Substrate profiling of protein tyrosine phosphatase PTP1B by screening a combinatorial peptide library

Article abstract of DOI:10.1021/ja071275i

Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. To date, the in vivo substrates and physiological functions of PTPs remain poorly defined. In this work, we have developed a novel combinatorial library method to systematically determine the substrate specificity of PTPs. A one-bead-one-compound peptide library containing five randomized residues, Fmoc-XXXXXpYAA (where X is norleucine or 17 proteinogenic amino acids excluding Tyr, Cys, and Met), was chemically synthesized on 90-μm TentaGel resin by the split-and-pool method. Limited treatment of the library with a PTP removed the phosphoryl group from beads that carry the most preferred substrates. The exposed tyrosine side chain was selectively oxidized into an orthoquinone by the treatment with tyrosinase in the presence of atmospheric oxygen. The orthoquinone was then selectively derivatized with biotin-hydrazide, followed by on-bead colorimetric assay. The positive beads were isolated and individually sequenced by partial Edman degradation/mass spectrometry to give the most preferred substrates of the PTP. Screening of the pY library against PTP1B confirmed the previously reported PTP1B specificity, but also revealed a second class of previously unrecognized peptide substrates. Several selected peptides were individually synthesized and assayed against PTP1B and the kinetic data confirmed the screening results. This method is generally applicable to studying the substrate specificity of other PTPs. Copyright

