(Received for publication, May 23, 1995; and in revised form, July 20, 1995)
From the
The active site substrate specificities of v-Abl and c-Src are compared and contrasted. Both enzymes catalyze the phosphorylation of a broad assortment of peptide-bound aliphatic and aromatic alcohols, such as achiral and simple straight chain residues. In addition, both protein kinases exhibit a ``dual specificity'' with respect to the ability to utilize D- and L-configurational isomers as substrates. However, c-Src and v-Abl are extremely inefficient as catalysts for certain structural arrangements, including secondary alcohols and primary alcohols containing large substituents in close proximity to the hydroxyl moiety. In addition to these similarities, these enzymes also display noteworthy differences in catalytic behavior. Whereas c-Src exhibits a modest preference for aromatic versus aliphatic alcohols, v-Abl does not. Most dramatic is the ability of c-Src to utilize short chain alcohols as substrates, an activity virtually absent from the catalytic repertoire of v-Abl. The implications of these observations are 2-fold. First, because both enzymes are able to accommodate a wide variety of structural variants within their respective active site regions, there exists a substantial degree of flexibility with respect to inhibitor design. Second, because these enzymes exhibit disparate active site specificities, it is possible that other tyrosine-specific protein kinases will display unique substrate specificities as well. Consequently, it may ultimately be possible to exploit these differences to generate inhibitors that precisely target specific protein kinases.
Protein phosphorylation plays a pivotal role in signaling pathways, which regulate such fundamental processes as cell growth, differentiation, and division(1, 2) . Consequently, protein kinase inhibitors that can be precisely directed to specific members of the kinase family could prove to be of decided utility in understanding and controlling these signaling pathways. Much of the effort devoted to the design and construction of agents that are targeted to individual protein kinases has focused on the identification of characteristic substrate sequence specificities. Because most protein kinases utilize short peptides as substrates(3, 4) , it may be possible to create inhibitors for specific protein kinases by employing peptides containing appropriate primary sequences. Unfortunately, this approach suffers from the limitation that many kinases exhibit broad overlapping sequence specificities with other family members. One way to overcome this obstacle is to employ additional recognition motifs in conjunction with the specific primary sequence to narrow the range of kinases that are impaired by inhibitory species.
We have developed a facile
method to rapidly assess the active site substrate specificity of
protein
kinases(5, 6, 7, 8, 9, 10, 11) .
Although protein kinases will generally phosphorylate only serine,
threonine, and/or tyrosine residues in intact protein substrates, we
have found that these enzymes will phosphorylate a diverse array of
alcohol-containing non-amino acid residues in synthetic peptides. This
approach has allowed us to investigate the range of functionality that
can be accommodated within and phosphorylated by the protein kinase
active site, an issue particularly germane to the development of such
inhibitory species as transition state analogs, affinity labels, and
mechanism-based inhibitors. In addition, although many
serine/threonine-specific protein kinases exhibit overlapping
specificities for conventional peptides, we have recently shown that
several of these enzymes will display remarkably different behavior
toward peptides bearing exotic alcohol-containing residues (9) . ()In short, it may ultimately be possible to
target specific members of the protein kinase family by coupling the
active site substrate specificity of an individual family member with
its characteristic sequence specificity.
We recently described the first active site substrate specificity analysis of a tyrosine-specific protein kinase(11) . The specificity of c-Src, like that of several serine/threonine-specific protein kinases, is not limited to amino acid residues found in proteins. Indeed, c-Src phosphorylates a broad assortment of aliphatic and aromatic alcohol-containing residues that are attached to active site-directed peptides. Do members of the tyrosine-specific protein kinase family, like their serine/threonine-specific counterparts, exhibit dissimilar active site specificities? If the answer to this question is in the affirmative, it may be possible to exploit these differences in specificity to create inhibitory agents that distinguish between otherwise closely related enzymes. As a consequence, we have investigated the active site specificity of a second ``tyrosine-specific'' protein kinase.
