A luminescent sensor for tyrosine phosphorylation

Article abstract of DOI:10.1021/ol701920x

We have developed a luminogenic probe for tyrosine phosphorylation based on a short peptide sequence containing an iminodiacetate moiety near the site of phosphorylation. In response to kinase activity, the probe provides a strong luminescence enhancement, resulting from the increased ability of the probe to bind and sensitize Tb³⁺ and Eu³⁺ ions upon phosphorylation.

Full text of DOI:10.1021/ol701920x

ORGANIC
LETTERS
2008
Vol. 10, No. 1
5-8
A Luminescent Sensor for Tyrosine
Phosphorylation
Matthew S. Tremblay, Minhee Lee, and Dalibor Sames*
Department of Chemistry, Columbia UniVersity, New York, New York 10027
sames@chem.columbia.edu
Received August 7, 2007
ABSTRACT
We have developed a luminogenic probe for tyrosine phosphorylation based on a short peptide sequence containing an iminodiacetate moiety
near the site of phosphorylation. In response to kinase activity, the probe provides a strong luminescence enhancement, resulting from the
+
+
increased ability of the probe to bind and sensitize Tb3 and Eu3 ions upon phosphorylation.
Phosphorylation and dephosphorylation of proteins and
peptides are the principal chemical mechanisms of intra-
cellular communication, and the protein kinases and phos-
phatases that control this chemistry have been implicated as
key players in a number of important signal transduction
pathways.1 Not surprisingly, these enzymes have been targets
of intense study, both for their therapeutic potential and their
role in fundamental biochemical processes. Although a
number of successful strategies for inhibition of both the
substrate2 and cofactor3 binding sites of protein kinases have
been developed, the design of optical reporters for measuring
and imaging kinase activity has generally presented a more
formidable challenge. On a supramolecular scale, protein
phosphorylation has profound consequences for protein
conformation and protein-protein interactions, from which
it derives its powerful signaling ability. On a local chemical
level, however, the event is relatively subtle with respect to
the π-electronic changes typically exploited in standard
approaches to optical switch development,4 making it a
relatively evasive property with regard to sensor design and
requiring more creative reporting mechanisms to be in-
vented.5,6
(4) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A.
J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997, 97,
1515-1566.
(5) For small molecule/short peptide conjugates, see: (a) Bowman, B.
F.; Peterson, J. A.; Stull, J. T. J. Biol. Chem. 1992, 267, 5346-5354. (b)
Higashi, H.; Sato, K.; Ohtake, A.; Omori, A.; Yoshida, S.; Kudo, Y. FEBS
Lett. 1997, 414, 55-60. (c) Chen, C.-A.; Yeh, R.-H.; Lawrence, D. S. J.
Am. Chem. Soc. 2002, 124, 3840-3841. (d) Shults, M. D. and Imperiali,
B. J. Am. Chem. Soc. 2003, 125, 14248-14249. (e) Chen, C.-A.; Yeh,
R.-H.; Yan, X.; Lawrence, D. S. Biochim. Biophys. Acta 2004, 1697,
39-51. (f) Rothman, D. M.; Shults, M. D.; Imperiali, B. TRENDS Cell
Biol. 2005, 15, 502-509. (g) Wang, Q.; Cahill, S. M.; Blumenstein, M.;
Lawrence, D. S. J. Am. Chem. Soc. 2006, 128, 1808-1809. (h) Balakrishnan,
S.; Zondlo, N. J. J. Am. Chem. Soc. 2006, 128, 5590-5591. (i) Wang, Q.;
Dai, Z.; Cahill, S. M.; Blumenstein, M.; Lawrence, D. S. J. Am. Chem.
Soc. 2006, 128, 14016-14017. (j) Tremblay, M. S.; Zhu, Q.; Mart´ı, A. A.;
Dyer, J.; Halim, M.; Jockusch, S.; Turro, N. J.; Sames, D. Org. Lett. 2006,
8, 2723-2726. (k) Choi, Y.; Ho, N.-H.; Tung, C.-H. Angew. Chem., Int.
