The vast majority of enzymes that participate in
growth-promoting signal transduction pathways are protein
kinases(1, 2) . Furthermore, the link between
carcinogenesis and certain constituitively activated pathways has been
clearly established(1, 2) . Consequently, it is not
surprising that there is a keen interest in the creation of protein
kinase inhibitors. However, in order for these inhibitors to be
effective they must be designed with specific criteria in mind. First,
the structural attributes of the inhibitory species must complement
those of the target enzyme. For example, if the mechanism of
inactivation is one that requires interaction with the enzyme active
site, then the inhibitory component must be readily accommodated and
properly positioned within the active site region. Second, protein
kinase inhibitors will only prove useful if they can be targeted
against specific kinases. This is a potentially difficult challenge,
since the reaction catalyzed by protein kinases, namely phosphoryl
transfer from ATP to an acceptor hydroxyl moiety in proteins, is a
relatively general one. Fortunately, most protein kinases will utilize
short peptides as substrates, and it is now apparent that substrate
recognition is often driven by the primary sequence associated with the
peptide undergoing phosphorylation(3) . In short, it may be
possible to target inhibitors for specific protein kinases by utilizing
peptides containing the appropriate primary sequence. Unfortunately,
many protein kinases exhibit overlapping sequence specificities,
thereby limiting the universal applicability of this approach.
Recently, we developed a facile method to rapidly assess the active
site specificity of protein kinases(4) . We have found that
protein kinases will phosphorylate a diverse array of
alcohol-containing non-amino acid
residues(4, 5, 6, 7, 8, 9) .
Most importantly, this approach allows us to address the specific
issues noted above concerning protein kinase inhibitor design. In
particular, the range of alcohol-bearing compounds that serve as
protein kinase substrates should provide a good assessment of the range
of functionality that can be readily accommodated within the active
site region. Second, we have recently shown that even those protein
kinases that exhibit overlapping substrate specificities for
conventional peptides will display remarkably distinct specificities
for peptides bearing unconventional alcohol-containing
residues(8) . In short, the active site substrate specificity
of protein kinases, coupled with their characteristic sequence
specificities, may ultimately provide a useful method for specifically
targeting protein kinases in the cell. Up until this point, our work
has dealt exclusively with serine/threonine-specific protein kinases.
However, in this paper we describe the first active site substrate
specificity analysis of a tyrosine-specific protein kinase, namely
pp60
. Much to our surprise, we have
found that the specificity of pp60
is extraordinarily broad and includes a unusually diverse
structural array of alcohol-containing residues.
MATERIALS AND METHODS
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), and the
Fmoc(
)-Leu-2-methoxy-4-alkoxybenzyl alcohol resin (Peninsula
Laboratories, Inc.). Phosphocellulose P-81 paper disks were purchased
from Whatman.
H NMR and
C NMR experiments were
performed at 400 MHz (Varian VXR-400S) and 22.5 MHz (Varian
Gemini-300), respectively. Chemical shifts are reported with respect to
tetramethylsilane. Fast atom bombardment (peptides) and chemical
ionization (aminoalcohols) mass spectral analyses were conducted with a
VG-70SE mass spectrometer.
Aminoalcohol Syntheses
All aminoalcohol derivatives were purchased from Aldrich,
except for (4-aminomethyl)benzyl alcohol, (3-aminomethyl)benzyl
alcohol, 7-amino-1-heptanol, 8-amino-1-octanol, 10-amino-1-decanol, and
12-amino-1dodecanol.
Protocol for the Synthesis of (3-Aminomethyl)benzyl Alcohol,
(4-Aminomethyl)benzyl Alcohol, 7-Amino-1-heptanol,
8-Amino-1-octanol
3-Cyanobenzaldehyde (0.5 g, 3.8 mmol) was
slowly added to a refluxing solution of suspended LiAlH
(1.0 g, 26.3 mmol) with vigorous stirring in anhydrous ethyl
ether (100 ml) maintained under an argon atmosphere. The solution was
heated at reflux for 10 h and then water was slowly added dropwise to
quench the reaction (under no further evolution of H
was
apparent). The solidified reaction mixture was vigorously stirred with
a 1:1 mixture of CHCl
/ethyl ether (3
80 ml), and
the combined extract was dried over Na
SO
and
filtered. Evaporation of the solvent furnished a slightly yellow
residue which was purified on a silica gel column (100-200 mesh),
employing a 70:25:5
CHCl
:CH
OH:NH
/H
O
solvent. Upon solvent evaporation, (3-aminomethyl)benzyl alcohol was
obtained as a white solid in 67% yield.