The protein product of v-abl, like that of c-src,
specifically phosphorylates tyrosine residues in protein substrates.
The oncogenic form of abl was first discovered as the
transforming gene of Abelson murine leukemia virus(12) . In
humans, the normal cellular homolog, c-abl, is involved in
chronic myelogenous leukemias and some acute lymphocytic leukemias
(reviewed in (13) ). In these leukemias, a chromosomal
translocation takes place in which 5` sequences of the bcr gene become fused to abl, generating an oncogenic Bcr-Abl
fusion protein(14) . In all cases, there is a strong
correlation between oncogenic transformation and tyrosine kinase
activity. Like abl, src was originally identified as
a retroviral oncogene; both transforming genes are altered versions of
normal cellular proto-oncogenes(15) . v-Abl and c-Src are
members of the nonreceptor tyrosine kinase family and, as such, share
many common features. Both contain SH2 and SH3
domains(16, 17) , and both Src and several forms of
Abl undergo myristoylation(18, 19) , which promotes
membrane association. These enzymes also share a strong sequence
homology within their respective active site
regions(20, 21) . Finally, both kinases exhibit
overlapping substrate specificities in vitro, ()a
feature that can render the design of kinase-specific inhibitors
problematic. In spite of these similarities, we now report that these
tyrosine-specific kinases can be distinguished from one another by
their active site substrate specificities.
All chemicals were obtained from Aldrich, except for
[-
P] ATP (DuPont NEN), piperidine (Advanced
ChemTech), protected amino acid derivatives (Advanced ChemTech and
Bachem), Liquiscint (National Diagnostics),
Fmoc-Leu-2-methoxy-4-alkoxybenzyl (
)alcohol resin
(Peninsula), glutathione-agarose (Molecular Probes), and N-lauroylsarcosine (Sigma). Phosphocellulose P 81 paper discs
were purchased from Whatman.
H NMR and
C NMR
experiments were performed at 400 (Varian VXR-400S) and 22.5 MHz
(Varian Gemini-300), respectively. Chemical shifts are reported with
respect to tetramethylsilane. Fast atom bombardment (peptides) and
electron impact (amino alcohols) mass spectral analyses were conducted
with a VG-70SE mass spectrometer.
Peptides 14 and 19 were synthesized by first coupling
Fmoc-protected L-serine and Fmoc-protected D-tyrosine, respectively, to the rink resin(24) . The
Fmoc protecting groups were then removed with piperidine. The free C
terminus of
Fmoc-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Leu-Glu(t-butyl)-Glu(t-butyl)-Leu-Leu
(40 mg, 0.014 mmol; see synthesis described above) was activated with
Bop (0.028 mmol) in 10 ml of
CHCl
/N,N-dimethylformamide
(1:1). The amino acid-substituted rink resins (0.042 mmol) were then
added to separate solutions containing the activated form of
Fmoc-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Leu-Glu(t-butyl)-Glu(t-butyl)-Leu-Leu.
The Fmoc group was removed (piperidine), and the undecapeptides were
cleaved (90% trifluoroacetic acid/10% thioanisole; 10 ml, 4 h) from the
rink resin. The peptides were isolated and purified as described above.
Both purified peptides gave satisfactory fast atom bombardment mass
spectral analyses.
Peptide substrates containing structurally disparate
alcohol-bearing residues, positioned at the site of phosphorylation,
can provide critical information concerning the range of compounds that
can be accommodated within and phosphorylated by the active site of
protein kinases(5) . Although it can be laborious to synthesize
peptides that contain exotic amino acid residues at internal sites, it
requires significantly less effort to prepare analogous peptides
containing nonstandard residues at either the N or the C
terminus(5) . Both c-Src and v-Abl phosphorylate the C-terminal
tyrosine residue in peptide 1 (Table 1). The five arginine
residues on the N terminus of this peptide are present in order to
promote binding of the phosphorylated peptide to phosphocellulose
filter paper. This provides a convenient method for following the
kinetics of phosphorylation. The C-terminal portion of the peptide,
Leu-Glu-Glu-Leu-Leu, is negatively charged, a feature that appears to
be important for substrate recognition by some tyrosine
kinases(15, 26, 27) . However,
whereas 1 is a substrate for both c-Src and v-Abl, it is clear
that this peptide is more efficiently phosphorylated by the former than
by the latter. This may be due, in part, to real differences in
sequence specificities between the two enzymes. For example, Src has a
special preference for peptide substrates that contain acidic residues
at the P-3 and P-4 positions(27) .