Ed. 2007, 46, 707-709. (l) Shults, M. D.; Janes, K. A.; Lauffenburger, D.
A.; Imperiali, B. Nature Methods 2005, 2, 277-284.
(1) A comprehensive issue on the role of protein phosphorylation in
signaling: (a) Chem. ReV. 2001, 101(8). The chemical biology of signal
transduction: (b) Acc. Chem. Res. 2003, 36 (6).
(2) (a) Shen, K.; Keng, Y.-F.; Wu, L.; Guo, X.-L.; Lawrence, D. S.;
Zhang, Z.-Y. J. Biol. Chem. 2001, 276, 47311-47319. (b) Yeh, R.-H.; Lee,
T. R.; Lawrence, D. S. J. Biol. Chem. 2001, 276, 12235-12240. (c) Lee,
J. H.; Nandy, S. K.; Lawrence, D. S. J. Am. Chem. Soc. 2004, 126, 3394-
3395. (d) Hah, J.-M.; Sharma, V.; Li, H.; Lawrence, D. S. J. Am. Chem.
Soc. 2006, 128, 5996-5997.
(6) For protein-derived reporters, see: (a) Post, P. L.; Trybus, K. M.;
Taylor, D. L. J. Biol. Chem. 1994, 269, 12880-12887. (b) Zhang, J.; Ma,
Y.; Taylor, S. S.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
14997-15002. (c) Ting, A. Y.; Kain, K. H.; Klemke, R. L.; Tsien, R. Y.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 15003-15008. (d) Wang, Q.;
Lawrence, D. S. J. Am. Chem. Soc. 2005, 127, 7684-7685.
(3) Bridges, A. J. Chem. ReV. 2001, 101, 2541-2572.
10.1021/ol701920x CCC: $40.75
© 2008 American Chemical Society
Published on Web 12/11/2007

Herein, we report the development of a tyrosine kinase
reporter substrate that provides a significant increase in
luminescence upon tyrosine phosphorylation (up to 10-fold).
This probe enables a straightforward and practical assay for
tyrosine phosphorylation where the optical readout can be
programmed to appear at a variety of wavelengths ranging
from ∼490 to 700 nm depending on the choice of lanthanide
metal.
As part of a program aimed at developing optical sensors
for metabolic and signaling enzymes,5j,7 we became interested
in designing a reporting system for protein phosphorylation
based on the luminescent lanthanide ions, whose sharp
emission lines and long radiative lifetimes facilitate their
spectral and temporal resolution from background fluores-
cence.8 Since the luminescent lanthanides absorb light poorly
(ꢀ ∼ 1 M-1 cm-1), sensitization of the lanthanide excited-
state via an organic photon antenna is required to achieve a
useful luminescence intensity. We sought to exploit this fact,
and the known ability of phosphates and phosphotyrosine9
to bind trivalent lanthanides, to design a small peptide-based
reporting domain whose ability to bind and sensitize a
trivalent lanthanide ion would be dependent on the phos-
phorylation state of a proximal tyrosine residue. The presence
of an appropriate sensitizing chromophore in the reporting
domain would effectively gate the luminescence intensity
as a function of peptide phosphorylation, allowing kinase
and phosphatase activity to be measured.
Figure 1. Design of a tyrosine kinase/phosphatase sensor that binds
to Ln3+ (Ln ) Tb or Eu) and sensitizes its luminescence as a
function of phosphorylation state.
Ser/pSer; the resulting domain binds and sensitizes Tb3+ as
a function of the Ser phosphorylation state, since only pSer
is an adequate steric and electronic Glu mimic.
