(Received for publication, March 6, 1995; and in revised form, June 1, 1995)
From the
The minimum length required for phosphorylation of a peptide by
pp60 tyrosine kinase (srcTK) was delineated in this work. Budde (M. D. Anderson
University of Texas, personal communication) suggested that the peptide
(FGE)
Y(GEF)
GD (peptide I) was a
``good'' srcTK substrate. Peptide I yielded a
251-fold higher k
/K
than RRLIEDAEYAARRG, a peptide substrate based upon the
autophosphorylation site of srcTK. This was due to a 38-fold
lower K
and a 6.6-fold increase in k
. N-terminal truncation of up to 8 residues in
a series of peptides yielded only a 3-fold decrease in activity.
Removal of the final N-terminal residue resulted in a 10-fold loss in
substrate activity, primarily as a result of an increase in the K
. C-terminal truncations ending in the
amide yielded no significant loss in activity until the Y+3
residue was removed, which resulted in a 73-fold decrease in k
/K
relative to
peptide I. The latter was due primarily to an increase in K
. The results from peptides truncated on
both termini suggest that subsite recognition N- and C-terminal
relative to the site of phosphorylation can be examined independently.
In addition, the observation that only 5 residues are required for
significant substrate activity suggests that small molecule inhibitors
based upon interactions with the phosphoacceptor site may be developed.
Protein tyrosine kinases (TKs) ()were initially
discovered as either oncogenes or proto-oncogene products, pointing to
their therapeutic potential as targets in cancer (for a review of
oncogenes, see Pimentel (1989a and 1989b)). The phosphorylation
equilibrium of protein tyrosine residues is regulated by cytokines and
growth factors, pointing to roles for these equilibria in signal
transduction pathways. Therefore, control of these equilibria has the
potential for therapeutic intervention in diseases including cancer,
inflammatory diseases, and diabetes, to name a few.
While initially
TKs were thought to be nonspecific, more recent work has demonstrated
that they do indeed phosphorylate specific substrates in vivo (for example, see Ogawa et al.(1994)). The study of these
enzymes has been hampered by the lack of specific peptide substrates.
The specificity requirements for amino acid sequences remain to be
elucidated. While several groups proposed that the specificity of TKs
was not governed by the amino acid sequence surrounding the tyrosine
(Tinker et al., 1988; Radziejewski et al., 1989),
more recent studies have suggested that there is specific recognition
of proximal residues. For example, Garcia et al.(1993)
reported differences in the peptide sequences recognized by
pp60(
)and v-abl TK. These workers found that substitution of N (
)for D
in the Y-1 (
)position of KKSRGDYMTMQIG, a peptide
based upon a phosphorylation site of insulin receptor substrate-1,
resulted in complete loss of substrate activity for both TKs.
Substitution of I for M in the Y+1 position resulted in a 4-fold
loss in catalytic efficiency for pp60
but a 10-fold increase in the catalytic efficiency versus v-abl-TK. Till et al.(1994) have
suggested that I is preferred by 6-fold over E and L at the Y-1
site of v-abl-TK substrates. Tinker et al.(1992)
suggested that acidic residues N-terminal to the tyrosine were
important for p56
activity. The Y-3 and
Y-4 positions displayed the greatest sensitivity to E versus A at these positions. A clear-cut correlation for pp60
activity was not obtained in those studies. Songyang et
al.(1995) derived some specificity requirements governing
substrates for 9 TKs using peptide libraries. These workers report that
the optimal substrate for srcTK would contain the sequence
EEIYGEFF, although the scoring of additional residues in various
positions suggests considerable flexibility. Barker et al.(
)suggested that srcTK prefers smaller
hydrophobic residues at the Y+1 position when angiotensin analogs
were used as substrates. This proposal is consistent with the
conclusion drawn from the peptide library studies. Wong and
Goldberg(1983) reported that pp60
displayed similar activity toward the two angiotensin
analogs. Knowledge of the sequence specificity of TKs could allow the
development of specific inhibitors that interact with the protein
binding subsites.
Typically, members of the srcTK family
have been assayed with either peptides based upon the
autophosphorylation site of srcTK (Hunter, 1982; Casnellie et al., 1982; Wong and Goldberg, 1983) or peptides based upon
angiotensin (Wong and Goldberg, 1983). The utility of these and other
reported peptide TK substrates is limited by inefficient kinetic
constants and the size of the peptides (from 8 to 13 residues). The
only relatively systematic examination of the length of peptide
required for TK substrate activity was reported by Cola et
al.(1989). These workers examined the lymphoid-derived TKs (lyn-TK and TPKIIB) and the abl-TK using truncated
versions of the octapeptide, EEKEYHAE, derived from the Tyr site of phosphorylation of the EGF receptor. These studies did
not clearly demonstrate a pattern since the minimal peptide EYH
displayed activity greater than some of the longer peptides. In the
case of the v-abl-TK, EYH was a 3-fold better substrate than
the heptapeptide, EKEYHAE. In the case of TPK-IIB, EYH was 10-fold less
active than the octapeptide, but the effects of truncations were not
additive. For example, EYHAE was 10-fold less active than the parent.
