Anion sensor-based ratiometric peptide probe for protein kinase activity

Article abstract of DOI:10.1021/ol9006508

A new fluorescent sensor consisting of Cd"-cylcen appended aminocoumarin and a substrate peptide for protein kinase A (PKA) has been designed. Upon phosphorylation by PKA, the metal complex moiety binds to a phosphorylated residue, which In turn displaces

Full text of DOI:10.1021/ol9006508

ORGANIC
LETTERS
2009
Vol. 11, No. 13
2732-2735
Anion Sensor-Based Ratiometric Peptide
Probe for Protein Kinase Activity
Kazuya Kikuchi,†,‡,§ Shigeki Hashimoto,†,⊥ Shin Mizukami,‡,§ and
Tetsuo Nagano*,‡
Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan and PRESTO, JST Corporation,
Kawaguchi, Saitama, Japan
tlong@mol.f.u-tokyo.ac.jp
Received April 7, 2009
ABSTRACT
A new fluorescent sensor consisting of CdII-cylcen appended aminocoumarin and a substrate peptide for protein kinase A (PKA) has been
designed. Upon phosphorylation by PKA, the metal complex moiety binds to a phosphorylated residue, which in turn displaces the coumarin
fluorophore, and this event results in ratiometric change of excitation spectrum in neutral aqueous solution.
Signal transduction pathways provide mechanisms for trans-
ducing external signals to intracellular biological responses.
Protein kinases modulate the activity of their target proteins by
phosphorylating serine, threonine and tyrosine residues within
the intact proteins in these pathways. A great number of kinases
have been discovered, and the characterization of their roles in
complicated signaling pathways is now a very active research
area.1 The development of an analytical tool that can enable
monitoring of the temporal and spatial dynamics of cellular
kinases would therefore contribute substantially to a better
understanding of signal transduction mechanisms.2
Various approaches to monitor the activities of protein
kinases have been made,3,4 one of which is the use of
fluorophore-labeled peptide substrates.5 Traditional peptide
probes contain a polarity-sensitive fluorophore near the site
(3) (a) Nagai, Y.; Miyazaki, M.; Aoki, R.; Zama, T.; Inouye, S.; Hirose,
K.; Iino, M.; Hagiwara, M. Nat. Biotechnol. 2000, 18, 313–316. (b)
Hofmann, R. M.; Cotton, G. J.; Chang, E. J.; Vidal, E.; Veach, D.;
Bornmann, W.; Muir, T. W. Bioorg. Med. Chem. Lett. 2001, 11, 3091–
3094. (c) Kurokawa, K.; Mochizuki, N.; Ohba, Y.; Mizuno, H.; Miyawaki,
A.; Matsuda, M. J. Biol. Chem. 2001, 276, 31305–31310. (d) Ting, A. Y.;
Kain, K. H.; Klemke, R. L.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 15003–15008. (e) Sato, M.; Ozawa, T.; Inukai, K.; Asano, T.;
Umezawa, Y. Nat. Biotechnol. 2002, 20, 287–294
.
(4) (a) Ohuchi, Y.; Katayama, Y.; Maeda, M. Analyst 2000, 125, 1905–
1907. (b) Ojida, A.; Inoue, M.; Mito-oka, Y.; Hamachi, I. J. Am. Chem.
Soc. 2003, 125, 10184–10185. (c) Ojida, A.; Mito-oka, Y.; Sada, K.;
† JST corporation.
‡ The University of Tokyo.
⊥ Present address: Faculty of Industrial Science and Technology, Tokyo
University of Science, Oshamanbe, Hokkaido 049-3514, Japan.
§ Present address: Graduate School of Engineering, Osaka University,
2-1 Yamada-oka, Suita City, Osaka 565-0871, Japan.
(1) Chem. ReV. 2001, 101, issue 8: Protein Phosphorylation and
Signaling.
Hamachi, I. J. Am. Chem. Soc. 2004, 126, 2454–2463.
(5) (a) McIIroy, B. K.; Walters, J. D.; Johnson, J. D. Anal. Biochem.
1991, 195, 148–152. (b) Post, P. L.; Trybus, K. M.; Taylor, D. L. J. Biol.
Chem. 1994, 269, 12880–12887. (c) Higashi, H.; Sato, K.; Omori, A.;
Sekiguchi, M.; Ohtake, A.; Kudo, Y. NeuroReport 1996, 7, 2695–2700.
(d) Higashi, H.; Sato, K.; Ohtake, A.; Omori, A.; Yoshida, S.; Kudo, Y.
FEBS Lett. 1997, 414, 55–60. (e) Yeh, R.-H.; Yan, X.; Cammer, M.;
Bresnick, A. R.; Lawrence, D. S. J. Biol. Chem. 2002, 277, 11527–111532.
(2) (a) Eisele, F.; Owen, D. J.; Waldmann, H. Bioorg. Med. Chem. 1999,
7, 193–224. (b) Lawrence, D. S. Acc. Chem. Res. 2003, 36, 401–409.
10.1021/ol9006508 CCC: $40.75
Published on Web 06/04/2009
 2009 American Chemical Society