In contrast,
v-Abl is much less dependent upon the presence of negatively charged
residues for substrate recognition(26, 27) . However,
it is also important to note that kinetic constants obtained for the
v-Abl-catalyzed phosphorylation of 1 compare favorably with
those associated with the best peptide substrates reported for this
enzyme (15) .
In short, the fact that peptide 1 is a 42-fold more efficient substrate for c-Src than for v-Abl may
simply be due to the innate ability of the former to serve as more
potent phosphoryl transfer catalyst than the latter. The salient point
is that both enzymes phosphorylate common substrates, a feature that
has been previously noted(15, 28) .
Both v-Abl and c-Src phosphorylate aromatic and benzylic alcohols (Table 1). For example, peptide 2, which contains an tyramine residue, is a substrate for both enzymes. Although it is significant that these tyrosine-specific kinases will phosphorylate an achiral residue, it is equally noteworthy that 2 displays an enhanced specificity for c-Src versus v-Abl relative to that of the tyrosine-containing peptide 1 (Table 2). In contrast, the aminomethylbenzyl alcohol 3 is almost identical to L-tyrosine in its ability to discriminate between these enzymes. Finally, the benzylic alcohol in 4 is less efficient than L-tyrosine in distinguishing between c-Src and v-Abl. The decrease in c-Src/v-Abl specificity as one moves from compound 2 to compound 3 to compound 4 is largely due to changes in activity of c-Src toward these substrates. All three peptides are virtually identical as substrates for v-Abl. In short, although c-Src does discriminate between aromatic and aliphatic alcohols (i.e.2versus3 or 4), v-Abl does not.
We previously demonstrated that the serine/threonine-specific
cAMP-dependent protein kinase will not catalyze the phosphorylation of
tyrosine in an active site-directed peptide(29) . However, this
protein kinase will catalyze the phosphorylation of aromatic alcohols
if the hydroxyl moiety can be positioned in the active site in a manner
comparable with that of serine(8, 10) . In addition,
although cAMP-dependent protein kinase (as well as protein kinase C)
will phosphorylate achiral aliphatic alcohols, this activity is highly
dependent upon chain length(5, 9) . There is a
dramatic loss in substrate efficacy if the number of methylene groups
on the residue to be phosphorylated is greater than two. This is not
particularly surprising, because the hydroxyl and amino groups in
serine are separated by two methylene groups as well. The active site
substrate specificity of serine/threonine-specific protein kinases is
therefore strictly determined by the relative orientation of and the
distance between the phosphorylatable alcohol moiety and the peptide
backbone to which it is attached. Is the active site specificity of
tyrosine-specific protein kinases as tightly regulated? The results in Table 1clearly demonstrate that both c-Src and v-Abl will
phosphorylate aromatic and benzylic (i.e. aliphatic) alcohols.
However, each of these substrates possesses an aromatic moiety.
Furthermore, the hydroxyl functionality in species 2-4 is oriented, relative to the peptide backbone, in a manner
reminiscent of that found in the tyrosine-containing peptide 1.
Is this structural alignment of functionality a requirement for
recognition by tyrosine-specific protein kinases? In order to address
this question, we prepared a series of peptides containing straight
chain aliphatic alcohols. In a broad sense, both v-Abl and c-Src
utilize these species as substrates (Table 3). Therefore, it is
evident that an aromatic moiety is not required for substrate
recognition by either of these enzymes. Furthermore, the distance
between the aromatic alcohol in tyrosine and the -amino group is
roughly equivalent to a chain containing 5-7 methylene groups.