C-terminal truncations had very little effect on the activity. The
potential for development of specific, small molecule inhibitors of TKs
requires obtaining the greatest binding energy and specificity from
minimal peptide subsite interactions. In this study, we delineate the
minimal substrate length required for srcTK.
Phosphorylation of peptides (FGE)Y(GEF)GD,
Ac-(FGE)
YGE-amide, and Ac-FGEYGEF-amide was confirmed by
HPLC-electrospray ionization mass spectrometry. In a typical
experiment, activated N-85-srcTK (0.125 µM) was
incubated at room temperature with 1 mM Ac-FGEYGEF-amide, 1
mM ATP, and 20 mM MgCl
in 100 mM HEPES, pH 7.5. After 5 min, a 4-µl aliquot of the reaction
mixture was added to 46 µl of 0.2% trifluoroacetic acid, and a
3-µl aliquot of this solution was injected onto a Poros R2/H
800-µm
10-cm perfusion column (LC Packings, San Francisco).
The unreacted peptide and phosphopeptide product were eluted at 80
µl/min (Hewlett Packard-1090 microbore pump system) using a
gradient consisting of 1% to 51% eluant B over 6 min (0.035%
trifluoroacetic acid in 90:10 acetonitrile:H
O; eluant A was
0.05% trifluoroacetic acid). The column eluant was monitored at 215 nm
with an Applied Biosystems Instruments model 788A UV-visible detector
equipped with a capillary Z-flow cell (LC Packings) and a API-III
triple quadrupole mass spectrometer equipped with an electrospray ion
source (PE-Sciex, Thornhill, Ontario). The mass spectrometer was
scanned from 300 to 1000 Da in 2.3 s using a 0.3-Da step size and a
1-ms dwell time. Mass spectra were acquired using an orifice potential
of 80 V and an ion multiplier voltage of -4000 V.
An absolute
requirement for residues C-terminal to the tyrosine was not evident
until the amino acid in the Y+3 position was removed. Removal of
this residue resulted in a 73-fold decrease in k/K
, relative to the value
obtained with peptide I. This was primarily due to a 34-fold increase
in K
.
Further removal of the Y+1
and Y+2 residues resulted in only slight additional decreases in
activity. There is not an obvious trend indicative of the relief of
nonproductive binding modes upon truncating the C terminus. This may
reflect the statistics of the effect of a single non-productive binding
mode (FGEF in Y+3 to Y+6) due to the C terminus of the
peptide relative to two to three possible nonproductive modes due to
the N-terminal sequence of these peptides.
Combining the results
from the N- and C-terminal truncations into two peptides produced
essentially additive results. AcEFGEYGEF-amide had a similar catalytic
efficiency to truncations on each terminus singularly. However, removal
of the Y-4 residue of AcEFGEYGEF-amide yielded approximately a
2-fold increase in K and a 2-fold lower k
/K
relative to the values
obtained with the octamer. A similar trend in the K
was seen when AcEFGEY(GEF)
GD is compared to
AcFGEY(GEF)
GD. Further N-terminal truncation of the 7-mer
to produce AcEYGEF-amide had no effect on the kinetic parameters,
although a similar truncation in the context of
AcFGEY(GEF)
GD to AcEY(GEF)
GD resulted in a
2-fold decrease in the K
. These data demonstrate
to a first approximation that substrate specificity can be explored by
assuming that subsite recognition, N- and C-terminal relative to the
site of phosphorylation, can be examined independently.
Songyang et al.(1995) suggested a preference for a hydrophobic residue,
particularly an isoleucine in the Y-1 position. AcFGEYGEF-amide
and AcEYGEF-amide were chosen with the hope that the E to I
substitution in the Y-1 position might restore optimal activity.
In the case of the 5-mer, this substitution resulted in a 2-fold
increase in k and k
/K
, but in the case of the
7-mer, the same substitution resulted in a 2-fold decrease in k
. In the latter case, k
/K
was only marginally
affected. Comparison of these results and those obtained from the
peptide library work suggests that the specificity for particular
subsites are affected by the presence and/or identity of residues
occupying other subsites N-terminal to the tyrosine. These results
suggest that the equivalent of local minima may be obtained when
determining subsite specificity using peptide libraries. In other
words, the optimal sequence determined may depend upon the context and
sequence length that the peptide library is based upon. Future work
will examine the subsite specificity of srcTK using peptide
libraries based upon Ac-FGEYGEF-amide and AcIYGEF-amide to explore this
possibility.
In conclusion, several short, highly active substrates for srcTK were developed. The observation that only 5-7 residues are required for significant substrate activity suggests that small molecule inhibitors based upon interaction with the phosphoacceptor site may be developed.