H NMR
(Me
SO-d
): 7.09-7.23 (m, 4H,
aromatic hydrogens); 4.44 (s, 2H, -CH
-OH); 3.65 (s, 2H,
-CH
-NH
).
C NMR
(Me
SO-d
): 144.4, 142.8, 128.2, 125.7,
125.6, and 124.7 (aromatic C); 63.2 (-CH
-OH); 45.8
(-CH
-NH
). CI-MS m/e calculated for C
H
NO: 137.2; found: 138.3
(M
). (4-aminomethyl)benzyl alcohol was obtained from
methyl 4-cyanobenzoate using the above protocol in 76% yield.
H NMR (Me
SO-d
): 7.24 (q,
4H, aromatic hydrogens); 4.44 (s, 2H, -CH
-OH); 3.67 (s, 2H,
-CH
-NH
).
C NMR
(Me
SO-d
): 142.9, 140.7, 127.0, 126.6
(aromatic C); 62.8 (-CH
-OH); 45.4
(-CH
-NH
). CI-MS m/e calculated for C
H
NO: 137.2; found: 138.1
(M
). 7-Amino-1-heptanol was obtained from
7-aminoheptanoic acid using the above protocol in 64% yield.
H NMR (CDCl
): 3.59 (t, 2H, -CH
-OH);
2.64 (t, 2H, -CH
-NH
); 1.30-1.51 (m, 10H,
remaining aliphatic hydrogens).
C NMR (CDCl
):
63.2 (-CH
-OH); 42.4 (-CH
-NH
); 33.9,
33.0, 29.5, 27.0, 25.9 (remaining aliphatic carbons). CI-MS m/e calculated for C
H
NO:
131.3; found: 132.2 (M
). 8-Amino-1-octanol was
obtained from 8-aminooctanoic acid using the above protocol in 55%
yield.
H NMR (CDCl
): 3.58 (t, 2H,
-CH
-OH); 2.64 (t, 2H, -CH
-NH
);
1.28-1.52 (m, 12H, remaining aliphatic hydrogens).
C
NMR (CDCl
): 63.2 (-CH
-OH); 42.4
(-CH
-NH
); 33.9, 33.0, 29.63, 29.59, 27.0, 25.9
(remaining aliphatic carbons). CI-MS m/e calculated
for C
H
NO: 145.3; found: 146.2
(M
).
Protocol for the Synthesis of 10-Amino-1-decanol and
12-Amino-1-dodecanol
A solution of potassium phthalimide (0.65
g, 3.5 mmol) and 10-bromo-1-decanol (0.71 g, 3 mmol) in DMF (10 ml) was
heated to reflux for 4 h. The reaction was allowed to cool and was then
filtered to remove the white precipitate, and the solvent was
subsequently removed in vacuo. The residue obtained was then
dissolved in 30 ml of ethanol containing 0.5 ml of 98% hydrazine (16
mmol). The mixture was heated to reflux for 4 h, cooled to room
temperature, and filtered to remove the precipitate (phthalhydrazide).
This precipitate was washed three times with chloroform (20 ml) and the
combined extracts added to the ethanol filtrate. A white solid was
obtained after evaporating the solvent in vacuo. The solid was
further purified via column chromatography on silica gel (100-200
mesh; 70:25:5
CHCl
:CH
OH:NH
/H
O) to
furnish 10-amino-1-dodecanol in 85% yield.
H NMR
(CDCl
): 3.60 (t, 2H, -CH
-OH); 2.65 (t, 2H,
-CH
-NH
); 1.25-1.52 (m, 16H, remaining
aliphatic hydrogens).
C NMR (CDCl
): 63.4
(-CH
-OH); 42.4 (-CH
-NH
); 33.7,
33.0, 29.7 (two peaks), 29.61, 29.57, 27.0, 25.9 (remaining aliphatic
carbons). CI-MS m/e calculated for
C
H
NO: 173.4; found: 174.4
(M
). 12-Amino-1-dodecanol was obtained from
12-bromo-1-dodecanol using the above protocol in 91% yield.