Consequently, one might predict that compounds 8-10 should serve as the most efficient substrates for v-Abl and c-Src
and that the remainder of the compounds listed in Table 3would
be extraordinarily inefficient substrates (if they are phosphorylated
at all). Somewhat surprisingly, this is not the case (Table 2).
For example, the octanolamine derivative 11 is the most
effective c-Src substrate. However, the k
/K
values for peptides 5-12 do not differ to any significant extent with
this enzyme. Only at a chain length of 12 carbon atoms is a drop-off in
substrate efficacy apparent. In this particular case, we were only able
to obtain a lower limit of the maximal velocity. This is due to the
fact that substrate saturation appears to occur at a relatively low
concentration (<150 µM) and that at these
concentrations the rate of phosphorylation of 13 is exceedingly
weak. This implies that the concentrations of 13 employed may
exceed the K
. As a consequence, we are able to
provide only an estimate of V
for this peptide,
as well for peptides 15-18 in Table 4.
In
contrast to the behavior displayed by c-Src toward peptides 5-13, v-Abl exhibits a definite dependence upon
chain length. Short chains, such as the ethanolamine (5) and
propanolamine (6) derivatives, are extremely poor v-Abl
substrates. Interestingly, our inability to obtain K and V
values in these two instances is not
due to the difficulties that we encountered with the c-Src-catalyzed
phosphorylation of peptide 13 described above. In the case of
v-Abl, although high concentrations (1-2 mM) of peptides 5 and 6 provide weak rates of phosphorylation, we did not
detect any evidence of substrate saturation. This implies that the K
values for peptides 5 and 6 are
quite large, a notion consistent with the results obtained for peptide 7, the next higher homolog. At substrate concentrations
significantly below the K
, the Michaelis-Menten
equation reduces to v = (k
/K
)[E][S],
which allows the ready acquisition of k
/K
. However, because we
are uncertain of the actual K
, the k
/K
values indicated for
peptides 5, 6, and 14 in Table 2should be
viewed as approximations. Finally, the v-Abl-catalyzed rates of
phosphorylation for peptides 15-18 are so weak that
we could only provide an estimate of k
/K
in these cases.
Based upon the k/K
value
extracted for peptide 5, it is evident that this species is a
significantly more potent substrate (>2,000-fold) for c-Src than for
v-Abl. As such, peptide 5 is the most accurate c-Src-targeted
substrate that we have evaluated to date. However, this bias in favor
of c-Src decreases in a dramatic fashion with increasing chain length.
This decrease is a consequence of an improvement in k
/K
for v-Abl with the
longer aliphatics, which in turn is due to a decrease in K
. Indeed, the K
for peptide 13 is even smaller than that obtained for the L-tyrosine peptide 1. Interestingly, these results are
consistent with previous sequence specificity studies reported for
these two kinases. v-Abl has been shown to prefer hydrophobic residues
on the N-terminal side of tyrosine to a greater extent than
c-Src(15) . This appears to correlate with the preference of
v-Abl for the longer and more hydrophobic straight chain alcohols of Table 3.
Because the ethanolamine-containing peptide 5 serves as a substrate for c-Src it is not too surprising that Arg-Arg-Arg-Arg-Arg-Leu-Glu-Glu-Leu-Leu-Ser-amide (14) is phosphorylated by this enzyme as well (Table 4). However, the ethanolamine derivative (5) is 4.5-fold more efficient than its serine-containing counterpart (14). One possible explanation for this observation is that the amide moiety, which is present in 14 but absent in 5, encounters unfavorable steric interactions with active site residues. If this supposition is correct, then substituents more sterically demanding than the amide functionality should produce even less effective substrates (Table 4). Indeed, this appears to be the case for the benzyl-substituted derivatives 15 and 16. These results are not particularly unexpected given the structural characteristics of the customary substrate of c-Src. In the case of tyrosine, the hydroxyl moiety protrudes from an aromatic framework, which is tied back in a sterically uncompromising cyclic fashion. In contrast, the amide and benzyl substituents of peptides 14-16 are more sterically prominent and obtrusive than the aromatic moiety of tyrosine, particularly near the hydroxyl functionality. Furthermore, secondary alcohols (17 and 18) also fail to serve as efficient c-Src substrates. All of these observations are consistent with the notion that sterically demanding functionality may encounter unfavorable interactions when forced to reside within the active site region of c-Src.
