed on solutions containing 0.75 µM C and either 5 or 12 µM 9AC, correspond to Kapp values for ATP of 12.5 ± 0.9 µM and 19.1 ± 1.0 µM, respectively. The dependence of Kapp on the concentration of 9AC is consistent with K3 > K2. Table I includes the values of both Kapp and Kcorr that were obtained from titrations with ATP, ADP, and AMP-PNP. Note that Kcorr is the approximation to the true value of K1 that is calculated from Equation 3 using K2 = 20.9 µM. This manipulation brings the dissociation constants for ATP into closer agreement, with small variations that are probably the result of experimental error. The corrected dissociation constants agree with the value (10.6 ± 1.1 µM) determined from fluorescence polarization measurements of the competitive displacement of linbenzoadenosine 5'-phosphate (32).


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  		Fig. 3.   Effect of ATP on 9-anthroylcholine binding by the isolated catalytic subunit of cAMP-dependent protein kinase. bullet , 0.75 µM C-subunit plus 12 µM 9AC; open circle , 0.75 µM C-subunit plus 5.0 µM 9AC; , 0.75 µM C, 0.75 µM RII, 15.9 µM cAMP, 5.0 µM 9AC; and black-square, 0.75 µM C, 0.75 µM RI, 2.6 µM cAMP, 5.0 µM 9AC. Additional measurements were performed up to 300 µM ATP. Apparent dissociation constants of 19.1 µM (bullet ), 12.5 µM ATP (open circle ), and ~10.5 µM ATP () were obtained by analysis of these data. The inset shows an expanded view of the titration of RI2C2 (curve is not theoretical). See text for details. Conditions: 0.10 M KCl, 5.0 mM MOPS, 0.5 mM EGTA, 5.0 mM Mg(CH3CO2)2, 1 mM dithiothreitol, pH 7.3, 25 °C. Path: 1 cm. Check legend to Fig. 1 for other conditions.

Fluorescence enhancements (F/F0) at the beginning of the titrations with nucleotide were in the range of 3.2 to 3.3 in the case of 5 µM 9AC. Table I shows that saturating levels of nucleotide produce 80-84% reversal of the original enhancement, with maximum effects deduced by extrapolation of the McClure-Edelman (53) plots to infinite nucleotide concentration. Fig. 3 shows normalized fluorescence intensities, with "100" representing the difference between the intensity determined prior to nucleotide addition and the extrapolated final intensity. This normalization was undertaken since the initial values of F/F0 vary among the different experiments shown.

Influence of Protein Kinase Substrates: Kemptide, Protamine, and Troponin I-- We next examined the effects of protein kinase substrates, Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), salmon protamine (salmine), skeletal muscle troponin I, and histone H2A, on the binding of 9-anthroylcholine by the isolated catalytic subunit. Fig. 4 illustrates the changes in 9AC fluorescence that take place upon the addition of Kemptide, a synthetic peptide containing the phosphorylation site of pyruvate kinase and protamine. Analysis of the results according to the noncompetitive model explained in the preceding section (with the peptide substrate replacing ATP in the equations) gives values for Kapp of 0.37 mM for Kemptide and of 4.7 µM for rabbit skeletal muscle troponin I (data not shown). The maximum reduction in fluorescence enhancement, which ranges from 79% with protamine to 63% with Kemptide (Table II), shows that a portion of the 9AC remains bound at saturating substrate concentrations (i.e. as 9AC·C·substrate complex). Note that a significant fraction of the protamine is apparently bound by the C subunit, leading to nonlinear McClure-Edelman plots that suggest a maximum value for Kapp of ~3 µg/ml. Histone H2A, tested at concentrations up to 0.27 mg/ml, exerted minimal effects (<10%) on the fluorescence. Control experiments, in which both intensity and anisotropy were examined, indicated that none of these basic proteins and peptides associates significantly with the cationic 9-anthroylcholine molecule.


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  		Fig. 4.   Influence of polypeptide substrates on 9-anthroylcholine binding by the isolated catalytic subunit of cAMP-dependent protein kinase. The titration with Kemptide corresponds to Kapp = 0.37 mM and F/Fo = 1.85 at infinite substrate concentration. The inset shows the results obtained with protamine (see text for details). Conditions: 0.9 µM C-subunit, 5.0 µM 9AC. Path: 1 cm. Refer to legend under Fig. 1 for remaining conditions.

                              
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Table II
Binding of nucleotide and polypeptide substrates of cAMP-dependent protein kinase analyzed in measurements of 9-anthroylcholine fluorescence
The following conditions were used: 0.10 M KCl, 5 mM MOPs, 0.5 mM EGTA, 1.0 mM dithiothreitol, pH 7.3, 25 °C. 5.0 mM magnesium acetate was present in the titrations with ATP, ADP, and AMP-PNP. The enzyme concentrations were 0.7 to 0.8 µM.

Reassociation of the Catalytic Subunit with the RI and RII Regulatory Subunits-- Since the regulatory subunits have been hypothesized to contain autoinhibitory sequences that are competitive with the protein substrates of the catalytic subunit (Corbin et al. (43); cf. see review by Soderling (54)), the effects of added RI or RII on the binding of 9-anthroylcholine by the C-subunit may resemble those produced by the substrates (Fig. 4). Figs. 5A and 6A illustrate the stoichiometric decline in 9AC fluorescence that occurs upon the introduction of either RI or RII into solutions containing the catalytic subunit.


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  		Fig. 5.   Reconstitution and dissociation of RI2C2 monitored in measurements of 9-anthroylcholine fluorescence. Panel A shows the results of stepwise addition of RI to a solution containing 0.4 µM C-subunit plus 5.0 µM 9AC. Panel B illustrates the cooperative recovery of 9AC fluorescence obtained upon the addition of cAMP to RI2C2 (0.4 µM in terms of protomer). Refer to legend to Fig. 1 for other conditions.

The dissociation of the holoenzyme that follows the binding of cAMP by the regulatory subunits (Equation 1) should result in the recovery of 9AC binding. The titration of RI2C2 with cAMP gives a sigmoid-shaped profile of fluorescence versus concentration (Fig. 5B) that is consistent with the cooperative cyclic nucleotide-binding properties of RI (cf. reviews by Hoppe and Wagner (55) and Smith et al. (56)). In the case of RII2C2, the recovery of 9AC fluorescence takes place over a wider range of cAMP concentrations (Fig. 6B). Cooperativity is not expected with RII, which contains two dissimilar noninteracting cAMP-binding sites (57, 58). The fact that the final fluorescence intensities are slightly less than the initial value, determined prior to the addition of RII, may be related to the incomplete dissociation of RII2C2 that was detected in sedimentation velocity experiments (30).


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  		Fig. 6.   Reconstitution and dissociation of RII2C2 monitored in measurements of 9-anthroylcholine fluorescence. Panel A illustrates the effects of stepwise addition of RII to a solution containing 0.67 µM C-subunit plus 5 µM 9AC. Panel B shows the recovery of 9AC fluorescence produced by the addition of cAMP to RII2C2 (0.67 µM in terms of protomer). See legend to Fig. 1 for other conditions.

The tetrameric structure of the RI2C2 holoenzyme is stabilized by the high affinity binding of MgATP (12, 59). ATP titrations of the reconstituted holoenzyme, carried out in the presence of 2.6 µM cAMP and 5 µM 9AC, demonstrate the high affinity binding that is characteristic of RI2C2 (Fig. 3, inset). The low concentrations of ATP required to produce the observed changes in 9AC fluorescence suggest an apparent dissociation constant of ~0.25 µM under these conditions. This value is 50 times smaller than the apparent dissociation constant (12.5 µM) determined for the isolated C subunit and 100-200 times larger than the dissociation constant obtained by Lew et al. (60) with RI2C2 in the absence of cAMP. Note that the apparent dissociation constant determined in the presence of cAMP is expected to be larger than the value obtained in its absence since cAMP has a destabilizing effect on the RI2C2 holoenzyme (5). The results of ATP binding experiments performed with RII2C2 plus 15.9 µM cAMP approach those found with the catalytic subunit alone, with Kapp ~ 10 µM (Fig. 3).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The isolated catalytic subunit of cAMP-dependent protein kinase resembles smooth muscle myosin light chain kinase in its interactions with 9-anthroylcholine. In both cases, binding of the dye is optimal with the catalytically active form of the enzyme, i.e. the free catalytic subunit (C) or the smooth muscle myosin light chain kinase-calmodulin complex. The stabilities of the enzyme·9AC complexes are in the micromolar range: 21 µM for C and 17 µM for MLCK, or 3.8 µM for MLCK·CaM (25, 26). Both enzymes exhibit similar relationships between nucleotide binding and 9AC fluorescence. The dissociation constant that we deduced from titrations of the C·subunit·9AC complex, 11 ± 1 µM MgATP, agrees with the value of 10.6 ± 1.1 µM obtained from competition experiments with linbenzoadenosine 5'-phosphate(32). We used the general noncompetitive model of McClure and Edelman (53) in analysis of the data. Small fluorescence enhancements of 16-20% remaining at saturating concentrations of the nucleotides (Table II) probably result from the existence of an unfavorable ternary complex (9AC·enzyme·nucleotide). The partial dissociation of the 9AC·enzyme complex demonstrated in sedimentation measurements that were performed in the presence of MgATP (Table I) is consistent with a noncompetitive model in which the binary complexes (MgATP·C-subunit and 9AC·C-subunit) are favored. Although we did not estimate a value for K3 in the case of MLCK, values of K3 that are 10 to 15 times larger than the values of K2 would have been within the realm of the observations.

The influence of peptide and protein substrates on the binding of 9AC is distinctly different with smooth muscle MLCK and the isolated C subunit, however. A 13-residue analogue of the 21-kDa smooth muscle myosin light chain exerts no detectable effect on the association of 9AC with MLCK alone but strongly facilitates the competitive displacement of the dye that takes place on the addition of AMP-PNP to the MLCK-calmodulin complex (26). The fact that substrate binding was detected only when AMP-PNP and calmodulin were both present is consistent with both the subsequent pseudosubstrate hypothesis (61) and the ordered sequential kinetic mechanism (in which ATP binds first) that was demonstrated for smooth muscle MLCK by Sobieszek (62). Polypeptide substrates alone, Kemptide, troponin I, protamine, and the two regulatory subunits have large effects on the fluorescence of solutions containing the isolated catalytic subunit of cAMP-dependent protein kinase and 9AC. The kinetic mechanism for the cAMP-dependent protein kinase includes two significant binary complexes: enzyme-ATP and enzyme-polypeptide (63). The action of more than one kind of ligand on the binding of 9AC to the isolated catalytic subunit is reminiscent of that found with glycogen phosphorylase. In that case, substrates and products (Pi and glucose 1-phosphate), an allosteric inhibitor (glucose 6-phosphate), and an allosteric activator (AMP) decrease the fluorescence of solutions containing ANS and phosphorylase b by as much as 85% (24). Later x-ray crystallographic studies revealed that most of the bound ANS occupies the AMP-binding site of phosphorylase (64).

The sensitivities of the 9-anthroylcholine complexes to both MgATP and the protein effectors of smooth muscle MLCK and cAMP-dependent protein kinase suggest that the bound 9AC molecules occupy homologous loci in the two enzymes. Their conserved kinase catalytic cores are possible 9AC-binding regions. Yet skeletal muscle MLCK and the 33-kDa active fragment of phosphorylase kinase do not associate appreciably with this probe, pointing to the existence of functionally significant differences among the enzymes that were not evident in the x-ray crystallographic studies. The distinction in 9AC binding is one of many large differences between the two myosin light chain kinases. They also vary in molecular weight: smooth = 107,534 (48), skeletal = 65,337 (49); immunological reactivity, specificity, pH optima (cf. review by Hartshorne and Siemankowski (65)) and in sequence, both within and outside of the proposed core (19, 48, 49). The 9AC-binding sites do not necessarily coincide with either the nucleotide or the peptide-binding sites. The above mentioned experiments with ANS and glycogen phosphorylase (24, 64) demonstrate the possibilities for communication within a network of differing ligand-binding sites.

9-Anthroylcholine is the only noncovalent probe that is known to undergo changes in fluorescence spectrum and quantum yield upon interaction with the isolated catalytic subunit of cAMP-dependent protein kinase. Lew et al. (60) have prepared a fluorescent covalent conjugate by reacting acrylodan with an engineered form of the catalytic subunit in which cysteine has replaced the normal Asn326. Since the conjugate is not subject to dissociation of the probe, it can be employed at lower concentrations than those used in the experiments with 9AC. However, the acrylodan-labeled enzyme is not highly sensitive to ligand binding. Protein or peptide substrates and the regulatory subunits have no direct effect on its total fluorescence intensity. Additions of ATP or AMP-PNP result in 9-10% and 19% quenching of the acrylodan fluorescence, respectively.

In summary, 9-anthroylcholine is highly sensitive to differences among closely related protein kinases and is responsive to the binding of substrate and effectors by either cAMP-dependent protein kinase or smooth muscle myosin light chain kinase. In view of these characteristics and the fact that it does not require covalent labeling or genetic manipulation, 9-anthroylcholine should find additional applications in the functional characterization of protein kinases.

    FOOTNOTES

* This work was supported by National Institutes of Health Research Grant DK13912.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 541-737-4486; Fax: 541-737-0481; E-mail: malencid{at}ucs.orst.edu.

The abbreviations used are: bis(ANS), 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate]; AMP-PNP, 5'-adenyl-beta ,gamma -imidodiphosphate; 9AC, 9-anthroylcholine; ANS, 8-anilino-1-naphthalenesulfonate; MOPS, 3-(N-morpholino)propanesulfonic acid; C, catalytic subunit of cAMP-dependent protein kinase; RI and RII, regulatory subunits of cAMP-dependent protein kinase; CaM, calmodulin; MLCK, myosin light chain kinase; F0, fluorescence intensity of a solution containing 9AC alone; F, fluorescence of a solution containing enzyme plus the same total concentration of 9AC used in the determination of F0; I0, fluorescence determined prior to the addition of a ligand (such as ATP) to a solution containing enzyme and 9AC; I, fluorescence determined after the addition of the indicated concentration of ligand.

2 The chymotryptic fragment of phosphorylase kinase is in fact a mixture of three fragments containing residues 1-290, 1-296, and 1-298 of the 44.7-kDa gamma -subunit (45). The overexpressed truncated form of the gamma -subunit contains residues 1-298 (18).

3 K3 was estimated from the observed value = 0.24 (Table I), the values of K1 (11 µM) and K2 (21 µM), and the concentrations X0 = 100 µM, P0 = 90.7 µM, and ATP0 = 1 mM. Since K1 << 1 mM ATP, we assume that the proportion of free subunit present is negligible. Thus [P0] = [C-9AC] + [C-ATP] + [9AC-C-ATP]; [X0] = [C-9AC] + [9AC] + [9AC-C-ATP]; [ATP0] = [ATP] + [C-ATP] + [9AC-C-ATP]; [ATP] = 1000 - [C-ATP] - [9AC-C-ATP]; n[P0] = [C-9AC] + [9AC-C-ATP]. By substituting the above values into these equations, we calculate, [C-ATP] = 68.9 µM and [9AC] = 78.2 µM; K3 [9AC-C-ATP] = 5390 µM (see Footnote 2); [9AC-C] = n[P0] - [9AC-C-ATP]; K1/K2 = [ATP] [C-9AC]]/([9AC] [C-ATP]); K1/K2 = (931 - [9AC-C-ATP])(21.8 - [9AC-C-ATP])/5390. Since K1/K2 = 0.524, solution of this equation for the value of [9AC-C-ATP] gives ~18.7 µM and K3 ~ 5390/18.7 = 288 µM. With the concentrations that were used, the calculated value of K3 is relatively insensitive to moderate variations in K1/K2. For example, when the value of K1/K2 is either 0.25 or 1.0, the calculated values of K3 is either 265 or 340 µM.

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Abstract
Introduction
Procedures
Results
Discussion
References

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