©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Precision Substrate Targeting of Protein Kinases
THE cGMP- AND cAMP-DEPENDENT PROTEIN KINASES (*)

(Received for publication, September 15, 1995; and in revised form, October 27, 1995)

Jason S. Wood (1) Xiongwei Yan (1) Marianne Mendelow (1) Jackie D. Corbin (2) Sharron H. Francis (2) David S. Lawrence (1)(§)

From the  (1)Departments of Chemistry and Medicinal Chemistry, Natural Sciences and Mathematics Complex, State University of New York, Buffalo, New York 14260 and the (2)Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The cAMP-dependent (PKA) and cGMP-dependent protein kinases (PKG) share a strong primary sequence homology within their respective active site regions. Not surprisingly, these enzymes also exhibit overlapping substrate specificities, a feature that often interferes with efforts to elucidate their distinct biological roles. In this report, we demonstrate that PKA and PKG exhibit dramatically different behavior with respect to the phosphorylation of alpha-substituted alcohols. Although PKA will phosphorylate only residues that contain an alpha-center configuration analogous to that found in L-serine, PKG utilizes residues that correspond to both L- and D-serine as substrates. The PKG/PKA selectivity of these substrates is the highest ever reported.


INTRODUCTION

Protein kinase cascades are the predominant component of signal transduction pathways(1) . These pathways transmit extracellular signals from the plasma membrane to distant intracellular sites in the cytoplasm and nucleus. Estimates have placed the potential number of protein kinases encoded by the mammalian genome at greater than 1,000 (2) . Consequently, defining the role of a particular protein kinase can be a daunting task given the size of this enzyme family and the plethora of pathways in which these phosphoryl transfer catalysts serve as participants. This task is rendered even more formidable by the general nature of the reaction catalyzed by these enzymes and by their nearly universal utilization of a common substrate, ATP. However, individual protein kinases presumably do exhibit unique properties in vivo, as exemplified by their ability to catalyze the phosphorylation of specific proteins. This precision is potentially regulated by several different parameters, including (i) the microenvironment to which the enzyme is confined within the cell(3) , (ii) the secondary and/or tertiary structure that encompasses the site of phosphorylation on the target protein substrate, (iii) the primary sequence that envelops the phosphorylatable amino acid residue, and (iv) the active site substrate specificity of the protein kinase, which is generally limited to serine, threonine, and/or tyrosine residues (although exceptions are known).

Can these four parameters be manipulated to design inhibitors that target specific protein kinases? Peptide-based substrates have been described for a number of protein kinases utilizing data from the specificity parameter (iii) described above(4) . These substrate specificity studies have lead to the creation of nonphosphorylatable reversible inhibitors: peptides in which the alcohol-containing amino acid has been replaced by a residue that lacks a hydroxyl moiety (e.g. alanine for serine exchange). Unfortunately, the ultimate utility of this approach is limited by the fact that many protein kinases exhibit overlapping substrate specificities. A case in point is the cAMP-dependent protein kinase (5, 6) and its closely related counterpart, the cGMP-dependent protein kinase(6, 7) . Both enzymes exhibit a special affinity for amino acid sequences that contain positively charged residues near the site of phosphorylation (the consensus sequence for these enzymes is Arg-Arg-Xaa-Ser-, where Xaa is generally a hydrophobic residue) (8, 9, 10) . However, these enzymes do exhibit some subtle, yet intriguing, differences in their substrate specificity. For example, both phosphorylate the peptide Val-Leu-Gln-Arg-Arg-Arg-Pro-Ser-Ser-Ile-Pro-Gln with similar K values(11) . However, whereas PKA specifically phosphorylates the serine closest to the N terminus, PKG phosphorylates both equally well. These enzymes also exhibit somewhat different kinetic behavior toward two closely positioned serine residues contained within histone H2B(9, 12) . In addition, a number of peptides have been identified that are moderately more efficient substrates for PKG (^1)than for PKA(10, 13, 14, 15, 16) and vice versa. However, while differences in specificity between PKA and PKG do exist, no substrates have been reported that are absolutely specific for PKG.