Although the ethanolamine-derivatized peptide 5 is an effective c-Src substrate, it is almost imperceptibly phosphorylated by v-Abl. If the steric arguments outlined above apply to v-Abl as well, then at the very least this enzyme should not be able to phosphorylate peptides 14-18 any more efficiently than 5. Indeed this is the case. Nevertheless, v-Abl does phosphorylate all of these peptides, albeit at an nearly indiscernible level that is barely above background.
Protein kinases are commonly classified by their ability to phosphorylate aliphatic (i.e. serine/threonine) or aromatic (i.e. tyrosine) hydroxyl groups in naturally occurring proteins. However, as is apparent from this as well as earlier(5, 6, 7, 8, 9, 10, 11) active site substrate specificity studies, these categories break down with synthetic substrates. Nonetheless, a new pattern is beginning to emerge with respect to the classification of protein kinases. We, as well as others, have shown that although cAMP-dependent protein kinase will only phosphorylate Lamino acid residues, protein kinase C will catalyze the phosphorylation of both L- and D-stereoisomers (5, 9, 30, 31) . More recently, cGMP-dependent protein kinase and c-Src (11) have been added to the latter category. We have now found that peptide 19, which contains a D-tyrosine residue, will also serve as a substrate for v-Abl. The following features concerning this activity are notable. First, although v-Abl will phosphorylate peptides containing either D- or L-tyrosine, the latter is a significantly more efficient (65-fold) substrate than the former. Second, the D-tyrosine-containing peptide (19) is a 5-fold poorer substrate than the corresponding achiral species (2). Consequently, it is clear that the inverted configuration associated with D-tyrosine in conjunction with the amide substituent actively interferes with the ability of this species to be recognized as a substrate for v-Abl. Third, peptide 19 serves as a 230-fold more efficient substrate for c-Src than for v-Abl, which may indicate that the former is able to accommodate D-amino acid residues more readily than the latter.
We have not yet completely unraveled the molecular mechanism that enables certain protein kinases to phosphorylate both L- and D-configurational isomers. However, we have previously proposed that the alcohol functionality of a D-residue can be presented to the target kinase in a manner analogous to that of an L-residue if the peptide were to bind to the enzyme surface in a aberrant C to N terminus fashion(9) . Although an analogous binding mode may be in operation for v-Abl and c-Src, other mechanisms may account for this form of active site specificity as well.
In summary, we have compared and contrasted the active site substrate specificities of v-Abl and c-Src. Although these enzymes are commonly referred to as tyrosine-specific, both species are able to phosphorylate a wide variety of aromatic and aliphatic alcohols, including a D-tyrosine residue. Both are also unable to phosphorylate certain structural arrangements, such as secondary alcohols and primary alcohols containing large substituents within close proximity to the hydroxyl moiety. Nevertheless, v-Abl and c-Src do exhibit certain subtle and gross differences in catalytic behavior as well. Although c-Src displays a moderate preference for aromatic versus benzylic alcohols, v-Abl does not. Although c-Src utilizes short straight chain alcohols as substrates, the v-abl-encoded protein kinase does not. Indeed, the former does not discriminate against aliphatic alcohols on the basis of chain length, whereas the latter exhibits a clear bias on these grounds. In short, these results are encouraging with respect to inhibitor design, because both enzymes are able to accommodate a range of variants within their active site regions. Also encouraging are the clear differences in the active site specificities of these enzymes. This particular feature, when employed in combination with preferred sequence specificities, may ultimately provide a means to precisely target and inactivate specific protein kinases.