Full text of DOI:10.1021/ja071275i

Published on Web 04/07/2007
Substrate Profiling of Protein Tyrosine Phosphatase PTP1B by Screening a
Combinatorial Peptide Library
Mathieu Garaud and Dehua Pei*
Department of Chemistry, The Ohio State UniVersity, 100 West 18th AVenue, Columbus, Ohio 43210
Received February 22, 2007; E-mail: pei.3@osu.edu
Reversible phosphorylation of proteins on tyrosyl residues is one
of the key events that mediate the execution and regulation of many
cellular processes. A proper level of phosphorylation is critical for
these processes and is controlled by the opposing actions of protein
tyrosine kinases, which catalyze the transfer of a phosphoryl group
from ATP to the p-hydroxyl of tyrosine, and protein tyrosine
phosphatases (PTPs), which hydrolyze the phosphotyrosine (pY)
back to tyrosine and inorganic phosphate. The human genome
encode ∼100 PTPs, which share a highly conserved ∼250-amino
acid catalytic domain and a common catalytic mechanism.1 In
Figure 1. Reactions involved in pY library screening against PTPs.
contrast to their well-established catalytic mechanism, the physi-
to remove the N-terminal Fmoc group, and individually sequenced
by partial Edman degradation/mass spectrometry (PED/MS).11
To test the effectiveness of tyrosinase-mediated derivatization
reaction, peptide RALYDNVPE was treated with mushroom
tyrosinase and biotin hydrazide and analyzed by HPLC. The
untreated peptide had a retention time of 18.8 min; after treatment,
the peak at 18.8 min disappeared and a new peak appeared at 16.5
min (Figure S1 in Supporting Information). MS analysis of the
reaction product revealed the addition of a single biotin hydrazide
molecule to the peptide (Figure S2 in Supporting Information). In
contrast, a pY peptide was not affected by the treatment. Next, a
pY peptide library TAXXpYXXXLNBBRM (library B, where X
is Nle or any of 18 proteinogenic amino acids except for Met and
Cys) was subjected to tyrosinase treatment and the screening
procedure, resulting in ∼20% colored beads. Twenty colored and
20 colorless beads were randomly selected from the library and
sequenced. While all 20 colored beads contained at least one Tyr
in their sequences, none of the colorless beads contained any Tyr
(Tables S1 and S2 in Supporting Information). Finally, when library
C (YXXXXXYBBRM) was subjected to the screening procedure,
all library beads became colored. These results demonstrate that
the oxidation/derivatization reaction is highly selective for Tyr and
efficient and that pY is completely resistant to modification under
the experimental conditions.
To assess the substrate specificity of PTP1B, a total of 150 mg
of library A (∼429000 beads) was screened against PTP1B at pH
7.4 in three 50-mg batches. The most colored beads, which should
represent the most preferred substrates of PTP1B, were removed
from the library and individually sequenced by PED/MS to give
12, 46, and 25 complete sequences, respectively (Tables S3-S5 in
Supporting Information). The selected sequences clearly fall into
two different classes, with consensus sequences of XXXEWpY
(class I) and [RK][AGST][LIV]XXpY (class II), where X can be
any amino acid (Figure 2). The same types of sequences were
selected from all three experiments, indicating that the library
screening method is highly reproducible. In addition to the intensely
colored beads, PTP1B treatment also produced medium (∼5% of
all beads) and lightly colored beads (∼30% of all beads). Fifty
beads from each color category were randomly selected for
sequencing (Tables S6 and S7 in Supporting Information). These
ological substrates and function of these PTPs remain poorly
defined. PTPs appear to have exquisite substrate specificity in vivo,
which is at least partially determined by the intrinsic sequence
specificity of the catalytic domain.2 A number of approaches have
been employed to define the sequence specificity of PTPs. Initial
studies involved kinetic assay of individually synthesized pY
peptides.3 This approach is inherently limited because PTPs can
interact with 4-5 residues on either side of pY.4 In later studies,
combinatorial peptide libraries were directly screened against PTPs
of interest. However, owing to the lack of stable association between
an active PTP and a pY peptide, library screening was typically
based on binding instead of catalysis, using either a catalytically
impaired PTP mutant or a nonhydrolyzable pY analogue.5,6 As a
result, none of the previous studies have led to comprehensive
specificity data on any PTP. Here we report a novel combinatorial
library method for the rapid determination of the substrate specificity
of PTPs and its application to PTP1B, the prototypical PTP and a
target for antidiabetic drug design.7
The main challenge in screening pY peptide libraries against a
PTP is how to differentiate a small amount of reaction product (i.e.,
Y peptides) from a large excess of unreacted pY peptides. Our
strategy is to selectively oxidize the tyrosine side chain into an
orthoquinone by the enzymatic action of tyrosinase (Figure 1).8
To test this strategy, a pY library containing five random residues,
Fmoc-XXXXXpYAALNBBRM-resin [library A, where X is nor-
leucine (Nle) or any of the 17 proteinogenic amino acids except
for Met, Cys, and Tyr; B ) â-alanine; theoretical diversity ) 1.9
× 106], was synthesized on 90-µm TentaGel resin by the split-
pool method.9 Limited treatment of the library with a small amount
of PTP should dephosphorylate those beads that carry the best
substrates of the PTP. The exposed Tyr side chain is then oxidized
into orthoquinone by treatment with an excess amount of tyrosinase
in the presence of atmospheric O2. The resulting orthoquinone is
selectively derivatized with biotin hydrazide. Subsequently, the
library is incubated with streptavidin-alkaline phosphatase, which
is recruited to any biotinylated beads. Upon the addition of 5-bromo-
4-chloro-3-indolyl phosphate, the dephosphorylated (and thus
biotinylated) beads should become turquoise colored.10 The positive
beads are manually removed from the library, treated with piperidine
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10.1021/ja071275i CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
two distinct classes of substrates; the class I substrates have been
reported in the literature,3,5,6 but the class II peptides have not
previously been recognized.
A dozen or so proteins have been reported as PTP1B substrates
in vivo.12 The pY sites recognized by PTP1B have been identified
in a few of these proteins. The majority of the pY sites including
those in insulin receptor,13 JAK2,14 and Tyk2 kinases14 contain (E/
D)(Y/pY)pY motifs and belong to class I substrates. STAT5a, a
transcription factor involved in cytokine receptor signaling, contains
a class II motif, KAVDGpY694VKP.15 These data suggest that both
classes of substrates selected from the peptide library are physi-
ologically relevant.
In summary, a novel combinatorial library method has been
developed to systematically assess the substrate specificity of PTPs.
Application of the method to PTP1B has revealed that its active
site can recognize at least two distinct classes of substrates, one of
which was previously unknown. Sequence specificity data of this
type should be useful for predicting the protein substrates of PTPs
and guiding the design of specific PTP inhibitors.
Acknowledgment. This work was supported by the National
Institutes of Health (Grant GM062820).
Supporting Information Available: Experimental details, peptide
sequences, HPLC and MS analysis results. This material is available
free of charge via the Internet at http://pubs.acs.org.
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Figure 2. Sequence specificity of PTP1B. Displayed are the amino acids
identified at each position from -5 to -1 relative to pY (position 0). Number
of occurrences on the y-axis represents the number of selected sequences
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Table 1. Catalytic Constants of PTP1B against Selected Peptides
1
1
peptide
bead color
k
cat (s-
)
KM
(µ
M)
k
cat/KM (M-1 s-
)
KAVFIpYAA
RTINEpYAA
RTIEWpYAA
QEDEPpYAA
EHTGHpYAA
DHVTQpYAA
RLKQQpYAA
intense
intense
intense
intense
medium
light
16 ( 2
13 ( 1
7.8 ( 0.1
8.3 ( 0.6
14 ( 2
23 ( 6
17 ( 2
26 ( 1
20 ( 5
290 ( 70
>200
700000
765000
300000
420000
47000
550
colorless
>200
138
sequences reveal that PTP1B accepts a wide variety of peptides as
substrates (albeit less efficiently), but has a general preference for
acidic residues N-terminal to pY (Figure S4 in Supporting Informa-
tion). To identify peptides that are resistant to PTP1B action, library
A (10 mg) was treated exhaustively with PTP1B, and 50 of the
colorless beads (∼40% of all beads) were randomly selected and
sequenced (Table S8 in Supporting Information). None of the
resistant peptides contained Trp at pY-1 position, and only five
peptides had a single acidic residue.
Seven representative peptides from different color categories were
individually synthesized, purified, and assayed against PTP1B in
solution (pH 7.4). All four peptides derived from intensely colored
beads are excellent substrates of PTP1B, with kcat/KM values in the
range of 3-8 × 105 M-1 s-1 (Table 1). There is a general
correlation between the color intensity of a bead and the kinetic
constants (kcat/KM) of the peptide it carries. Thus, PTP1B recognizes
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