Although a number of studies have appeared that report differences in the substrate specificities of PKA and PKG, nearly all of these reports have focused on the role of primary sequence in substrate recognition (i.e. parameter (iii))(10, 13, 14, 15, 16) . However, as noted above, there are at least three other ways in which substrate recognition can be specified. In particular, PKA and PKG may exhibit some differences in terms of their active site specificities (i.e. parameter (iv)). At first glance, this seems unlikely since both enzymes phosphorylate only serine and threonine residues in proteins. However, we have demonstrated that the active site specificities of several protein kinases are not merely limited to serine, threonine, and/or tyrosine(17, 18, 19, 20, 21, 22, 23) . Indeed, PKA will phosphorylate a diverse array of alcohol-containing compounds, including phenols. Utilizing this newly delineated activity of protein kinases, we have recently shown that the active site substrate specificities of PKA and PKC are radically dissimilar, in spite of the fact that both enzymes are serine/threonine-``specific'' protein kinases(21) . We report herein an assessment of the active site substrate specificity of PKG. Unlike the closely overlapping substrate sequence specificities of PKA and PKG, we have found that these two enzymes exhibit astonishingly dramatic differences in their active site specificities. Indeed, we have identified several peptides that serve as efficient substrates for PKG, but fail to be phosphorylated by PKA.


MATERIALS AND METHODS

All chemicals were obtained from Aldrich, except for [-P]ATP (DuPont NEN), cGMP (Sigma), cAMP (Fluka), bovine serum albumin (Sigma), protected amino acid derivatives (Advanced ChemTech or Bachem California), and Liquiscint (National Diagnostics). Dialysis tubing was purchased from Spectrum, and Affi-Gel Blue was obtained from Bio-Rad. Phosphocellulose P-81 filter papers were purchased from Whatman.

cAMP-dependent Protein Kinase Preparation

The catalytic subunit was purified to homogeneity using a previously described method(24) . Purity was assessed by SDS-polyacrylamide gel electrophoresis, which showed a single band at 41-kDa molecular mass. Ellman's reagent titrated the cysteine residues to 2.05-2.10 sulfhydryls/molecule of enzyme which is in excellent agreement with the known primary structure of the enzyme(25) .

cGMP-dependent Protein Kinase Preparation

PKG-Ialpha was isolated and purified to homogeneity as described previously(26) .

Peptide Synthesis

t-Butoxycarbonyl-Leu-Arg-Arg-Arg-Arg-Phe- was prepared on Kaiser's oxime resin utilizing the t-butoxycarbonyl methodology, and displacement from the resin was afforded with various amino alcohols according to previously published methods(27, 28, 29, 30, 31) . Ac-Ser-Phe-Arg-Arg-Arg-Arg-Lys-NH(2) was synthesized on the benzhydrylamine resin and cleaved with HF. All crude peptides were partially purified via ion exchange chromatography on Sephadex CMC-25 (0.4-1.2 M KCl gradient in a 50 mM NaOAc, pH 3.5 buffer, 200-300 ml total volume). The peptides were then simultaneously desalted and purified further using three Waters radial compression modules (25 times 10 cm) connected in series ((solvent A: 0.1% trifluoroacetic acid in water; solvent B: 0.1% trifluoroacetic acid in acetonitrile): 0-3 min (100% A); a linear gradient from 3 min (100% A) to 30 min (75% A and 25% B); a steep final gradient to 90% B for column cleansing purposes). All peptides gave satisfactory fast atom bombardment mass spectral analysis.

