Angewandte
Chemie
distal to the phosphorylation site.[14] For example, ERK1 and
2 phosphorylate the transcription factor Ets-1 at Thr38 within
a short ThrPro (TP) consensus motif.[15] Since this short
sequence would be the target of multiple kinases, ERK
recognition of Ets-1 depends an adjoining N-terminal pointed
(PNT) domain[16] to dock the substrate specifically to ERK1/
2, which engages the phosphorylation machinery.[17] With this
docking-domain strategy, PNT-based substrates demonstrate
good affinity for ERK1/2 (KM ꢀ 6–9 mm),[16] in stark contrast
to short peptide substrates derived only from the TP sequence
(KM > 200 mm).[16,18] Owing to the size of the PNT domain
(11 kDa), these docking-domain interactions cannot be
exploited with the types of synthetic peptide-based sensors
that have been reported. However, in light of its importance
in cellular homeostasis and the prominence of ERK disregu-
lation in cancer, we set out to construct a chimeric sensor for
ERK1/2 that combines the advantageous reporting properties
of the Sox fluorophore with the outstanding specificity
provided by the native protein domain-based recognition
(Figure 1a). Most importantly, to ensure facile and high-
throughput analysis of ERK1/2 activity, our ultimate require-
ment is that the probe be highly selective in cell lysates where
it would be exposed to hundreds of other active kinases.
Herein we describe the semisynthesis of a chimeric Sox-
based ERK1/2 sensor through a key native chemical ligation
(NCL) reaction that efficiently conjugates the recombinat
PNT domain of Ets-1 to a synthetic ERK1/2 consensus
sequence including the Sox sensing module. The extended
PNT recognition element confers the ERK1/2 sensor with
excellent selectivity, as demonstrated by comparative quanti-
tative analyses with a panel of related recombinant enzymes
and in unfractionated lysates from four different cell lines.
Most importantly, the docking-domain-based sensor design
should be generally applicable to the development of
selective sensors for other medically important kinases.
The new sensor was assembled as illustrated in Figure 1,
using NCL[19] to ligate the synthetic Sox-containing peptide
thioester with the expressed PNT domain, comprising Ets-1
residues 46–138 (Figure 1b). The peptide thioester was
synthesized using Fmoc-based solid-phase peptide synthesis
(SPPS; Fmoc = fluorenylmethyloxycarbonyl) on highly acid-
labile TGT resin, with subsequent off-bead thioesterification
of the protected peptide.[20] The Sox chromophore was
introduced as the amino acid C-Sox.[13] An optimized
phosphorylation motif based on the ERK2 phosphorylation
sequence within the myelin basic protein (MBP;[18]
TPGGRR) was used in place of the phosphorylated region
of Ets-1 (TPSSKE). When the Sox chromophore was
incorporated into these short peptides, preliminary studies
indicated that the MBP-based sequence had better fluores-
cent properties (Table S1 in the Supporting Information). The
distance between the TP recognition sequence and the PNT
domain in the wild-type protein was preserved in the sensor
(Table S2 in the Supporting Information). This design intro-
duced residue replacements in the unstructured N-terminal
region of Ets-1, thereby minimizing perturbations to the
overall secondary structure. Additionally, the C-terminal
residue, Met44, was changed to Gly to eliminate the
possibility of epimerization during thioesterification and to
increase ligation efficiency.[21] The expressed C-terminal
fragment of the sensor, GST-PNT, was proteolyzed to reveal
Cys-PNT. After ligation of Cys-PNT to the peptide thioester
in nondenaturing conditions, the ERK1/2 probe Sox-PNTwas
isolated in good yield (24%; the accurate mass of the isolated
material was based on the molar absorptivity emax of the Sox
chromophore; see the Supporting Information). The corre-
sponding phosphoprotein (pThr38) pSox-PNT was con-
structed using analogous methods.
Initial spectroscopic studies with Sox-PNT and pSox-PNT
revealed a robust threefold enhancement in fluorescence
upon phosphorylation (Figures S2 and S3 in the Supporting
Information). Moreover, similar to Sox-based peptide sen-
sors,[13] Sox-PNT was found to have an excellent Z’ factor
value (0.81), which is a statistical quality parameter used to
evaluate and validate performance of assays, with useful
ranges being 0.5–1.[22] Subsequent in vitro assays determined
Sox-PNT to be an efficient substrate for ERK2 when
compared with the corresponding Sox peptide (Ac-VP-
CSox-LTPGGRRG-OH; Figure 2a and Figure S3 in the
Figure 2. In vitro characterization of Sox-PNT. a) The efficiency of
phosphorylation by recombinant ERK2 (11 ng) of the Sox-PNT probe
was compared to that of the Sox peptide under identical conditions.
b) The kinetic parameters for Sox-PNT were obtained with ERK2
(10 ng) from a direct fit of n vs. [S] plots using the Briggs–Haldane
equation. Plotted values indicate the mean standard error of measure-
ment (Æs.e.m.) for triplicate measurements. c) Promiscuity of Sox-
PNT (5 mm) was tested with a panel of related kinases at 15 nm (black
bars) and 150 nm (white bars) of each enzyme. Inset: a representative
plot of the change in the fluorescence signal over time obtained with
15 nm enzyme in the fluorescence plate reader. Plotted values for
15 nm enzyme indicate the mean Æs.e.m. for triplicate measure-
ments.
Angew. Chem. Int. Ed. 2009, 48, 6828 –6831
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim