A Light-Activated Probe of Intracellular Protein Kinase Activity

Article abstract of DOI:10.1021/ja037801x

The first example of a photoactivated probe of intracellular enzymatic activity is described. The caged derivative of a fluorescent protein kinase C peptide-based sensor was prepared by modifying the free hydroxyl group of a phosphorylatable serine moiety with a photolabile appendage that blocks phosphoryl transfer. We have demonstrated that the caged sensor allows one to (1) sample PKC activity with exquisite temporal precision, (2) control the relative amount of active sensor available for phosphorylation, and (3) examine protein kinase activity at multiple time points. Copyright

Full text of DOI:10.1021/ja037801x

Published on Web 10/11/2003
A Light-Activated Probe of Intracellular Protein Kinase Activity
Willem F. Veldhuyzen,† Quan Nguyen,‡ Gary McMaster,‡ and David S. Lawrence*,†
Department of Biochemistry, The Albert Einstein College of Medicine, 1300 Morris Park AVenue,
Bronx, New York 10461, and GenoSpectra, 6519 Dumbarton Circle, Fremont, California 94555
Received August 7, 2003; E-mail: dlawrenc@aecom.yu.edu
Scheme 1
Signal transduction pathways are driven, in large part, by the
intracellular action of protein kinases. Members of this large enzyme
family (>500) catalyze the transfer of the γ-phosphoryl group of
ATP to serine, threonine, and tyrosine residues in a wide variety
of protein substrates. These enzymes play an important role in nearly
every aspect of normal cellular function. In addition, nearly every
human malady has been linked, in some manner, to abnormal
protein kinase activity. Not surprisingly, there has been and
continues to be intense interest in the development of protein kinase
inhibitors as potential therapeutic agents. In addition, several protein
kinase sensors have recently been described that furnish a visual
readout of protein kinase activity in living cells. These probes not
only reveal the activity of protein kinases in response to some
environmental stimulus, but they also offer a realistic cell-based
assessment of protein kinase inhibitor efficacy. Several investigators
have described genetically encoded protein kinase substrates that
possess appended green fluorescent protein analogues for visual
readout.1 In addition, we’ve recently developed several peptide-
based species that respond to phosphorylation in a fluorescently
sensitive fashion.2 For example, compound 1, which contains an
environmentally sensitive fluorophore positioned within a few
angstroms of the phosphorylatable serine hydroxyl moiety, displays
a robust response to protein kinase C (PKC)-catalyzed phosphor-
ylation.2a However, none of the fluorescent reporters described to
date allow the investigator to control when protein kinase activity
sampling is performed. The latter property would be extremely
valuable in a number of instances. For example, cells harboring
constitutively active protein kinases can render the intracellular
loading of a kinase sensor and the subsequent observation of activity
at a well-defined time point problematic. Furthermore, protein
kinases can exhibit intermittent activity as a function of some
cellular event, such as with PKC, which appears to be activated at
several distinct stages during mitosis.3 In general, the ability to
control when protein kinase activity is measured with respect to
multiple cellular signposts provides the opportunity to collect a large
series of parallel temporally offset samplings of protein kinase action
within the context of a single experiment. We report herein the
first example of a light-activated (“caged”) probe (2) of protein
kinase activity.
should be resistant to PKC-catalyzed phosphorylation until activated
by light. Alternatively, the presence of a single photolytically
sensitive substituent on the phosphorylatable serine hydroxyl should
likewise preclude PKC-catalyzed phosphoryl transfer. The latter
offers the advantage, relative to a peptide containing multiply caged
residues, that only a single functional group need be photolytically
liberated (peptide 2) to generate the active protein kinase fluorescent
reporter. The caged serine was prepared as outlined in Scheme 1.6
The key step, benzylation of the serine side-chain hydroxyl, was
achieved using the trichloroacetimidate 4 in the presence of a
catalytic amount of triflic acid.7 The Fmoc derivative 6 was
subsequently employed to create the active site-directed peptide 2
via solid-phase peptide synthesis (see Supporting Information).
As would be expected for a peptide lacking a free hydroxyl
group, compound 2 fails to serve as a substrate for PKC (Figure
1). Two potentially useful attributes of the caged substrate include
(1) sampling of protein kinase activity at a time of the investigator’s
choosing and (2) control over the amount of active substrate
available for phosphorylation. The former is illustrated in Figure
1, where the caged substrate 2 is incubated with active PKC for
various time intervals (10, 20, and 30 min) and then subsequently
photoactivated (Hg arc lamp for 90 s). A broad band-pass filter
(300-400 nm with λmax @ 360 nm) was employed to protect the
nitrobenzofurazan fluorophore from photobleaching, and an IR filter
was used to shield PKC from heat inactivation. Peptide 2 is
completely inert as a PKC substrate irrespective of PKC incubation
time. Furthermore, identical robust fluorescence responses are
immediately observed following photolysis, irrespective of the pre-
photolysis PKC incubation time.
HPLC analysis revealed that the maximal conversion of caged
to uncaged substrate is approximately 60% (see Supporting
Information). The quantum yield for photolytic conversion is 0.06
as determined by actinometry.8 Although a 90 s irradiation time is
required for maximal in vitro formation of the uncaged substrate,
intracellular uncaging should proceed more rapidly due to an
enhanced photon flux through a comparatively smaller cellular
volume. Furthermore, as illustrated in Figure 2, total photon flux
can be used to control the amount of free protein kinase probe
liberated.
Both the timing and relative amount of sensor release can be
controlled in a single experiment (Figure 1, insert). Approximately
half of compound 2 was photochemically converted to the active
probe 1 in the presence of PKC, as indicated by the observed change
in fluorescence. Subsequent illumination of the reaction mixture
afforded additional free sensor, which likewise furnished a fluo-
rescent response. These experiments demonstrate that it is not only
We envisioned two different strategies for the construction of
caged protein kinase sensors. PKC is known to recognize peptides
containing appropriately positioned arginine residues.4 Conse-
quently, a substrate harboring multiply caged arginine moieties5
† The Albert Einstein College of Medicine.
‡ GenoSpectra.
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C O M M U N I C A T I O N S
Figure 3. Intracellular fluorescence change as a function of time following
irradiation and/or TPA treatment. HeLa cells were microinjected with peptide
2 and subsequently (A) irradiated and treated with TPA, (B) treated with
TPA in the absence of light, and (C) irradiated in the absence of TPA
Figure 1. Time-dependent change in fluorescence before and after in situ
illumination of caged peptide. The caged peptide 2 was incubated at 30 °C
with PKCR and the change in fluorescence measured for 10 (A), 20 (B), or
30 (C) min. Samples were then irradiated at the indicated time points. (Insert)
Partial photolysis of 2 followed by a second exposure to brief illumination.
See Supporting Information for details.
In summary, we have prepared a caged protein kinase sensor
via introduction of a serine moiety containing a photolytically labile
side-chain appendage. The presence of the latter affords control
over both the timing and amount of active sensor release. To the
best of our knowledge, compound 2 represents the first example
of a caged fluorescent reporter of intracellular enzymatic activity.
Acknowledgment. We thank Dr. Aigou Zhang and Mr. Yusuf
Ali for technical support and the NIH for funding.
Supporting Information Available: Experimental details of the
synthesis, characterization, photo-uncaging, and in vitro and in vivo
assays of compound 2 (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
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Figure 2. Fluorescence change as a function of irradiation time. Peptide 2
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