H NMR (CDCl
): 3.59 (t, 2H, -CH
-OH);
2.64 (t, 2H, -CH
-NH
); 1.23-1.52 (m, 20H,
remaining aliphatic hydrogens).
C NMR (CDCl
):
63.3 (-CH
-OH); 42.3 (-CH
-NH
); 33.7,
33.0, 29.75 (two peaks), 29.71 (two peaks), 29.62, 29.58, 27.0, 25.9
(remaining aliphatic carbons). CI-MS m/e calculated
for C
H
NO: 201.5; found: 202.5
(M
).
Peptide Synthesis
Fmoc-Arg[4-methoxy-2,3,6-trimethylbenzenesulfonyl
(Mtr)]-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Leu-Glu(t-butyl)-Glu(t-butyl)-LeuLeu-resin
was prepared on the 2-methoxy-4-alkoxybenzyl alcohol resin
(substitution level = 0.45 mmol/g of resin) with
fluorenylmethoxycarbonyl (Fmoc) amino acids(10, 11) .
A standard Fmoc peptide synthesis protocol was employed using an
automated peptide synthesizer (Advanced ChemTech Act 90). Upon
completion of peptide synthesis, the side chain-protected peptide was
cleaved from the resin using 1% trifluoroacetic acid in
CH
Cl
(20 ml/g of resin; 15 min). The resin was
filtered and the trifluoroacetic acid/CH
Cl
solvent (containing the peptide) was collected. This process was
repeated three additional times. 100 ml of water was subsequently added
to the combined trifluoroacetic acid/CH
Cl
extracts. Triethylamine was added to furnish a neutral solution
and then all but 15 ml of the solvent was removed under reduced
pressure. The precipitated peptide was collected via filtration and
washed with ethyl ether (50 ml). Typically, 1 g of resin provided 400
mg of side chain-protected peptide. The free C terminus of
FmocArg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Leu-Glu(t-butyl)-Glu(t-butyl)-Leu-Leu
(40 mg, 0.014 mmol) was activated via treatment with
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate
(Bop; 0.028 mmol) in 3 ml of CH
Cl
/DMF (1:1) for
3 min. Individual amino alcohols (0.14 mmol) were introduced and
allowed to react with the activated peptide for 1 h. Subsequently, the
N-terminal Fmoc protecting group was removed via treatment with
piperidine for 30 min, and the N terminus deprotected peptide alcohol
was then precipitated from solution by addition of ethyl ether (50 ml).
The heterogeneous solution was filtered in order to isolate the
peptide. The remaining protecting groups (Mtr and t-butyl
ester) were removed by dissolving the peptide alcohol in 90%
trifluoroacetic acid, 10% thioanisol (6 ml). After stirring for 6 h,
the peptide was precipitated from solution (50 ml of ethyl ether) and
subsequently isolated via filtration. Each peptide alcohol was then
purified via a two-step sequence. First, the individual peptides were
ion-exchanged on CM-Sephadex C-25 (0.4 M KCl to a 1.2 M KCl gradient using a 50 mM potassium acetate, pH 3.5,
buffer). The desired peptide alcohol typically eluted at 0.8-1.0 M KCl was subsequently collected and lyophilized. The peptides
were then desalted via preparative HPLC using three Waters radial
compression modules (2.5
10 cm) connected in series (gradient
(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 5 min (80% A and 20% B); 5-20 min
(20% B to 50% B); 20-25 min (50% B to 80% B). All of the purified
peptides gave satisfactory fast atom bombardment mass spectral
analyses.Peptides 1-5 were prepared as C terminus
amides on the benzhydrylamine resin with t-butyloxycarbonyl (t-Boc) amino acids employing a previously described
protocol(12) . The peptides were simultaneously deprotected and
cleaved from the resin by sequential treatment with 50% methyl sulfide,
50% HF (30 min) and 10% anisole, 90% HF (45 min). All of the crude
peptides were purified by cation-exchange chromatography on CM-Sephadex
C-25, followed by preparative HPLC as described above.
Peptides 21 and 26 were synthesized by first coupling
Fmoc-protected L-serine and Fmoc-protected D-tyrosine, respectively, to the rink resin(13) . 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 CH
Cl
/DMF (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 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.
Human Recombinant pp60

Human pp60
was purchased from
Upstate Biotechnology Inc. The enzyme was expressed by recombinant
baculovirus containing the human c-src gene in SF9 insect
cells.