Our general design was inspired by work done in the
Lawrence group,5c which showed that a phosphorylated
amino acid could serve as an iminodiacetate surrogate in
ethylene glycol tetraacetate (EGTA)-type cooperative che-
lation of a divalent cation. Since the tricationic, oxophilic
lanthanides can also be expected to bind with high affinity
to such a motif,8a we envisioned a peptide containing an
iminodiacetate moiety that would bind tightly to Tb3+ or Eu3+
and sensitize its luminescence as a function of nearby
tyrosine phosphorylation (Figure 1). The intervening peptide
sequence should contain an appropriate organic photon
antenna, and the domain surrounding the key tyrosine residue
should contain kinase recognition elements. Balakrishnan and
Zondlo5h recently reported a strategy for monitoring serine
phosphorylation that involves replacing a key glutamate
residue in a Ca2+ (or Tb3+) binding EF-hand domain with
With these design features in mind, a pair of Tyr/pTyr-
containing peptides were prepared by standard Fmoc-based
solid-phase peptide synthesis (1 and p1, Scheme 1). Custom
Scheme 1
(7) (a) Chen, G.; Yee, D. J.; Gubernator, N. G.; Sames, D. J. Am. Chem.
Soc. 2005, 127, 4544-4545. (b) Froemming, M. K.; Sames, D. Angew.
Chem., Int. Ed. 2006, 45, 637-642. (c) Yee, D. J.; Balsanek, V.; Bauman,
D. R.; Penning, T. M.; Sames, D. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
13304-13309. (d) Tremblay, M. S.; Halim, M.; Sames, D. J. Am. Chem.
Soc. 2007, 129, 7570-7577. (e) Halim, M.; Tremblay, M. S.; Jockusch,
S.; Turro, N. J.; Sames, D. J. Am. Chem. Soc. 2007, 129, 7704-7705.
(8) For a general review, see: (a) Parker, D.; Dickins, R. S; Puschmann,
H.; Crossland, C.; Howard, J. A. K. Chem. ReV. 2002, 102, 1977-2010.
(b) For a review of lanthanide-based switches, see: Gunnlaugsson, T.;
Leonard, J. P. Chem. Commun. 2005, 3114-3131. For recent examples of
biologically relevant switches, see: (c) Lee, K.; Dzubeck, V.; Latshaw, L.;
Schneider, J. P. J. Am. Chem. Soc. 2004, 126, 13616-13617. (d) Terai, T.;
Kikuchi, K.; Iwasawa, S.-y.; Kawabe, T.; Hirata, Y.; Urano, Y.; Nagano,
T. J. Am. Chem. Soc. 2006, 128, 6938-6946.
(9) (a) dos Santos, C. M. G.; Ferna´ndez, P. B.; Plush, S. E.; Leonard, J.
P.; Gunnlaugsson, T. Chem. Commun. 2007, 3389-3391. (b) Atkinson,
P.; Bretonniere, Y.; Parker, D.; Muller, G. HelV. Chim. Acta 2005, 88, 391-
405. (c) Ringer, D. P.; Etheredge, J. L.; Dalrymple, B. L.; Niedbalski, J. S.
Biochem. Biophys. Res. Commun. 1990, 168, 267-273. (d) Galvan, B.;
Christopoulos, T. K. Clin. Biochem. 1996, 29, 125-131.
building blocks (t-Bu)2NTA-OH and Fmoc-Csa-OH (NTA
) nitrilotriacetic acid; Csa ) carbostyril aspartamide) were
readily synthesized from commercially available amino acid
derivatives and facilitated the introduction of the desired
iminodiacetate and organic photon antenna components,
6
Org. Lett., Vol. 10, No. 1, 2008

respectively (Scheme 1). The carbostyril 124 chromophore
in Csa provides highly efficient sensitization of Tb3+
luminescence and also sensitizes Eu3+, allowing the readout
of this system to be extended to ∼700 nm.10 The sequence
C-terminal to the Tyr residue contains recognition elements
common to both the src and abl protein tyrosine kinases
(PTKs).