of phosphorylation, and this serves to signal the change of
environment upon phosphorylation. The groups of Lawrence
and Imperiali have developed chelator-appended fluorescent
peptides for monitoring kinase activities.6 Upon phospho-
rylation, these peptides show a significant fluorescence
intensity increase owing to the formation of divalent alkaline
earth metal complexes coordinated to the newly generated
phosphate group and the fluorophore.
Fluorescence measurement at a single wavelength without
much shift of either the excitation or emission wavelength
can be influenced by artifacts associated with the microscopic
imaging system. To reduce the influence of such factors,
ratiometric measurement is utilized, namely, simultaneous
recording of the fluorescence intensities at two wavelengths
and calculation of their ratio.7 For this approach, probes that
signal phosphorylation via a shift of either excitation or
emission wavelength are required.
CdII of the cyclen complex is coordinated by the four nitrogen
atoms of cyclen and the aromatic 7-amino group of cou-
marin.9 When a negatively charged phosphate group coor-
dinates to CdII as the fifth ligand, the aromatic 7-amino group
is displaced from the metal. The anion sensor signals this
replacement, because the increase of electron density of the
7-amino group induces a red shift of the excitation spectrum.
We have designed peptide sensor 1 for protein kinase A
(PKA) as shown in Scheme 2. The sequence of the peptide
Scheme 2
.
Sequence of Peptide Sensor and its Phosphorylated
Standard Employed in This Study
We have designed a fluorescent anion sensor, consisting of
7-aminotrifluoromethylcoumarin as a fluorescent reporter and
CdII-cyclen (1,4,7,10-tetraazacyclododecane) as an anion host.8
This sensor molecule can detect phosphate anion species, such
as pyrophosphate, with high sensitivity in aqueous neutral
solution. As an extension of the anion sensor concept, we have
newly designed an anion sensor-appended peptide substrate for
protein kinases. Here we describe the sensing of a kinase-
mediated phosphorylation event by a fluorescent peptide sensor.
This novel class of peptide probe exhibited a shift of excitation
spectrum upon phosphorylation, enabling ratiometric measure-
ment of kinase activity. This technique can provide more precise
data than measurement at a single wavelength, canceling out
the influence of variations in instrument efficiency, content of
effective dye, and so forth.
sensor is known as Kemptide and has been shown to be a
good substrate for the kinase.10 The sensing moiety is
positioned at the N-terminus of the peptide through an alkyl
tether, enabling recognition of a phosphorylated serine
residue. We also designed a phosphorylated sensor 1P to
estimate preliminarily the extent of spectral change upon
phosphorylation.
The operational concept of the peptide sensor is schemati-
cally presented in Scheme 1. This peptide sensor consists of
The cyclen-appended 7-aminocoumarincarboxylic acid
2 was synthesized according to the established procedure.8
The peptide sequence was synthesized using Fmoc solid-
phase chemistry on an automated peptide synthesizer and
the ligand 2 was manually coupled to the amino linker.
The resulting peptide conjugate was metalated with
Cd(ClO4)2 to give the desired peptide sensor 1. Phospho-
rylated peptide sensor 1P was prepared by protein kinase-
mediated phosphorylation of the peptide conjugate fol-
lowed by metalation with CdII. The structures of 1 and
1P were confirmed by MALDI-TOF MS (matrix assisted
laser desorption/ionization-time-of-flight mass spectrom-
etry) and quantitative amino acid analysis.
Scheme 1
.
Schematic Representation of our Peptide Sensor for
Phosphorylation
We tested the sensing ability of peptide sensor 1 by
comparing the excitation spectrum with that of the
phosphorylated product, 1P (see Supporting Information).
Upon phosphorylation, the excitation intensity at 360 nm
decreased, whereas the intensity at 410 nm increased. The
ratio of the excitation intensities (410 nm/360 nm) changed
an anion sensor and a phosphorylation target peptide
sequence. The sensing moiety is positioned near the target
hydroxyl amino acid residue. In neutral aqueous solution,
(6) (a) Chen, C. A.; Yeh, R. H.; Lawrence, D. S. J. Am. Chem. Soc.
2002, 124, 3840–3841. (b) Shults, M. D.; Imperiali, B. J. Am. Chem. Soc.
2003, 125, 14248–14249.
(9) (a) Koike, T.; Watanabe, T.; Aoki, S.; Kimura, E.; Shiro, M. J. Am.
Chem. Soc. 1996, 118, 12696–12703. (b) Aoki, S.; Kaido, S.; Fujioka, H.;
Kimura, E. Inorg. Chem. 2003, 42, 1023–1030.
(7) (a) Tsien, R. Y.; Harootunian, A. T. Cell Calcium 1990, 11, 93. (b)
Kikuchi, K.; Takakusa, H.; Nagano, T. Trends in Anal. Chem. 2004, 23,
407–415.
(10) (a) Kemp, B. E.; Graves, D. J.; Benjamini, E.; Krebs, E. G. J. Biol.
Chem. 1977, 252, 4888–4894. (b) Kemp, B. E. J. Biol. Chem. 1980, 255,
2914–2918.
(8) Mizukami, S.; Nagano, T.; Urano, Y.; Odani, A.; Kikuchi, K. J. Am.
Chem. Soc. 2002, 124, 3920–3925.
Org. Lett., Vol. 11, No. 13, 2009
2733