Kinase Assay
Assays were performed in triplicate at pH 7.5 and
thermostatted in a water bath maintained at 22 °C. Final assay
volume totaled 40 µl and contained 20 mM Hepes, 20 mM MgCl
, 0.125 mg/ml bovine serum albumin, 100 µM (Na
VO
), and 1.55 nM pp60
(except for peptides 8 and 9, where [pp60
] =
0.775 nM). For the determination of kinetic constants the
following concentrations were employed: 100 µM [
-
P]ATP (1000-2000 cpm/pmol) and
a substrate concentration over a 10-fold range around the apparent K
. Phosphorylation reactions were initiated by
addition of 10 µl of pp60
diluted from a
concentrated stock solution (6.2 nM (or where appropriate, 3.1
nM) in 1 mM dithiothreitol and 20 mM Hepes,
pH 7.5) to a solution containing 15 µl of peptide (20 mM Hepes, pH 7.5) and 15 µl of assay buffer (20 mM Hepes, 0.33 mg/ml bovine serum albumin, 53.33 mM MgCl
, and 233 µM Na
VO
). Reactions were terminated after
20.0 min by spotting 25-µl aliquots onto 2.1-cm diameter
phosphocellulose paper disks. After 10 s the disks were immersed in 10%
glacial acetic acid and allowed to soak with occasional stirring for at
least 1 h. The acetic acid was decanted, and the disks were
collectively washed with 4 volumes of 0.5% H
PO
,
1 volume of water, followed by a final acetone rinse. The disks were
air-dried and placed in plastic scintillation vials containing 6 ml of
Liquiscint prior to scintillation counting for radioactivity.
Determination of Kinetic Constants
The apparent K
(± S.D.) and V
(± S.D.) values were determined from
initial rate experiments. The data from these experiments were plotted
using the Lineweaver-Burk procedure, and the corresponding plots proved
to be linear.
RESULTS AND DISCUSSION
We have recently shown that the active site substrate
specificity of both the cAMP-dependent protein kinase
(``PKA'') as well as protein kinase C (``PKC'') is
not limited to serine and threonine
moieties(4, 5, 6, 7, 8, 9) .
Indeed, these protein kinases will phosphorylate a variety of non-amino
acid residues, if these residues are properly positioned on active
site-directed peptides. This has allowed us to assess which structural
features can (and cannot) be readily tolerated within the active site
of these enzymes. In addition, we were surprised to find that the
active site specificity of PKA and PKC dramatically differ with respect
to non-amino acid residues(8) . This is in spite of the fact
that both of these protein kinases exhibit overlapping substrate
specificities with conventional synthetic peptides. These results
suggest that protein kinases can be distinguished from one another on
the basis of their active site specificities, even if their sequence
specificities are nearly identical. We have now applied this analysis
to a tyrosine-specific protein kinase. The range of structurally
diverse species that are phosphorylated by pp60
is extraordinarily broad, much more so than that of PKC and PKA.
Consequently, the remarkably accommodating nature of the
pp60
active site should afford considerable
flexibility in terms of inhibitor design.
We have previously noted
that an active site specificity analysis requires ready access to a
family of peptides containing structurally dissimilar residues at the
site of phosphorylation(4) . In the majority of protein kinase
peptide substrates that have been described to date, the
phosphorylatable residue typically resides at a nonterminal position
within the peptide(3) . Unfortunately, the preparation of
peptides containing exotic residues at internal positions can be
synthetically challenging, particularly if the residue of interest
contains hypersensitive functionality that would not ordinarily survive
the harsh conditions of solid phase peptide synthesis. In addition,
even if peptides of this sort could be readily prepared, for every
peptide-based substrate candidate to be investigated a complete peptide
would have to be synthesized. These time-consuming synthetic obstacles
can be circumvented if the alcohol-bearing residue can be directly
attached to the intact peptide at either the N or C terminus. Under
these circumstances, the key residue is not exposed to the acidic
and/or basic conditions associated with peptide synthesis, and in
addition, the intact peptide precursor need be synthesized only once.
