Long-wavelength fluorescent reporters for monitoring protein kinase activity

Article abstract of DOI:10.1002/anie.201309691

In vivo optical imaging must contend with the limitations imposed by the optical window of tissue (600-1000 nm). Although a wide array of fluorophores are available that are visualized in the red and near-IR region of the spectrum, with the exception of proteases, there are few long wavelength probes for enzymes. This situation poses a particular challenge for studying the intracellular biochemistry of erythrocytes, the high hemoglobin content of which optically obscures subcellular monitoring at wavelengths less than 600 nm. To address this, tunable fluorescent reporters for protein kinase activity were developed. The probing wavelength is preprogrammed by using readily available fluorophores, thereby enabling detection within the optical window of tissue, specifically in the far-red and near-IR region. These agents were used to monitor endogenous cAMP-dependent protein kinase activity in erythrocyte lysates and in intact erythrocytes when using a light-activatable reporter. Searching far and near: Fluorescent reporters for protein kinase activity were developed. The probing wavelength is preprogrammed by using readily available fluorophores, thereby enabling detection within the optical window of tissue, specifically in the far-red and near-IR region. These agents were used to monitor endogenous cAMP-dependent protein kinase activity 1) in erythrocyte lysates and 2) in intact erythrocytes when using a light-activatable reporter.

Full text of DOI:10.1002/anie.201309691

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DOI: 10.1002/anie.201309691
Fluorescent Probes
Long-Wavelength Fluorescent Reporters for Monitoring Protein
Kinase Activity**
Nathan P. Oien, Luong T. Nguyen, Finith E. Jernigan, Melanie A. Priestman, and
David S. Lawrence*
Abstract: In vivo optical imaging must contend with the
limitations imposed by the optical window of tissue (600–
1000 nm). Although a wide array of fluorophores are available
that are visualized in the red and near-IR region of the
spectrum, with the exception of proteases, there are few long
wavelength probes for enzymes. This situation poses a partic-
ular challenge for studying the intracellular biochemistry of
erythrocytes, the high hemoglobin content of which optically
obscures subcellular monitoring at wavelengths less than
600 nm. To address this, tunable fluorescent reporters for
protein kinase activity were developed. The probing wave-
length is preprogrammed by using readily available fluoro-
phores, thereby enabling detection within the optical window of
tissue, specifically in the far-red and near-IR region. These
agents were used to monitor endogenous cAMP-dependent
protein kinase activity in erythrocyte lysates and in intact
erythrocytes when using a light-activatable reporter.
Protein kinases catalyze phosphoryl transfer from ATP to
hydroxy residues in proteins and peptides. Although a variety
of fluorescent sensors for protein kinases have been de-
scribed,[7,8] strategies have not yet been developed that can
tune sensors to specific wavelengths within the optical
window of tissue. One appealing approach is to take
advantage of the commercial availability of far-red and
near-IR fluorophores. Enzyme-catalyzed unquenching of
fluorescence through the separation of a fluorophore from
a nearby fluorescent quencher has been successfully applied
to proteases.[5] Although we previously used such a strategy
for protein kinases, the “unquenching” required the presence
of a third party, namely stoichiometric amounts of a protein
that sequesters the phosphorylated product.[9] We herein
describe a much simpler and more robust alternative in which
the newly introduced phosphate serves as a molecular trigger
that drives the release of the fluorescent quencher. This
provides access into the biologically useful far-red/near-IR
wavelength realm as exemplified by visualization of kinase
activity in the optically challenging intracellular domain of
erythrocytes.
I
n vivo optical imaging must contend with the limitations and
opportunities imposed by the optical window of tissue (600–
1000 nm).[1] Tissue transparency at wavelengths less than
600 nm is limited by the presence of hemoglobin and melanin
whereas water is the chief culprit above 1000 nm. Fortunately,
a wide array of fluorophores are available that are visualized
in the red and near-IR[2] region of the spectrum and several of
these have been covalently attached to antibodies for
diagnostic purposes.[3,4] However, with the prominent excep-
tion of proteases,[5] there are few long wavelength probes for
enzymes, a situation that is especially problematic with
respect to intracellular erythrocyte biochemistry.[6] Unlike
other mammalian cells, the high hemoglobin content of
erythrocytes optically obscures subcellular monitoring at
wavelengths less than 600 nm. To address the need for
observing biochemical pathways in these cells, and with an
eye on potential applications for tissue-based studies, red and
far-red probes of protein kinase activity were designed.
This strategy is outlined in Scheme 1. Our initial efforts
focused on the cAMP-dependent protein kinase (PKA)
because of its central role in erythrocyte behavior and the
life cycle of the malarial parasite Plasmodium Falciparum.[10]
PKA efficiently phosphorylates a diverse array of serine-
containing positively charged sequences, and we employed
two of these sequences in this study: Aoc-GRTGRRFSY-
amide[11] and KRRRLASLAA-amide.[12] Fluorophores were
[*] N. P. Oien, L. T. Nguyen, Dr. F. E. Jernigan, Prof. M. A. Priestman,
Prof. D. S. Lawrence
Department of Chemistry, Division of Chemical Biology and
Medicinal Chemistry, and the Department of Pharmacology
University of North Carolina, Chapel Hill, NC 27599 (USA)
E-mail: lawrencd@email.unc.edu
Scheme 1. General strategy for the protein kinase catalyzed unquench-
ing of fluorescent kinase substrates. A positively charged fluorescent
kinase substrate is quenched upon exposure to a negatively charged
quencher dye (Q). Kinase-catalyzed phosphorylation releases Q as
a result of favorable intramolecular electrostatic interactions between
the newly introduced phosphate and positively charged substrate
residues. Note: the nonfluorescent Q/kinase substrate complex in this
study does not exist in a 1:1 stoichiometry.
[**] We thank the National Institutes of Health for financial support
(RO1 CA79954), Prof. Christian Doerig (Monash University) for
bringing to our attention the role of PKA in erythrocyte function, and
Prof. Karl Drexhage (ATTO-TEC GmbH; Universitꢀt Siegen).
Supporting information for this article (including experimental
details) is available on the WWW under http://dx.doi.org/10.1002/
anie.201309691.
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Table 1: The PKA-catalyzed fluorescence increase (Fl-fold) of the fluo-
rophore-substituted peptides (2.5 mm) in in buffer [Tris-HCl pH 7.5
(25 mm), MgCl2 (1 mm), where [1] is variable, see Table S6] and in 10%
erythrocyte lysates [PBS buffer pH 7.4, MgCl2 (5 mm), Halt protease and
phosphatase inhibitor cocktail (1X), leupeptin (1 mm), 1 (150 mm)] with
ATP (1 mm) and PKA (10 nm). Structures of the fluorophores are
provided in Table S1.
appended to the N termini of both peptides. The amino
octanoic acid (Aoc) moiety was used as a spacer in one of
these to reduce any potential unfavorable steric clashes
between the large fluorophores and the kinase active site. As
noted below, this proved to be an unnecessary precaution
because all of the peptides in this study serve as PKA
substrates. A total of 14 fluorophores were examined that
encompass a nearly 250 nm wavelength range throughout the
red/far-red region (lex = 494–727 nm, lem = 530–752 nm). For
comparative purposes, the absorbances of five fluorophore-
Aoc-GRTGRRFSY-amide peptides are shown relative to
that of hemoglobin (Figure 1). We assessed the ability of
Fluorophore–peptide
lem, lex
Cond.
Fl-fold›
[nm]
5Fam-Aoc-GRTGRRFSY
TAMRA-Aoc-GRTGRRFSY
Atto620-Aoc-GRTGRRFSY
Atto633-Aoc-GRTGRRFSY
Red681-Aoc-GRTGRRFSY
492, 518
550, 580
619, 643
635, 655
670, 706
Buffer
Lysate
Buffer
Lysate
Buffer
Lysate
Buffer
Lysate
Buffer
Lysate
(2.5Æ0.1)
(1.3Æ0.1)
(104Æ24)
(14.6Æ0.6)
(16.6Æ0.8)
(12.8Æ0.8)
(30.2Æ9.1)
(3.9Æ0.2)
(11.2Æ0.6)
(3.4Æ0.1)
Figure 1. Relative wavelength-dependent absorbances of erythrocyte
lysate (red) and fluorophore-Aoc-GRTGRRFSY-amide peptides where
the fluorophore is 5Fam (green), TAMRA (violet), Atto620 (cyan),
Atto633 (blue), or Red681 (black).
Scheme 2. Structures of quenchers 1 and 2.
