Affinity-based probes based on type II kinase inhibitors

Article abstract of DOI:10.1021/ja306035v

Protein kinases are key components of most mammalian signal transduction networks and are therapeutically relevant drug targets. Efforts to study protein kinase function would benefit from new technologies that are able to profile kinases in complex prote

Full text of DOI:10.1021/ja306035v

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Article
Affinity-Based Probes Based on Type II Kinase Inhibitors
Pratistha Ranjitkar, B. Gayani K. Perera, Danielle L Swaney, Sanjay B. Hari, Eric T.
Larson, Ratika Krishnamurty, Ethan A Merritt, Judit Villen, and Dustin James Maly
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 22 Oct 2012
Downloaded from http://pubs.acs.org on November 6, 2012
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Affinity-Based Probes Based on Type II Kinase Inhibitors
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Pratistha Ranjitkar,†,# B. Gayani K. Perera,†,# Danielle Swaney,‡ Sanjay B. Hari,† Eric T. Larson,∥
Ratika Krishnamurty,† Ethan A. Merritt,∥ Judit Villén,‡ and Dustin J. Maly†,*
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†Department of Chemistry, University of Washington, Seattle, Washington, USA
‡Department of Genome Sciences, University of Washington, Seattle, Washington, USA
∥Department of Biochemistry, University of Washington, Seattle, Washington, USA.
Corresponding Author
maly@chem.washington.edu
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Abstract: Protein kinases are key components of most mammalian signal transduction networks
and are therapeutically relevant drug targets. Efforts to study protein kinase function would
benefit from new technologies that are able to profile kinases in complex proteomes. Here, we
describe active siteꢀdirected probes for profiling kinases in whole cell extracts and live cells.
These probes contain general ligands that stabilize a specific inactive conformation of the ATPꢀ
binding sites of protein kinases, as well as trifluoromethylphenyl diazirine and alkyne moieties
that allow covalent modification and enrichment of kinases, respectively. A diverse group of
serine/threonine and tyrosine kinases were identified as specific targets of these probes in whole
cell extracts. In addition, a number of kinase targets were selectively labeled in live cells. Our
chemical proteomics approach should be valuable for interrogating protein kinase active sites in
physiologically relevant environments.
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Introduction
Protein phosphorylation cascades mediate a wide array of intraꢀcellular signaling events
in eukaryotic cells.1,2 The diversity of processes regulated by this postꢀtranslational modification
is reflected by the large number of protein kinases (> 500) encoded by the human genome.3
While significant efforts have been made to functionally characterize protein kinases, the roles
of many of these enzymes in complex biological environments have yet to be interrogated. For
this reason, there is a great deal of interest in the development and application of reagents that
allow the global analysis of the protein kinase family. For example, immobilized ATPꢀ
competitive inhibitors have proven to be useful chemoꢀproteomic tools for studying protein
kinases because they allow the enrichment of these low abundance enzymes. These reagents
have provided valuable insight into specific signaling events and have facilitated exhaustive
inhibitor selectivity screens in cell lysates.4ꢀ16 Despite the widespread utility of affinity matrices
that specifically target kinases, these methods are not ideal because they require cell lysis and the
use of homogenous lysate preparations, which may disrupt signaling complexes. Furthermore,
nonꢀcovalent affinity methods are not able to discriminate between proteins that are directly
bound to an inhibitor of interest or are instead associated with an enriched protein complex.
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Affinityꢀ and activityꢀbased probes that are able to label protein kinases based on conserved
active site features overcome many of the limitations of nonꢀcovalent affinity reagents.17ꢀ22
However, the arsenal of labeling reagents that is available for studying protein kinases is limited
compared to other enzyme families (for example, serine hydrolases),23ꢀ25 and there is still a clear
need for additional chemical tools that allow their functional interrogation. Active siteꢀdirected
probes that facilitate the in situ labeling of protein kinase active sites are especially needed.