In the presence of Tb3+ or Eu3+, peptide 1 exhibits
negligible emission, while its phosphorylated counterpart p1
sensitizes both Tb3+ and Eu3+ luminescence (Figure 2a,b),
with src PTK and ATP for 20 h, a ∼10-fold increase in Tb3+
luminescence was observed that was dependent on the
presence of src PTK (Figure 3a). Detailed analysis of the
Figure 3. Phosphorylation of 1 catalyzed by Src PTK (a) and
dephosphorylation of p1 catalyzed by PTP1B (b). Conditions: (a)
250 µM peptide, 1.8 µg/mL src PTK, 1 mM ATP, 50 mM Tris‚
HCl, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 0.01% Brij 35,
pH 7.5, 30 °C, 20 h; then diluted 10× with 10 mM HEPES (pH
7.4) containing TbCl3 (750 µM); (b) 250 µM peptide, 1.0 µg/mL
PTP1B, 5 mM HEPES, 100 mM NaCl, 5 mM DTT, pH 7.4, 37
°C, 1.5 h; then diluted with TbCl3 as described for (a). Excitation
at 340 nm; luminescence is the integrated emission intensity from
530-560 nm region. Error bars are standard deviations from three
runs.
phosphorylation rate afforded the kinetic parameters for probe
1 (Km ) 56 ( 13 µM; Vmax ) 0.69 ( 0.09 µmol min-1
mg-1), which are comparable to those obtained with the
optimized peptide (Km ) 33 µM; Vmax ) 0.8 µmol min-1
mg-1).12 This data shows that the nonphysiological amino
acid derivatives present in peptide 1 are well tolerated by
the Src PTK. Hydrolysis of p1 by protein tyrosine phos-
phatase 1B (PTP1B) also proceeded smoothly, giving a
9-fold decrease in luminescence intensity in the presence of
the enzyme after 1.5 h (Figure 3b).13
Figure 2. Emission spectra of 1 and p1 in the presence of Tb3+
(a, gray and green lines, respectively) and Eu3+ (b, gray and red
lines, respectively). Titration of 1/p1 with Tb3+ (gray and green
diamonds, respectively) and Eu3+ (white and red circles, respec-
tively) (c) Conditions: 50 µM peptide, 1500 µM LnCl3 [Ln ) Tb
(a) or Eu (b)] or 0-1500 µM LnCl3 (c), 10 mM HEPES, pH 7.4;
excitation, 340 nm; emission, 530-560 nm (Tb3+) or 580-625
nm (Eu3+). Error bars are derived from standard deviations between
four independent trials.
Since the src and abl PTK families phosphorylate similar
consensus peptide sequences, we also examined the reaction
of 1 with abl PTK. Indeed, a time-course assay revealed
an increase in luminescence that corresponded to phospho-
rylation of 1 as confirmed by HPLC and MALDI-MS
analysis (Figure 4a, gray diamonds), and the kinetic param-
eters were obtained (Km ) 26 ( 6 µM; Vmax ) 0.8 ( 1
pmol min-1 unit-1). Although the Km value for probe 1 is
several fold higher than the published value for an optimized
peptide (Km ) 4 µM, EAIYAAPFAKKK),12 the catalytic
activity is similar as judged by comparison to the number
provided for the optimized peptide by the commercial
constituting an excellent phosphorylation-responsive lumi-
nescent switch. The peptides were titrated with Tb3+ and
Eu3+, and as expected, the phosphorylated peptide p1
exhibited a dramatic increase in Tb3+ and Eu3+ luminescence,
which could be correlated to an apparent dissociation constant
(Kd) of 160 µM (Figure 2c).11 The nonphosphorylated peptide
1 showed no significant luminescence even after addition
of 30 equiv of Tb3+. While p1[Tb3+] is more than an order
of magnitude brighter than p1[Eu3+] (compare parts a and b
of Figure 2), the latter is still desirable for its red emission
band centered at 696 nm.
(12) Songyang, Z.; Carraway, K. L.; Eck, M. J.; Harrison, S. C.; Feldman,
R. A.; Mohammadi, M.; Schlessinger, J.; Hubbard, S. R.; Smith, D. P.;
Eng, C.; Lorenzo, M. J.; Ponder, B. A. J.; Mayer, B. J.; Cantley, L. C.
Nature 1995, 373, 536-539.