cAMP-dependent Protein Kinase Assay

Assays were performed in triplicate at pH 7.1 and thermostatted in a water bath at 30 °C. Final assay volume totaled 50 µl and contained 100 mM MOPS, 150 mM KCl, 12.5 mM MgCl(2), 0.125 mg/ml bovine serum albumin, and 1.5-10.0 nM PKA catalytic subunit. 150 µM [-P]ATP (300-2000 cpm/pmol depending on the substrate efficacy) and a substrate concentration that varied over a 6-fold range around the apparent K(m) were employed for the determination of kinetic constants. Phosphorylation reactions were initiated by the addition of 10 µl of catalytic subunit from a stock solution (1.5 mg/ml in 100 mM MOPS, 1 mM dithiothreitol, and 0.125 mg/ml bovine serum albumin at pH 7.1). Reactions were terminated after 5 min by spotting 25-µl aliquots onto 2.1-cm phosphocellulose paper disks. After 10 s, the disks were immersed in 10% glacial acetic acid (30 ml per disk) and allowed to soak for at least 60 min. The acetic acid was decanted and the disks were washed collectively with 4 volumes of 0.5% phosphoric acid, 1 volume of water and a final acetone rinse (2 ml/disk). The disks were air-dried and placed in plastic scintillation vials containing 6 ml of Liquiscint prior to scintillation counting for radioactivity.

cGMP-dependent Protein Kinase Assay

Assays were performed in triplicate at pH 7.4 and thermostatted in a water bath at 30 °C. Final assay volume totaled 50 µl and contained 10 mM Tris, 2 mM MgCl(2), 0.3 mg/ml bovine serum albumin, 20 µM cGMP, and between 1.0 and 4.0 nM PKG. 200 µM [-P]ATP (400-600 cpm/pmol depending on the substrate efficacy) and a substrate concentration that varied over a 10-fold range around the apparent K(m) were employed for the determination of kinetic constants. Phosphorylation reactions were initiated by the addition of 10 µl of PKG from a stock solution (0.136 mg/ml in 10 mM Tris, 25 mM 2-mercaptoethanol, 1 mM EDTA, and 1 mg/ml bovine serum albumin at pH 7.4). Reactions were terminated after 10 min by spotting 25-µl aliquots onto 2.1-cm phosphocellulose paper disks, and the paper disks were washed and counted as described for the PKA assay.

Determination of Kinetic Constants

The apparent K(m) (± S.D.) and V(max) values (±S.D.) for all peptides were determined from initial rate experiments, and the k and k/K(m) values were calculated accordingly. The data from these experiments were plotted using the Lineweaver-Burk procedure, and the corresponding plots proved to be linear in all cases.


RESULTS AND DISCUSSION

Although PKA and PKG share a strong primary sequence homology (32) , these enzymes exhibit differences that sweep the spectrum from the biological to the structural. Their biological distribution differs in a dramatic fashion. While PKA is nearly ubiquitous, its cGMP-dependent counterpart is located primarily in the lung, cerebellum, and smooth muscle(7) . In addition, very high levels of PKG have been found in blood platelets. PKA serves as the main receptor, by far, for cAMP in mammals, although certain ion channels may be cAMP-gated(6) . In contrast, PKG is only one of several proteins whose biological activity is dependent upon cGMP(7) . These cyclic nucleotide-dependent protein kinases differ in a structural sense as well. The holoenzyme form of PKA is a tetramer composed of two regulatory and two catalytic subunits(5, 6) . The latter are noncovalently coordinated to the regulatory subunit dimer. Upon interaction with cAMP, the catalytic subunits dissociate from the holoenzyme and are then free to catalyze the phosphorylation of protein substrates. On the other hand, PKG is a dimeric species. Each subunit contains covalently linked regulatory and catalytic domains. Consequently, when cGMP activates PKG, the catalytic and regulatory components remain physically attached. In contrast to these rather dramatic differences, these enzymes exhibit a pronounced overlapping substrate specificity. This is not a surprising observation given the strong primary sequence homology shared by PKA and PKG, particularly within their active site regions. However, the substrate specificities of these enzymes are not identical. Consequently, there have been attempts to take advantage of some subtle differences in specificity to create inhibitors that are targeted for only one of the two enzymes. Interestingly, the primary success in this regard is based upon work with PKI, a naturally occurring inhibitor of PKA. A number of truncated derivatives of PKI have been examined, and nearly all of these inhibitors exhibit a clear affinity for PKA relative to that of PKG (33) . In contrast, peptide-based inhibitors that are PKG-specific have not been reported. Since 1982, several laboratories have attempted to construct PKG-selective substrates. Unfortunately, only a few peptides have been found to serve as somewhat more efficient substrates for PKG than for PKA(10, 13, 15, 16) . However, we have recently demonstrated that several protein kinases can also be distinguished by their active site specificities(21) . We now report that PKA and PKG can be differentiated precisely on this basis.