GRTGRRFSY-amide, the EQ50 values are (2.14 Æ 0.05) mm
for 1 and (0.15 Æ 0.05) mm for 2 (Figures S11–S13).
a library of 48 negatively charged dyes to quench the
fluorescence of the fluorophore-substituted peptides
(Table S5 in the Supporting Information). Upon subsequent
addition of PKA and ATP, fluorescence recovery ranging
from modest to dramatic was observed (Table 1 and
Tables S6–S8 in the Supporting Information). We had pre-
viously shown that a phosphorylated residue in a PKA
phosphopeptide product interacts with nearby arginine res-
idues.[13] As a working hypothesis, we propose that this
intramolecular electrostatic interaction displaces the
quencher dye (Q) from the peptide, thereby resulting in the
observed increase in fluorescence.
A few representative reporters are shown in Table 1.
These agents respond to phosphorylation at wavelengths that
span the green, red, far-red, and near-IR regions. Some of the
most responsive fluorescence changes are observed when
Acid Blue 80 (1; Scheme 2) is paired with either fluorophore-
Aoc-GRTGRRFSY-amide or fluorophore-KRRRLASLAA-
amide (Table S6). Although the fold changes observed with
Evans Blue (2; Scheme 2) as the quencher are somewhat less
dramatic, this dye appears to have a greater affinity for its
peptidic counterparts than 1. Since a Job plot analysis
(Figure S10) revealed that the stoichiometry of the
quencher/fluorophore–peptide pairs is not 1:1, we are
unable to assign Kd values. Instead, we employed EQ50 as
an assessment of affinity; that is, the [Q] that generates
a fluorescence intensity halfway between [Q] = 0 and satu-
rating Q. For example, in the case of TAMRA-Aoc-
As validation of the presumed ability of these reporters to
detect inhibitory activity, we employed fluorophore-Aoc-
GRTGRRFSY-amide/2 to examine the potency of two well-
known inhibitors of PKA, H89 and KT5720. Both inhibitors
block the phosphorylation of the Atto633-Aoc-
GRTGRRFSY-amide substrate and, under the experimental
conditions (1 mm ATP), they display IC50 values of (0.43 Æ
0.15) and (0.61 Æ 0.25) mm, respectively (Figure S14).
PKA plays key roles in erythrocyte senescence, localized
vasodilation, and deformation.[14] Given the high hemoglobin
concentration ( ꢀ 5 mm) in erythrocytes,[15] clean observation
of endogenous PKA activity should be more pronounced at
wavelengths beyond 600 nm. We initially evaluated the ability
of the fluorophore-Aoc-GRTGRRFSY-amide peptides to
monitor endogenous PKA activity in 10% erythrocyte
lysates. 5Fam-Aoc-GRTGRRFSY-amide displays very
modest fluorescent enhancements upon phosphorylation in
the presence of quenchers 1 (1.3-fold, Table 1) or 2 (1.04-fold,
Table S10). By contrast, the fluorescence enhancement is
significantly more robust with TAMRA-Aoc-GRTGRRFSY-
amide (1: 14.6-fold; 2: 7.3-fold) or Atto633-Aoc-
GRTGRRFSY-amide (1: 3.9-fold; 2: 6.3-fold).
Although the TAMRA-labelled peptide responds vigo-
rously to phosphorylation in terms of absolute fluorescence
fold change, the Atto633 species displays a superior signal to
noise ratio (64:1 versus 12:1 for the TAMRA–peptide) for the
progress curve (Figure 2). Indeed, whereas 90% of the
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Scheme 3. Photolysis of probe 3.
in the absence of photolysis. By contrast, upon illumination,
the anticipated fluorescence increase is observed (Fig-
ure S19). In addition, the fluorescence response of the
photochemically activated sensor is blocked in the presence
of a PKA inhibitor.
Peptide 3 (1 mm) and quencher 2 (2 mm) were simulta-
neously introduced into erythrocytes under hypotonic con-
ditions. Loading of the fluorescent peptide alone was
confirmed by flow cytometry and confocal microscopy
(Figures S20,S21). Erythrocytes loaded with peptide 3/
quencher 2 display a weakly fluorescent interior and a mod-
erately fluorescent membrane (Figure 3). We suspect that
during sensor loading, a portion of the positively charged
peptide binds to and remains associated with the exterior of
the cell membrane. The surface of erythrocytes is negatively
charged as a result of proteins adorned with sialic acid.[17]
Indeed, this property has been used to decorate red blood
cells with a variety of positively charged nanoparticles.[18] The
fluorescent ring that outlines the cell presumably reflects the
Figure 2. Reaction progress curve of the PKA-catalyzed phosphoryla-
tion of fluorophore-Aoc-GRTGRRFSY-amide (1 mm) with quencher 2
(2 mm) in 10% erythrocyte lysates where the fluorophore is Atto633
(black) or TAMRA (grey). Insert: in 100% lysate.