All protein kinases contain a conserved, biꢀlobal catalytic core that consists of 250ꢀ350
residues.26,27 The site of phosphate transfer is located between these two lobes, with ATP
occupying a narrow hydrophobic cleft. The adenine ring of ATP makes a number of hydrophobic
contacts in the ATPꢀbinding cleft and forms at least one hydrogen bond with the backbone of a
peptide linker ꢀ referred to as the hinge region ꢀ that connects the N- and C-terminal lobes. A
number of conserved catalytic residues that are necessary for phosphate transfer line the ATPꢀ
binding sites of protein kinases. The active sites of protein kinases are highly dynamic and the
movement of key residues allows kinases to interconvert between catalytically active and
inactive forms.28,29 While the spatial arrangement of active site residues within the ATPꢀbinding
sites of kinases that are catalytically competent are highly conserved, there is more
conformational heterogeneity for kinases in an inactive state. Indeed, structural studies of protein
kinases have revealed a number of catalytically incompetent ATPꢀbinding site configurations.
Despite this variability, there appears to be several distinct classes of inactive conformations that
are observed throughout the kinome.30,31 These inactive forms are distinguished by structural
elements that are topologically distinct. One inactive conformation that is accessible to a number
of kinases is the DFGꢀout conformation.32ꢀ34 This inactive form is characterized by the
movement, and almost 180° rotation, of the highly conserved AspꢀPheꢀGly motif (DFGꢀmotif),
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which is located at the base of the activation loop, relative to the active conformation. This
displacement results in the Asp of the DFGꢀmotif being unable to coordinate a magnesium ion
that is important for phosphate transfer and prevents the Phe side chain from participating in a
regulatory network of hydrophobic residues.35 A number of ligands that specifically complement
the topology of kinases in the DFGꢀout conformation have been identified (Figure 1A).32ꢀ34,36
These inhibitors, commonly referred to as type II inhibitors, contain moieties that make similar
contacts as the adenine ring of ATP and hydrophobic substituents that occupy the pocket created
by the movement of the Phe side chain in the DFGꢀmotif. In general, type II inhibitors are
conformationꢀselective ligands because they are sterically incompatible with the active forms of
protein kinases. This feature has made type II inhibitors useful reagents for probing the ATPꢀ
binding site conformations of protein kinases.
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Here we report the development of a new class of kinaseꢀdirected photoꢀcrosslinking
reagents. The kinaseꢀtargeting elements of these probes are general type II inhibitors that
stabilize the DFGꢀout inactive conformation of protein kinases. To allow labeling of the ATPꢀ
binding sites of protein kinases and visualization/enrichment of covalentlyꢀmodified targets,
these probes contain a trifluoromethylphenyl diazirine photoꢀcrosslinker and alkyne tag,
respectively. We show that these reagents can be photoꢀactivated to selectively label protein
kinases that adopt the DFGꢀout conformation. Use of these probes in combination with stableꢀ
isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry resulted in the
identification of a number of selectively enriched protein kinase targets. Furthermore, we
demonstrate that a novel kinase target that is specifically labeled by our conformationꢀselective
probes is indeed able to adopt the DFGꢀout conformation. Finally, we show that these reagents
are cell permeable and able to specifically label protein kinases in situ.
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Results and Discussion
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Design and synthesis of photo-affinity probes derived from type II ATP-competitive
inhibitors.
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Affinity reagents that are able to selectively label the active sites of protein kinases must
contain three components: 1) a high affinity ligand that selectively recognizes the active sites of
protein kinases, 2) a reactive group that is able to covalently modify probeꢀbound kinases, and 3)
a tag that allows visualization and/or purification. We first focused on identifying general ATPꢀ
competitive inhibitors that are able to target the active sites of protein kinases over other ATPꢀ
binding proteins. Ligands that are able to stabilize specific active site conformations of protein
kinases are of particular interest because they have the potential to provide information on the
functional state of these dynamic enzymes. Therefore, we pursued two general scaffolds based
on type II kinase inhibitors, which stabilize an inactive form of the ATPꢀbinding sites of protein
kinases called the DFGꢀout conformation (Inhibitors 1 and 2 in Figure 1B).37ꢀ39 Both of these
ligands contain structural features that make specific contacts with kinases that are in the DFGꢀ
out conformation, including a heteroꢀaromatic moiety that mimics adenine, and additional
functionalities that make a characteristic set of hydrogen bonds with a conserved glutamate in
helixꢀαC and the backbone amide of aspartate in the catalyticallyꢀimportant DFGꢀmotif (Figure
1B). Furthermore, 1 and 2 both possess 3ꢀtrifluoromethylphenyl groups that occupy the
hydrophobic pocket created by the movement of the Phe side chain from the DFGꢀmotif.
Because protein kinases do not possess any conserved residues that are highly nucleophilic, a
photoꢀcrosslinker was incorporated into our active siteꢀdirected probes. A trifluoromethylphenyl
diazirine was selected due to the relatively small size of this moiety and its ability to be
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Figure 1. Binding interactions of type II inhibitors and generation of photoꢀcrosslinking probes.
(A) A schematic representation of the interaction of type II inhibitors with the ATPꢀbinding
pocket of protein kinases. The residue numbering shown is for human cꢀSRC. Four hydrogen
bonds are made in the binding pocket; a pair of hydrogen bonds to the hinge region, one to the
conserved glutamic acid on helixꢀαC (Glu310) and one to the backbone of the DFGꢀmotif. A
heterocyclic moiety occupies the adenine pocket (shown in mint) while the 3ꢀtrifluoromethyl
phenyl group (shown in purple) accesses the pocket (DFGꢀout pocket) generated by movement
of the DFGꢀmotif. (B) Chemical structures of two general type II inhibitors (1 and 2) and their
corresponding probes (3 and 4) that contain a photoꢀcrosslinking group and an alkyne tag. The 3ꢀ
trifluoromethylphenyl groups (purple) of inhibitors 1 and 2 were replaced with 3ꢀ
trifluoromethylphenyl diazirine moieties. In addition, the 4ꢀanilino positions of these inhibitors
were modified with alkyne tags. (C) IC50 values of 1ꢀ4 against a panel of protein kinases. N/T =
not tested. The values shown are the average of assays run in triplicate or quadruplicate +/ꢀ SEM.
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photoꢀactivated at a wavelength (365 nm) that causes minimal photochemical damage to proteins
and cells.40 To allow the introduction of this crosslinking group without disrupting the interaction
of the core scaffold with protein kinase ATPꢀbinding sites, the trifluoromethyl groups of
inhibitors 1 and 2 were replaced with trifluoromethyl diazirines. Substitution at this position
places the crosslinking moiety deep within the hydrophobic pocket created by the movement of
the DFGꢀmotif. Finally, the solventꢀexposed 4ꢀanilino moieties of both inhibitors were
derivatized with an alkyne tag that allows visualization and enrichment of probeꢀlabeled
proteins.
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To determine whether probes 3 and 4 retain their ability to interact with the ATPꢀbinding
sites of protein kinases that adopt the DFGꢀout conformation, their inhibitory activities against a
small panel of protein kinases was tested (Figure 1C). ABL, CSK, EPHA3, HCK, LCK, p38α,
and SRC have been structurally characterized in the DFGꢀout conformation and are sensitive to
type II inhibitors.33,39,41ꢀ46 In contrast, IRAK4, MAP3K5, PAK4, PAK5, PKA, and p38δ have not
been observed in this inactive conformation and are not inhibited by type II inhibitors.45,46 Like 1
and 2, probes 3 and 4 inhibit kinases that adopt the DFGꢀout conformation and have very little
activity against IRAK4, MAP3K5, PAK4, PAK5, PKA, and p38δ (Figure 1C). While probes 3
and 4 have similar potencies as their parent ligands 1 and 2 for some kinases, they generally have
> 5ꢀfold higher IC50s for most. The observed loss in potency is most likely due to the
trifluoromethyl diazirine groups of 3 and 4 being less well accommodated in the hydrophobic
pocket created by the movement of the DFGꢀmotif, relative to the trifluoromethyl groups of 1
and 2. Regardless, the affinity of both probes for DFGꢀout adopting kinases is sufficient for their
use as kinaseꢀdirected probes.
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Photo-labeling of protein kinases in mammalian cell lysate.
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Probes 3 and 4 were first tested for their ability to label protein kinases in mammalian
cell lysate (Figure 2A). The DFGꢀoutꢀadopting protein kinases SRC, p38α, and EPHA3, were
incubated with either probe 3 (1 ꢁM) or 4 (1 ꢁM). Samples were then irradiated with 365 nm
light, conjugated to rhodamineꢀazide using copperꢀmediated click chemistry conditions,
separated by SDSꢀPAGE, and analyzed by inꢀgel fluorescence scanning. Robust labeling of
SRC, p38α and EPHA3 was observed with both probes (Figure 2A, lane 1). In contrast, no
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Figure 2. Labeling of DFGꢀout adopting kinases with probes 3 and 4. (A) His6ꢀtagged protein
kinases (90 nM) in mammalian cell lysate (0.2 mg/mL) were incubated with probes 3 (1 ꢁM) or
4 (1 ꢁM) in the absence (lanes 1 and 3) or presence (lane 2) of an active site competitor (10 ꢁM).
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The samples shown in lanes 1 and 2 were then irradiated with UV light for 15 minutes, while
those shown in lane 3 were not. All samples were then tagged with rhodamineꢀazide, resolved by
SDSꢀPAGE, and labeled proteins were detected with inꢀgel fluorescence scanning (top blots).
Immunoblots were performed with an antiꢀHis6 tag antibody (Cell Signaling) to ensure equal
amounts of kinase were present (bottom blots). (B) The effect of UV irradiation time on photoꢀ
crosslinking efficiency. The tyrosine kinase SRC was incubated with probes 3 (1 ꢁM) or 4 (1
ꢁM) and then irradiated with 365 nm light for 3, 5, 7, or 15 minutes. Normalized fluorescence
units versus irradiation time are shown. (C) Labeling of an activated and an inactive HCK
construct with probe 3. Activated or inactive HCK (50 or 100 nM) in mammalian cell lysate (0.2
mg/mL) were incubated with probe 3 (1 ꢁM). Samples were then irradiated with UV light for 15
minutes, conjugated to rhodamineꢀazide, resolved by SDSꢀPAGE, and labeled proteins were
detected with inꢀgel fluorescence scanning. Fluorescence intensities were quantified and
normalized to a fluorescent standard. The values shown are the average of assays run in triplicate
± SEM.
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fluorescent signal was observed if photoꢀcrosslinking was performed in the presence of an excess
of the active site competitors 3 or 4 (10 ꢁM) (Figure 2A, lane 2), indicating specific labeling of
kinase active sites. In addition, the type II inhibitors Ponatinib and Rebastinib, which are based
on different chemical scaffolds, are able to block active site labeling of SRC (SI, Figure S1).
Furthermore, no labeling was observed in the absence of UV light (Figure 2A, lane 3). To
determine whether probes 3 and 4 are able to selectively label kinases that adopt the DFGꢀout
conformation, photoꢀcrosslinking experiments were performed in the presence of two kinases,
MAP3K5 and PAK5, that have not been characterized to adopt this inactive form and are not
sensitive to type II inhibitors (Figure S2). The presence of only a single fluorescent band,
corresponding to SRC, EPHA3, or p38α, under these conditions further demonstrates the
specificity of probes 3 and 4 (Figure S2). Before performing experiments in more complex
protein mixtures, the effect of irradiation time on kinase crosslinking efficiency was explored.
For both probes, labeling was found to be linear up to 7 minutes, with maximum signal observed
within 10 minutes (Figure 2B).
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An interesting application of photoꢀaffinity probes based on type II inhibitors is their use
to interrogate how specific postꢀtranslation modifications and proteinꢀprotein interactions affect
the active site conformation of protein kinases. It has previously been demonstrated that some
protein kinases, like the tyrosine kinase ABL, have lower affinities for ligands that stabilize the
DFGꢀout conformation when their activation loops are phosphorylated.47 While this is not true
for all kinases (for example, the phosphorylation status of the activation loop of p38α does not
affect its sensitivity to most type II inhibitors)44, our photoꢀaffinity probes can serve as valuable
reagents for testing this relationship. To explore this possibility, we determined the labeling
efficiency of probe 3 for two different activation states of the tyrosine kinase HCK (Figure 2C).
An activated construct of HCK, which contains a phosphorylated activation loop and two
mutations that disrupt an autoinhibitory intraꢀmolecular SH3 domain interaction, and an inactive
HCK construct, which contains an unphosphorylated activation loop and two mutations that
strengthen an autoinhibitory intraꢀmolecular SH3 domain interaction, were tested at two different
concentrations (50 and 100 nM). At both kinase concentrations tested, probe 3 more readily
labeled the inactive HCK construct than the activated HCK construct. This result is consistent
with the ATPꢀbinding site of the inactive HCK construct showing an increased sampling of the
DFGꢀout conformation. The ability to discriminate between kinase activation states should allow
our probes to profile the interꢀrelationship between protein kinase activation states and ATPꢀ
binding site conformations.
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Proteome-wide labeling of protein kinases in A431 cell lysate.
To determine the full labeling profiles of both probes, photoꢀcrosslinking experiments
were performed in lysate derived from A431 cells (Figure 3A). A431 cells are a human epithelial
carcinoma cell line that overexpress epidermal growth factor receptor and are a model for
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studying growth factorꢀstimulated signaling. Because kinases are low abundance enzymes that
can often be obscured during proteomic analysis by more abundant cellular proteins, we used a
quantitative proteomics approach to identify the specific targets of probes 3 and 4. SILAC
methodology was used to generate lysate proteins that contain either “light” (natural isotopic
abundance) arginine and lysine or “heavy” (Uꢀ13C) arginine and lysine.48 Photoꢀcrosslinking was
performed in the heavy lysate with either probe 3 (1 ꢁM) or 4 (1 ꢁM). The same labeling
experiments were performed with the light lysate in the presence of an excess of an active site
competitor (10 ꢁM). After UV irradiation, both the heavy and light lysate were combined,
conjugated to biotinꢀazide, and enriched with streptavidinꢀconjugated beads. The streptavidinꢀ
conjugated beads were then washed extensively and subjected to onꢀbead digestion with trypsin.
Eluted peptides were separated by reverseꢀphase chromatography and online analyzed in a
hybrid LTQꢀOrbitrap mass spectrometer. Mass spectrometric analysis revealed peptide identities
and relative peptide abundances between the heavy and light samples. Proteins with a heavyꢀtoꢀ
light ratio (H/L) greater than 2ꢀfold were deemed as specific targets of the probes. Due to the
stringent wash conditions (1% SDS (3x) and 6M urea (3x)) used in the pullꢀdown experiments,
we felt it was unlikely that any protein targets would be enriched nonꢀcovalently. However, a
model study was performed to confirm that this is most likely the case (Figure S3). A cell lysate
supplemented with 90 nM HCK was incubated with probes 3 (1 ꢁM) or 4 (1 ꢁM) but not
irradiated with UV light. Samples were then conjugated to biotinꢀazide, subjected to the pullꢀ
down and wash protocol described above, and bound proteins were eluted from the streptavidin
beads with SDS. Under these conditions, no HCK could be detected in the eluted samples by
Western blot analysis (Figure S3). In contrast, HCK could readily be detected when samples
were irradiated prior to performing the same pullꢀdown experiment. This indicates that any
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proteins that are enriched in the SILAC experiments described above have most likely been
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covalently modified by probes 3 and 4.
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Figure 3. Labeling of endogenous protein kinases in A431 cell lysate with probes 3 and 4. (A) A
schematic representation of the photoꢀlabeling experiments that were performed in A431 cell
lysate with probes 3 and 4. A431 cells were metabolically labeled with arginine (13C6ꢀArg) and
lysine (13C6ꢀLys) and the lysate generated was photoꢀcrosslinked with probes 3 or 4. In parallel,
the same photoꢀlabeling experiments were performed with probes 3 or 4 in the presence of an
ATPꢀbinding site competitor (inhibitors 1 or 2) with cell lysate that contains “light” Lys and Arg.
After photoꢀcrosslinking, both labeling experiments were combined and the subsequent mixture
was subjected to copperꢀmediated click chemistry with biotinꢀazide. Streptavidinꢀconjugated
beads were used to enrich biotinꢀlabeled proteins. These beads were then washed extensively and
subjected to digestion with trypsin. Tryptic peptides were identified by tandem mass
spectrometry and quantified to obtain a heavyꢀtoꢀlight peptide ratio. The results of these
experiments are shown in Table 1. (B) Venn diagram showing total and overlapping proteins
enriched by probes 3 and 4. (C) Venn diagram showing total and overlapping protein kinases
enriched by probes 3 and 4.
A summary of the data that was obtained in the A431 cell lysate labeling experiments is
shown in Figure 3 and Table 1. For probe 3, 35 proteins were specifically labeled, with 21 of
those being protein kinases. For probe 4, 55 proteins were specifically labeled, with 28 of those
being protein kinases. There is a 45% overlap in the protein kinases that were enriched by the
two probes. A diverse group of protein kinases were specifically labeled, with tyrosine kinases
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representing the largest percentage (42%) of enriched kinases. Of the 15 protein kinases that
were selectively enriched by both probes 3 and 4, 10 are tyrosine kinases. Specifically enriched
tyrosine kinases include several SRCꢀfamily members, such as SRC, FRK and SRM, which are
Table 1. Kinases enriched in A431 lysate with probes 3 and 4.
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Protein
Probe
H/L
Ratio
(Probe 3)
# of
Peptides
Identified
(Probe 3)
H/L
Ratio
(Probe 4)
# of
Peptides
Identified
(Probe 4)
Kinase
Group
BRK
CSK
EPHA2
EPHB2
EPHB4
FAK
3 + 4
3 + 4
3 + 4
3 + 4
3 + 4
3 + 4
3 + 4
3 + 4
3 + 4
3 + 4
3 + 4
3 + 4
3 + 4
3 + 4
3 + 4
3
3
3
3
3
3
4
4
4
7.7
2.0
15
15
35
2
8
12
6
6
1
1
12
7
12
59
3
6
18
2
2
3
4
4
8
3
> 28
8.8
5.0
35
71
37
> 110
6.3
15
11
3.9
27
2
3
27
12
5
4
1
2
4
6
28
2
2
7
Tyrosine
Tyrosine
Tyrosine
Tyrosine
Tyrosine
Tyrosine
Tyrosine
CMGC
CMGC
STE
2.5
> 100
1.9
2.5
2.7
2.7
1.9
1.9
14
9.9
2.1
19
3.6
5.1
3.9
2.0
FRK
JNK1
JNK3
LOK
PYK2
SLK
smMLCK
SRC
SRM
CDC2
ERK2
GSK3β
p38α
PIP4K2C
TAO1
EGFR
EPHB3
ERBB4
HGK
KHS1
MST1
MST3
MST4
PAK2
PITSLRE
RIPK1
SYK
Tyrosine
STE
9.2
> 32
> 67
CAMK
Tyrosine
Tyrosine
CMGC
CMGC
CMGC
CMGC
PIP5K2
STE
Tyrosine
Tyrosine
Tyrosine
STE
STE
STE
STE
STE
STE
CMGC
TKL
Tyrosine
STE
1
6.1
> 22
11
> 17
12
4.6
2.4
3.8
2.3
15
1
1
3
2
20
1
1
7
2
1
4
4
4
4
4
4
4
4
11
> 14
3.2
6
1
1
4
4
TNIK
known to adopt the DFGꢀout conformation. With probe 3, a 10ꢀfold or higher heavyꢀtoꢀlight
peptide ratio was observed for both SRC and SRM, while only the heavy peptides were detected
for FRK. Similar results were observed with probe 4. In addition, probes 3 and 4 also selectively
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labeled a number of Ephrinꢀactivated receptor tyrosine kinases including EPHA2, EPHB2,
EPHB3, and EPHB4. This family of receptor tyrosine kinases has been structurally characterized
to adopt the DFGꢀout conformation and is believed to play prominent roles in many forms of
cancer.46 Of the 18 remaining enriched protein kinase targets that are not tyrosine kinases, 44%
are from the STE Group. Unlike the tyrosine kinases, a majority of the serine/threonine kinase
targets were labeled by only one probe. For example, only probe 3 selectively enriched the
mitogenꢀactivated protein kinase p38α (H/L ratio = 5.1), which has been well characterized to
adopt the DFGꢀout conformation. These results demonstrate that probes 3 and 4 are able to
selectively label expected as well as novel protein kinase targets. While selective labeling of a
kinase target is not definitive proof that a kinase can adopt the DFGꢀout conformation, these