An active site specificity analysis requires ready synthetic access to a family of peptides containing a diverse array of structurally disparate residues at the site of phosphorylation. We have previously noted that peptides containing unusual amino acid residues (with exotic and/or chemically sensitive functionality) at internal sites can be both time-consuming and difficult to prepare(17) . In contrast, it is significantly easier to construct closely analogous peptides with the unnatural residue of interest positioned at either the N or C terminus. Remarkably enough, protein kinases will phosphorylate residues located at the terminal portion of a peptide as long as that site is uncharged (i.e. the N-terminal amino group is acetylated or the C-terminal carboxyl group is amidated). We have found this to be the case with PKG as well.

PKG and PKA have a special affinity for peptides that contain positively charged residues on the N-terminal side of the phosphorylatable residue(10, 13, 15, 16) . In addition, a hydrophobic residue at the P-1 position is a common structural motif in substrates for both enzymes as well. Consequently, we prepared the peptide Leu-Arg-Arg-Arg-Arg-Phe-Ser-amide. As is apparent from Table 1, this peptide is efficiently phosphorylated by PKG. As one might predict (based on known overlapping substrate specificities), Leu-Arg-Arg-Arg-Arg-Phe-Ser-amide is a superb PKA substrate as well. Therefore, a family of peptides of the general form, Leu-Arg-Arg-Arg-Arg-Phe-Xaa, was prepared via the oxime resin protocol as described previously (17) and subsequently evaluated as substrates for both PKA and PKG.



While both PKA and PKG can phosphorylate common residues, these enzymes also exhibit several profound differences in their active site substrate specificity. The differences and similarities are presented below.

Achiral Residues Serve as Substrates for Both PKA and PKG

We have previously demonstrated that PKA will phosphorylate achiral residues positioned on the C terminus of the peptide Gly-Arg-Thr-Gly-Arg-Arg-Asn-Xaa(17) . In addition, PKA exhibits this behavior toward achiral residues appended to the C terminus of Leu-Arg-Arg-Arg-Arg-Phe-. The ethanolamine-containing peptide 2 is phosphorylated more efficiently by PKA than are the corresponding propanolamine (3) and butanolamine (4) derivatives (Table 1). These results are not too surprising given the fact that the hydroxyl moiety in 2 is the same distance from the peptide backbone as that for its serine-containing counterpart in 1. In contrast, the span between alcohol and peptide bond in 3 and 4 is greater than in 1 and 2. This is an apparently deleterious feature since the catalytic efficiency of 3 is 30-fold less than that for 2. PKG also phosphorylates achiral residues. The ethanolamine-containing peptide 2 is an efficient PKG substrate, exhibiting a k/K(m) that is essentially identical with that found for PKA. However, in contrast to the results obtained for PKA, the propanolamine derivative 3 is actually a better substrate for PKG Ialpha than is its shorter chain counterpart 2. In contrast, the butanolamine derivative 4 is a significantly weaker PKG substrate than its propanolamine-containing counterpart.

PKA and PKG Are Distinguished by Their Ability to Phosphorylate Chiral Residues with alpha-Substituents