1.8-fold (from a value of 0.54 to 0.96), demonstrating that
peptide phosphorylation can be detected with an anion
sensor.11
To investigate the utility of the compound as a
fluorescent probe for protein kinases, we measured the
time-dependent change of the excitation spectrum of 1
treated with ATP (adenosine 5′-triphosphate) and PKA
catalytic subunit (Figure 1). The phosphorylation reaction
this organic polyanion had no significant effect on the
excitation spectrum under the conditions employed.12 As
can be seen from Figure 1, the excitation spectra of 1
changed ratiometrically; the intensity at 360 nm decreased
with a concomitant intensity increase at 410 nm. The ratio
of excitation intensity (410 nm/360 nm) increased 1.7 fold
after 30 min reaction time, and this is similar to the value
obtained by comparison of the excitation spectra of
authentic 1 and 1P. The phosphorylation reaction was
accelerated by increasing the quantity of kinase employed
for the reaction (see Supporting Information).
We further carried out an inhibition experiment using the
heat-stable inhibitor protein of PKA (PKI), which acts
competitively with respect to the phosphoryl-accepting
substrate (Figure 2).13 Dose-dependent inhibition of kinase
Figure 2. Titration of PKA-mediated phosphorylation of 1 with
PKI peptide. The phosphorylation reaction was carried out in 50
mM HEPES (pH 7.4), 5 mM Mg(OAc)2 containing 1.0 µM 1, 4.3
nM catalytic subunit, 3.3 µM ATP and various amounts of PKI
peptide at 23 ( 0.1 °C. Extent of phosphorylation (%) was
determined by comparing the intensity ratio increase at the early
phase of the reaction (0-5 min) with that of the control.
activity by PKI peptide was observed for phosphorylation
of 1 with an IC50 (half maximal inhibitory concentration) of
ca. 15 nM under the conditions employed. This result
indicates that the ratiometric spectral change is caused by
PKA-mediated phosphorylation of 1.14
In conclusion, we have developed a new fluorescent probe
for protein kinase based on the anion sensing principle. It
has been demonstrated that this peptide probe can be used
Figure 1. (a) Time course of the excitation spectra of 1 treated
with PKA catalytic subunit. The peptide sensor 1 (1.3 µM) was
incubated in 50 mM HEPES (pH 7.4), 5 mM Mg(OAc)2
containing 4.3 nM catalytic subunit and 3.3 µM ATP at 23 (
0.1 °C. (b) Plot of intensity ratio (410 nm/360 nm) versus
reaction time.
was initiated by the addition of ATP to a mixture of 1
and the catalytic subunit. Though ATP strongly coordi-
nates to the CdII complex of the anion sensor, addition of
(12) The addition of more than three equivalents of ATP to the peptide
sensor 1 induced an excitation change at two wavelengtth (360 and 410
nm), which indicates the coordination of ATP to the metal complex
moiety.
(11) A significant shift of the absorption peak was also observed for
the CdII complex of methylated compound 2 upon addition of pyrophosphate
anion. Titration of the complex with pyrophosphate gave the Kd value of
53 µM.
(13) (a) Cheng, H. C.; Kemp, B. E.; Pearson, R. B.; Smith, A. J.;
Misconi, L.; Van Patten, S. M.; Walsh, D. A. J. Biol. Chem. 1986, 261,
989–992. (b) Glass, D. B.; Cheng, H. C.; Mueller, L. M.; Reed, J.; Walsh,
D. A. J. Biol. Chem. 1989, 264, 8802–8810.
2734
Org. Lett., Vol. 11, No. 13, 2009

to continuously monitor kinase-mediated phosphorylation
through intensity measurements at two wavelengths. This
peptide sensor might serve as the basis for a range of anion
sensor-based phosphorylation probes for many different
protein kinases.
Acknowledgment. We thank Prof. H. Mihara and Dr. T.
Takahashi at the Tokyo Institute of Technology for technical
assistance in peptide synthesis.
Supporting Information Available: Synthesis of anion
sensor and peptide conjugate, fluorescence experiment,
protein kinase assay. This material is available free of charge
via the Internet at http://pubs.acs.org.
(14) Actual phosphorylation of peptide sensor 1 was confirmed by
analyzing the reaction mixture, using C18 reverse-phase HPLC (high-
performance liquid chromatography). Time-dependent production of phos-
phorylated product was observed when the sensor 1 was exposed to PKA
catalytic subunit. The phosphorylated product co-eluted with authentic
standard 1P from the HPLC column.
OL9006508
Org. Lett., Vol. 11, No. 13, 2009
2735