However, in order for this type of approach to be successful, the
target protein kinase must be able to phosphorylate residues at a
terminal position in synthetic peptides. Indeed, we have recently
identified a number of potent substrates for various protein kinases (e.g. PKA, PKC, cdc2
, CaM-kinase Ia(
))
in which a serine or threonine residue is positioned at the C or N
terminus. Furthermore, Pinna and his colleagues have recently shown
that several tyrosine-specific protein kinases (members of the src kinase family) isolated from spleen will phosphorylate peptides
containing tyrosine located at either the N or C terminus(14) .
Consequently, it seemed likely that pp60
itself would exhibit analogous behavior.
We examined the
relative rates of phosphorylation of peptides 1-5 by
pp60
. Previous work by others have shown
that acidic residues can be important determinants for substrate
recognition by pp60
(14, 15, 16) . In addition, since we
chose to utilize the phosphocellulose binding assay, we attached
multiple arginine residues to these peptides in order to promote
efficient coordination of the phosphorylated product to the
phosphocellulose disc. On the basis of the results in Table 1we
decided to employ peptide 5 as the parent substrate. (
)
We previously prepared peptides containing
alcohol-bearing residues situated at the C terminus by utilizing an
oxime resin for solid phase peptide synthesis(4) .
Unfortunately, we have found that this synthetic strategy leads to
by-products as a consequence of the reactivity of the protected
glutamic acid moieties contained within the peptide. (
)Consequently, all of the peptide alcohols described herein
were prepared via activation of the protected peptide 6 with the
condensing agent 7 (Fig. 1). The desired peptide alcohols
were obtained upon treatment of the activated form of 6 with
various amino alcohols (followed by sequential deprotection with
piperidine and trifluoroacetic acid).
Figure 1:
Synthesis of the peptide-based
pp60
substrates. Fmoc-protected
amino acids were employed during solid phase peptide synthesis. The
synthetic peptide was cleaved from the 2-methoxy-4-alkoxybenzyl alcohol
resin with side chain-protecting groups intact using 1% trifluoroacetic
acid in CH
Cl
. The protected peptide was then
coupled to the desired aminoalcohols listed using the Bop (7)
activating agent. Deprotection furnished the peptides 9-25. See
``Materials and Methods'' for the preparation of peptides 2
and 26.
We note the following
concerning the active site substrate specificity of
pp60
.
Achiral Aromatic and Benzylic Alcohols Serve as
pp60
Substrates
We have previously
demonstrated that both PKA and PKC will phosphorylate achiral residues (8) . In an analogous fashion, pp60
will not only phosphorylate the aromatic alcohol of tyrosine, but
the hydroxyl moiety of achiral residues as well. For example, the
tyramine-containing peptide 9 exhibits a somewhat better K
than that of the parent peptide 8 (Table 2). This behavior is in marked contrast to that
displayed by PKA and PKC, where peptide substrates containing achiral
residues exhibit elevated K
values relative to
their chiral amino acid counterparts(8) . We have also
previously shown that PKA will phosphorylate both aliphatic and
aromatic alcohols(4, 9) . Consequently, we
investigated the ability of pp60
to catalyze
the phosphorylation of the benzylic alcohol-containing peptides 10 and 11. Both peptides serve as substrates, with species 10 exhibiting a relatively impressive K
but
a modest V
. There are several noteworthy
features concerning these results with respect to inhibitor design.
Inhibitors, particularly transition state analogs or suicide
substrates, often contain elaborate functionality whose preparation can
be time-consuming. The latter is especially true if the key
functionality of interest must be incorporated onto a chiral residue.
The fact that pp60
will recognize and
catalyze the phosphorylation of structurally simple achiral alcohols
should simplify the synthesis of novel inhibitory species. In addition,
the active site of pp60
appears to be
particularly accommodating since the enzyme phosphorylates both
aromatic and aliphatic alcohols, including the two structurally
dissimilar benzylic alcohols 10 and 11.