Both enzymes are able to phosphorylate alpha-substituted alcohol-bearing residues if the configuration at the stereocenter is analogous to that present in L-serine. PKA and PKG exhibit nearly identical K(m) values for both of the L-serine-containing peptides 5 and 6 (Table 2). Interestingly, the aryl-containing peptide 5 turns over an order of magnitude more rapidly with PKA than with PKG. This difference in k is nearly eliminated with substrate 6, a species that lacks the phenyl substituent. In addition, both enzymes catalyze the phosphorylation of peptides 7-12. These substrates contain alcohol-bearing residues that contain a variety of substituents appended to the alpha-position, in the same configurational sense, as that found in in L-serine itself (cf. 5-6 with 7-12). In contrast to compounds 5 and 6, substrates 7-12 do not possess an amide carbonyl directly attached to the chiral center of the phosphorylatable residue. For PKG, the absence of the carbonyl group has little impact on K(m), but a somewhat more significant effect on k. For example, the alpha-methyl (11), ethyl (10), and isobutyl (12) derivatives all exhibit turnover numbers that are an order of magnitude less than their serine-containing counterparts 5 and 6, whereas the K(m) values for these peptides are nearly identical. However, the aryl-substituted derivatives, particularly 8 and 9, do turn over more rapidly than the corresponding alkyl-substituted derivatives (i.e10-12). A similar trend is apparent with PKA. Nevertheless, it is evident that peptides 7-12 are phosphorylated an order of magnitude more rapidly by PKA than by PKG.



We, as well as others, have previously demonstrated that PKA will not phosphorylate D-serine or analogously configured residues(17, 34, 35) . We initially established this behavior with residues attached to the C terminus of Gly-Arg-Thr-Gly-Arg-Arg-Asn- (17) . The results from Table 3indicate that this active site specificity is independent of the particular amino acid sequence to which the alcohol-containing compound is appended (in this study the sequence Leu-Arg-Arg-Arg-Arg-Phe- was employed). In short, PKA is incapable of phosphorylating residues that bear a stereochemical resemblance to D-serine. (^2)In stark contrast, PKG does not suffer from this limitation, as exemplified by its ability to catalyze the phosphorylation of peptides 13-16.We know of no other substrates that exhibit such an absolute bias for PKG over that of PKA.



PKA and PKG Are Distinguished by Their Ability to Phosphorylate Chiral Residues with beta-Substituents

PKA will phosphorylate both primary (e.g. serine) and secondary alcohols (e.g. threonine). In the latter case, the configuration at the beta-center is a key recognition motif (17) In short, PKA will not phosphorylate residues that contain stereochemistry at the beta-carbon opposite to that present in L-threonine. Consequently, while peptide 17 is a PKA substrate, peptide 18 is not (Table 4). Once again, PKG is significantly more accommodating than its cAMP-dependent counterpart. Indeed, PKG does not exhibit a strong preference for one stereoisomer over the other.



Peptides 13-16 and 18 are the first examples of PKG substrates that fail to serve in a similar capacity for PKA. The results are extraordinary given the fact that both PKG and PKA phosphorylate common serine/threonine residues in intact proteins and that these two protein kinases share a strong sequence homology, particularly in the active site region. However, the ability of PKG to phosphorylate both configurational isomers at either the alpha or beta positions is not unique. Indeed, PKC exhibits an analogous form of active site specificity(21, 36) . Are these differences surprising given the presumed similarity of active site structure throughout the protein kinase family? We have previously addressed the issue of protein kinase active site specificity with PKA, an enzyme whose three-dimensional structure is known(37, 38, 39, 40, 41) . We proposed that the structural basis for the inability of this enzyme to catalyze the phosphorylation of D-amino acid residues (or non-amino acid residues containing a configuration that corresponds to D) is due to the presence of an active site threonine residue (i.e. Thr-201). In short, molecular modeling experiments suggest that a portion of the peptide attached to the D-amino acid would experience severe steric interactions with Thr-201 if a D-amino acid is forced to occupy that region of the active site required for phosphoryl transfer. Although this explanation remains to be verified, it is important to note that the sequence alignments of the isoforms of both PKC and PKG indicate that a threonine residue is present at this position in the primary structure of these enzymes as well. If this critical threonine is present in the same location of the active site region in PKC and PKG, then why are these kinases able to accommodate a D-amino acid residue (or close analogs thereof) whereas PKA is not? One obvious possibility is that our suggestion that Thr201 plays an important role in controlling the active site specificity of protein kinases (particularly PKA) is incorrect. However, there are two other explanations that could account for the different active site substrate specificities of PKA, PKC, and PKG. These are illustrated in Fig. 1for the alpha-substituted methyl derivatives 11 and 16. Fig. 1A depicts the likely fashion in which the PKA substrate 11 is accommodated within the active site. In contrast, the configurational isomer 16 fails to serve as a substrate for PKA, possibly due to deleterious steric interactions between the Thr-201 side chain and the alpha-methyl substituent (Fig. 1B). Although 16 is not a substrate for PKA, it is phosphorylated by PKC and PKG. This may be due to subtle differences in active site architecture between the latter two and that present in PKA. For example, the orientation of the active site threonine in PKG and PKC may not completely impede binding of residues containing the D-configuration (Fig. 1C). Alternatively, the peptide substrate may be able to associate with PKG or PKC in a nonclassical C-to-N terminus mode (Fig. 1D). In this arrangement, the ``disallowed'' configurational isomer would be presented to the enzyme in an allowed conformational sense. We addressed these possibilities by preparing Ac-Ser-Phe-Arg-Arg-Arg-Arg-Lys-NH(2). This peptide serves as an excellent substrate for PKC (K(m) = 0.81 ± 0.04 µM; V(max) = 11.0 ± 2.1 µmol/min/mg), results which are compatible with the binding mode in Fig. 1D. Indeed, PKC is known to recognize protein and peptide substrates that possess positively charged residues both upstream and downstream from the phosphorylatable residue (42) . Newton and her colleagues (43) recently modeled the three-dimensional structure of PKC, and it is apparent that there are regions of negative charge density enveloping both ends of the active site groove. In contrast, PKG is unable to utilize Ac-Ser-Phe-Arg-Arg-Arg-Arg-Lys-NH(2) as a substrate. These preliminary data suggest that PKC and PKG may accommodate D-isomers by different mechanisms. However, additional experiments are required to resolve this issue in an unequivocal fashion.


Figure 1: Possible kinase-bound orientations of peptides 11 and 16. A, the PKA substrate 11 coordinates to the active site in the conventional N to C terminus fashion (based on x-ray crystallography results(37, 38, 39, 40, 41) ). B, peptide 16 fails to serve as a PKA substrate as a consequence of proposed deleterious steric interactions between the alpha-substituent on the phosphorylatable residue and the side chain of Thr-201. C, the active site architecture of PKG may be subtly altered relative to that of PKA, which would allow the alpha-substituent of 16 to be accommodated within the active site region. D, peptide 16 binds to PKG in a nonclassical N to C terminus sense.



In summary, we have found that active site substrate specificities of PKG and PKA are remarkably distinct. In this study, several peptide pairs have been synthesized that possess the same amino acid sequence, yet differ in stereochemistry at a single critical chiral center. The chirality of the alpha-center on the phosphorylatable residue is a key recognition element for PKA, with only the appropriate stereoisomer serving as a substrate. On the other hand, the nature of this chiral center does not appear to be crucial for substrate recognition by PKG. Consequently, PKG can now be readily distinguished from PKA on the basis of active site specificity. This appears to augur well for the creation PKG-specific inhibitors, compounds that should be especially useful as agents for the further elucidation of the biological consequences of PKG action.


FOOTNOTES

*
This work was supported by Research Grants GM45989 (to D. S. L.) and DK40029 (to J. D. C.). 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.

§
To whom correspondence should be addressed. Tel.: 716-645-6800 (Ext. 2170); Fax: 716-645-6963.

(^1)
The abbreviations used are: PKG, cGMP-dependent protein kinase; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PKI, heat-stable inhibitor protein of cAMP-dependent protein kinase; MOPS, 4-morpholinepropanesulfonic acid.

(^2)
We do observe, with some of the residues whose configuration corresponds to D-serine, a minute amount of phosphopeptide generated by PKA. However, in those instances in which we followed the time course of phosphorylation, phosphopeptide formation levels off at significantly less than 1% of the total peptide present. This implies that a small amount of impurity (more than likely the L-isomer) is present.


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