fluorescence signal is lost for the FAM–peptide and 98% for
the TAMRA–peptide, only 10% of the signal is compromised
in the case of the Atto633–peptide (Figure S17). Further-
more, the FAM and TAMRA fluorescence signals are
completely obliterated in 100% erythrocyte lysates, whereas
50% of the original Atto633 signal is still present (Figure 2,
insert). We also examined the effect of the dye quenchers
1 and 2 on erythrocyte viability and discovered that cellular
integrity is markedly reduced upon exposure to 1 (30 mm), but
remains unperturbed in the presence of 2 (30 mm; Figure S18).
Consequently, all subsequent studies were performed with the
Atto633-Aoc-GRTGRRFSY-amide (1 mm)/2 (2 mm) sensor
pair.
Since erythrocytes possess a number of protein kinases,[6]
we investigated whether the observed fluorescence response
displayed by Atto633-Aoc-GRTGRRFSY-amide/2 is due to
endogenous PKA activity. The moderately selective PKA
inhibitor H89 blocks the observed fluorescence change with
an IC50 of 1.14 Æ 0.17 mm in 10% lysates (Figure S15 and Table
S11). The highly selective PKA inhibitor KT5720 likewise
prevents the phosphorylation-induced fluorescence response
(IC50 1.59 Æ 0.36 mm). Finally, a PKA-specific antibody was
employed to clear PKA from the lysates and phosphoryl
transferase activity was then assessed. The 80% decrease in
the phosphoryl transferase activity of the precleared lysate
directly corresponds with the amount of PKA removed from
the lysate (Figure S16). These results confirm that the
fluorescence response in the lysate is driven by PKA activity.
There are a number of challenges associated with assess-
ing intracellular enzymatic activity in cells, including control-
ling the start point (t = 0) of sensor phosphorylation. Light-
activatable analogues provide a means to achieve this end.[16]
The serine hydroxy moiety in Atto633-Aoc-GRTGRRFSY-
amide was modified with a light sensitive 4,5-dimethoxy-2-
nitrobenzyl (DNMB; Scheme 3) functional group to furnish
probe 3. As expected, both in buffer and in erythrocyte
lysates, the modified serine residue in 3 is not phosphorylated
Figure 3. Confocal images of 2/3-loaded erythrocytes a) before photo-
lysis and b) 300 s after photolysis of the encircled cell. Photolysis was
performed with a 50 mW, 405 nm laser (2% power level) in tornado
mode with a dwell time of 20 ms/pixel and 25 frames. Imaging was
performed with a 100X objective by using 635 nm at 4% laser power.
Scale bar: 5 mm. c) Fluorescence fold change as a function of time
where photolysis at 405 nm is applied at the third time point (filled
circles), in the presence of KT5720 (triangles), and under depleted ATP
(open circles). See Figure S22 for visual snapshots of the data plotted
in part (c).
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partial or complete displacement of the negatively charged
quencher from the surface-bound fluorophore-labelled pep-
tide.
PKA activity in intact erythrocytes was assessed by using
confocal microscopy. Since UV illumination ( ꢀ 360 nm) is
known to have an immediate untoward impact on hemoglo-
bin biochemistry,[19] we employed a 405 nm laser to convert
the DNMB–peptide 3 to its phosphorylatable counterpart.
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the interior of erythrocytes (Figure 3 and Figures S22–S25).
By contrast, the membrane fluorescence remains unchanged
as a function of time, a result that is expected if the
fluorophore–peptide is simply appended on the exterior
surface. In addition, the PKA-selective cell-permeable inhib-
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otherwise observed in the erythrocyte interior. Finally, under
conditions in which the intracellular content of the erythro-
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phosphorylates Atto633-GRTGRRFSY-amide.
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Aoc-GRTGRRFSY-amide/2 is sufficient to construct a series
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Received: November 7, 2013
Revised: December 20, 2013
Published online: March 6, 2014
Keywords: biosensors · enzymes · fluorescent probes ·
.
peptides · signal transduction
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