Straight Chain Achiral Aliphatic Alcohols Serve as
pp60
Substrates
PKA will not catalyze
the phosphorylation of tyrosine(7) . However, this
``serine/threonine''-specific kinase will phosphorylate
aromatic alcohols as long as the hydroxyl moiety on these residues can
be positioned in the active site in a manner comparable with that of
serine or threonine. In an analogous fashion, one might predict that if
an aliphatic side chain can properly orient a hydroxyl functionality
into the active site of a tyrosine-specific protein kinase, then
phosphoryl transfer should ensue as well. To a rough approximation,
this corresponds to a residue containing a side chain length of six
carbon atoms (see Fig. 2). Therefore, one might expect that
chain lengths much smaller or larger than this ideal would not be able
to optimally position the phosphoryl acceptor hydroxyl moiety in the
appropriate region of the enzyme active site. In spite of this
eminently reasonable assumption, it is clear that
pp60
does not discriminate in the expected
fashion on the basis of chain length (Table 3). For example,
pp60
phosphorylates peptide 12, in
spite of the fact that the distance from the hydroxyl moiety to the
peptide backbone is identical to that present in serine and
significantly shorter than that in tyrosine. Furthermore, there is
little variance in k
/K
from
the two carbon-bearing alcohol (i.e.12) up to the ten
carbon-containing species (i.e.19). Indeed, only with
peptide 20, which possesses a side chain of 12 carbon atoms, is
there a significant loss in substrate effectiveness. These observations
are certainly not in accord with those from our earlier analysis of the
substrate specificity of PKA and PKC. We previously found that both
enzymes will phosphorylate a variety of alcohol-containing residues, as
long as the hydroxyl moiety in these substrates is positioned the
appropriate distance from the peptide backbone (i.e. the same
as that present in serine and threonine)(8) . For example, a
dramatic reduction in substrate efficacy is observed if the side chain
is altered from the ideal length by as little as two carbon atoms.
Furthermore, this dependence upon side chain length is not just limited
to the activity of PKA and PKC. For example, Barford et al. (17) have recently proposed that, on the basis of the
three-dimensional structure of protein tyrosine phosphatase 1B, the
depth of the active site plays a critical role in enabling
tyrosine-specific protein phosphatases to distinguish between target
phosphotyrosine residues and those of phosphoserine and
phosphothreonine. Clearly, pp60
does not
conform to the limitations displayed by these related enzymatic
species. What is the structural basis for this behavior? In addition,
if the tyrosine-specific pp60
is able to
phosphorylate 12, shouldn't it be able to phosphorylate
serine as well?
Figure 2:
Comparison of the side chain lengths of
the tyrosine- and 6-amino-1-hexanol-containing
peptides.
An L-Serine-containing Peptide Serves as a
Substrate for pp60

Since
ethanolamine-bearing peptide 12 is a substrate for
pp60
, we investigated the possibility that
the corresponding serine-containing peptide 21 could also serve
as a substrate for this ``tyrosine-specific'' protein kinase.
Indeed, the latter is a substrate (Table 4), albeit a
significantly poorer one than the former. Nevertheless, this
observation is clearly reminiscent of the behavior of a small group of
protein kinases; namely those that are apparently able to phosphorylate
serine, threonine, and tyrosine(18) . What is the
structural basis for this dual specificity? One possibility is that the
active site is inherently flexible and is able to assume a conformation
that structurally complements that of the active site-bound residue.
However, this does not account for the difference in k
/K
values between peptides 12 and 21. An alternative explanation for this ``dual
specificity'' assumes a more conventional conformationally fixed
active site. In the model illustrated in Fig. 3, the peptide is
able to occupy any of several closely positioned loci on the enzyme
surface, each of which lies a characteristic distance from the
phosphorylation site. In this scenario, an alcohol moiety positioned on
a ``shorter-than-ideal'' side chain (e.g.12)
can assume the proper location in the active site if a portion of the
peptide backbone can be accommodated in the active site region. For an
alcohol functionality located on a ``longer-than-ideal'' side
chain (e.g.20), the peptide backbone would be
positioned at a greater distance from the active site. Implicit
within this model is the assumption that the interaction between the
peptide substrate and the enzyme surface is not fixed to a single
prescribed site, but instead fluctuates according to the structural
nature of the substrate. For example, this behavior might be
observed if the enzyme surface is characterized by an extended region
of positive charge density. Under these circumstances, the peptide
could favorably associate with the enzyme at a multitude of sites. In
addition, this model may offer an explanation for the disparate K
values associated with the phosphorylation of
peptides 12 and 21. While incorporation of the alcohol
moiety of 12 into the active site should not be structurally
demanding, insertion of the hydroxyl functionality of serine (i.e.21) by necessity, positions a structurally obtrusive amide
group in the active site. If this notion is correct, then a residue
that contains a substituent even larger than the amide moiety should
produce a poorer substrate. Indeed, peptide 22, which possesses
a benzyl substituent at the
-position, is such a poor substrate
that we were unable to obtain accurate kinetic constants. We also
examined the ability of peptide 24, which contains a methyl
substituent at the
position, to serve as a substrate for
pp60
. This peptide proved to be an
exceedingly poor substrate as well. Finally, we prepared the
diastereomers of 22 (i.e.23) and 24 (i.e.25). These peptides also failed to serve as
efficient substrates. In short, it is clear that substituents at the
or
positions of the phosphorylatable residue dramatically
interfere with the ability of short chain alcohols to serve as a
substrates for pp60
. While these
observations are certainly consistent with the model depicted in Fig. 3, it remains to be seen whether this type of analysis is
applicable to the several dual specificity protein kinases that have
recently been identified(18) .