(13) Other NTA-Csa-peptide conjugates were investigated and also
exhibited a marked increase in luminescence upon phosphorylation;
however, the shape of the titration curve differed and the amount of excess
metal added needs to be optimized for each sequence.
We next turned our attention to monitoring the enzyme-
catalyzed interconversion of 1 and p1. After incubation of 1
(10) Li, M.; Selvin, P. R. J. Am. Chem. Soc. 1995, 117, 8132-8138.
(11) A nonlinear least squares regression analysis was performed using
Microscoft Excel. See: Long, J. R.; Drago, R. S. J. Chem. Educ. 1982, 59,
1037-1039.
Org. Lett., Vol. 10, No. 1, 2008
7

Kinase inhibition can be readily evaluated using the
reporter system. This was demonstrated by addition of
imatinib mesylate (Gleevec), a known inhibitor of the abl
kinase family and a successful drug for the treatment of
chronic myelogenous leukemia,15 which completely inhibited
the phosphorylation reaction (no luminescence enhancement,
Figure 4b).
In summary, we have developed a luminescent sensor for
tyrosine kinase and phosphatase activity based on a peptide
whose ability to bind and sensitize Tb3+ and Eu3+ is enhanced
upon phosphorylation. Detection of src and abl kinase
activity, as well as PTP1B-catalyzed phosphotyrosine hy-
drolysis, is facilitated by a dramatic 8-10-fold change in
luminescence intensity that accompanies the interconversion
of 1 and p1. Additionally, the luminescent output can be
programmed to appear at a variety of wavelengths depending
on the choice of lanthanide ion. The Eu3+ complex of p1
has the longest wavelength emission of any phosphorylation
sensor yet reported. The kinetic parameters show that probe
1 is comparable to the optimized peptide substrates or peptide
reporters developed by others, and thus, the assay format
described herein is suitable for in vitro evaluation of
inhibitors and competitive substrates. The long radiative
lifetimes of Tb3+ and Eu3+ will enable highly sensitive,
background-free detection of kinase activity using time-
resolved emission measurement.8d
Figure 4. Time-course assay of the phosphorylation of 1 by abl
kinase, followed by in situ dephosphorylation of p1 by PTP1B (gray
diamonds): 1 without abl (black squares) (a) Inhibition of phos-
phorylation of 1 by abl kinase with Gleevec (b). Conditions: 250
µM peptide, 350 units/mL abl PTK, 1 mM ATP, 50 mM Tris‚
HCl, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 0.01% Brij 35,
pH 7.5, 30 °C, 0-24 h; at 24 h, 2.5 µg PTP1B added; aliquots
removed and diluted 10× with 10 mM HEPES (pH 7.4) containing
TbCl3 (1.25 mM). Excitation: 340 nm, emission: 530-560 nm
(luminescence was measured as the integrated emission intensity
over this region). Error bars are derived from standard deviations
between three independent trials.
vendor.14 When the reaction was judged complete by
comparison to a positive control, PTP1B was added to the
assay mixture and a sharp decrease in luminescence intensity
was observed that corresponded to complete hydrolysis of
p1 as judged by comparison to the negative control (Figure
4a, black squares). This experiment demonstrates that revers-
ible protein phosphorylation can be probed in situ by the
luminescent switching pair 1/p1 in an operationally straight-
forward procedure that involves simply diluting an aliquot
of the enzyme assay in buffer containing Tb3+ or Eu3+.
Acknowledgment. We gratefully acknowledge financial
support from the G. Harold & Leila Y. Mathers Charitable
Foundation.
Supporting Information Available: Detailed synthetic
procedures, spectral and photophysical characterization, and
assay protocol. This material is available free of charge via
the Internet at http://pubs.acs.org.
OL701920X
(15) Wolf, D.; Tilg, H.; Rumpold, H.; Gastl, G.; Wolf, A. M. Curr.
Cancer Drug Targets 2007, 7, 251-258.
(14) Src PTK was obtained from New England Biolabs.
8
Org. Lett., Vol. 10, No. 1, 2008