(Received for publication, September 15, 1995; and in revised form, October 27, 1995)
From the
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 -substituted
alcohols. Although PKA will phosphorylate only residues that contain an
-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.
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 (
)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.
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.
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.
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. ()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.
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 or
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
-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
-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
. This peptide serves as
an excellent substrate for PKC (K
= 0.81
± 0.04 µM; V
= 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
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
-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
-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 -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.