Figure 3:
Interactions of alcohol-containing
peptides with pp60
. A, the
tyrosine-bearing peptide 8 is aligned on the enzyme surface to properly
position the aromatic alcohol in the active site. B, since the
ethyl side chain of 12 is short relative to that in 8, the P-1 residue
must be partially inserted into the active site in order for the
hydroxyl functionality to be correctly situated in the active site. C, the side chain of 20 is of sufficient length so that the
peptide backbone may be located at a greater distance from the active
site than that in B. D, unfavorable steric
interactions between active site residues and the
-amide moiety of L-serine may arise upon interaction of 21 with
pp60
.
pp60
Phosphorylates a D-Tyrosine Residue in an Active Site-directed
Peptide
Although it is clear that substituents at either the
or
positions in the ethylalcohol-based species (12)
markedly lower substrate efficacy (cf. 21-25),
this is obviously not the case with the aromatic alcohol-based peptides 8 and 9. Indeed, the
-amide substituent in 8 is actually beneficial in terms of k
/K
. Evidently,
-substituents can be accommodated near the active site as long as
these substituents are positioned some distance from the alcohol that
suffers phosphorylation. This is also consistent with the model
depicted in Fig. 3. While it is clear that an
-substituent
can have favorable consequences in terms of substrate efficacy for
aromatic alcohols (e.g.8), we wondered whether the
configuration at the
-center played any role in substrate
recognition. We(8) , as well as
others(19, 20) , have shown that PKC will catalyze the
phosphorylation of both D- and L-residues, whereas
the specificity of PKA is strictly limited to L-residues.
Indeed, the D-tyrosine-containing peptide 26 does serve
as a substrate for pp60
, although not as
efficiently as its' isomeric counterpart 8. We have not
yet conclusively resolved how a D-amino acid residue can be
recognized by the active sites of PKC and
pp60
. In particular, if the active site
regions of these enzymes are analogous to that of PKA, then the
Michaelis complex of a D-amino acid residue should experience
severe steric interactions that would preclude the requisite alignment
of the hydroxyl moiety in the active site(8) . However, we have
previously proposed that the alcohol functionality in a D-residue could be presented to the active site of PKC in a
structurally acceptable fashion if the peptide is able to bind to the
enzyme surface in a retrograde C to N terminus sense(8) . An
analogous binding mode may be in operation for
pp60
as well.In summary,
pp60
exhibits an unusually broad active site
substrate specificity. This protein kinase is able to catalyze the
phosphorylation of achiral residues, including aromatic and aliphatic
alcohols. Furthermore, whereas the substrate specificity of PKA and PKC
is limited to straight chain aliphatic alcohols of four carbons or
less, pp60
phosphorylates aliphatic alcohols
that vary from two to twelve carbon atoms in length. Although, short
chain aliphatic alcohols containing substituents at either the
and
positions are extraordinarily poor substrates, aromatic
alcohols containing
-substituents are superior substrates (cf. 8 and 9). Most interestingly, the
configuration at the
position is not a critical element in
substrate recognition. Peptides containing either D- or L-tyrosine serve as pp60
substrates. These observations may very well have profound
implications in terms of inhibitor design. For example, it should be
possible to synthesize inhibitors of pp60
from readily available compounds, since this enzyme recognizes
and phosphorylates simple achiral residues. In addition, since
pp60
is able to utilize a variety of
structural variants as substrates, the potential flexibility associated
with inhibitor design should be equally as great. Finally, since the
synthetic approach employed in this study does not expose key residues
to the conditions of solid phase peptide synthesis, elaborate
functionality that might not ordinarily survive solid phase synthesis
can be easily incorporated into the active site-directed peptide.