studies provide a number of interesting candidates for future biochemical and structural
characterization. In addition to protein kinases, a number of other diverse protein targets were
selectively enriched by probes 3 and 4 (Table S2, SI). Unlike the protein kinases, there are not
multiple members of any one protein family represented. Perhaps the most interesting of the
enriched nonꢀkinase targets are retinal rod rhodopsinꢀsensitive cGMP 3',5'ꢀcyclic
phosphodiesterase subunit delta (PDE6D), reticulonꢀ3 (RTNꢀ3), and a member of the
cytoplasmic epoxide hydrolase 2 family (EPHX2), which are all selectively labeled by probes 3
and 4. We are currently exploring whether type II inhibitors perturb the functions of these targets
and if these proteins contain binding sites that are similar to the DFGꢀout conformation of
protein kinases.
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LOK adopts the DFG-out conformation when bound to type II inhibitor 1.
Of the targets that were selectively enriched by our photoꢀprobes, we were particularly
interested in the STE kinase LOK [probe 3 (H/L ratio = 2.7); probe 4 (H/L ratio = 11)] because it
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is distantly related to kinases that have already been characterized to adopt the DFGꢀout
conformation. We have also previously demonstrated that this kinase binds to affinity reagents
based on the type II triazoloꢀpyridine inhibitor scaffold.38 As confirmation of our proteomics
results, labeling experiments with the purified catalytic domain of LOK were performed (Figure
4C). As expected, probe 4 was found to efficiently label the active site of LOK. Furthermore, no
crosslinking was observed in the presence of an active site competitor. Next, we obtained a
crystal structure of a type II ligand (inhibitor 1) bound to the catalytic domain of LOK. Analysis
of the LOKꢀ1 complex clearly shows that the ATPꢀbinding site of LOK is in the DFGꢀout
conformation (Figure 4B). The conformation of LOK when bound to inhibitor 1 is strikingly
similar to that of the tyrosine kinase SRC bound to a similar triazoloꢀpyridine inhibitor.39 The
molecule sits deep within the catalytic cleft, spanning the adenine and DFGꢀout pockets. In
addition to the two hydrogen bonds that 1 forms with the hinge region of LOK, this inhibitor
makes two hydrogen bond contacts that are characteristic of type II ligands. These two hydrogen
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A
B
C
Probe 4
+
ꢀ
+
+
Competitor 2
LOK
αꢀHis6
Figure 4. The STE kinase LOK adopts the DFGꢀout conformation. (A) Crystal structure of the
catalytic domain of LOK bound to the type I inhibitor SU11274 (PBD 2j7t). The DGFꢀmotif of
LOK (shown in green) is in the active conformation. (B) Crystal structure of the LOKꢀinhibitor 1
complex. The DFGꢀmotif of LOK (shown in green) adopts the DFGꢀout conformation when
bound to inhibitor 1. Inhibitor 1 makes all of the characteristic contacts of type II inhibitors. (B)
In vitro labeling of the purified catalytic domain of LOK with probe 4.
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bonds are both between the amide group of the 3ꢀtrifluoromethylbenzamide moiety and LOK:
the nitrogen of 1 with Gluꢀ81 in helixꢀαC (equivalent to Gluꢀ310 in Figure 1A) and the carbonyl
of 1 with the backbone of the DFG motif. These results demonstrate that if a kinase is selectively
labeled by probes 3 or 4 it is most likely able to adopt the DFGꢀout conformation.
In situ activities of probes 3 and 4.
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An advantage of using small molecule probes to profile protein kinases is they have the
potential to label their targets in situ. To test the cell permeability of photoꢀcrosslinkers 3 and 4,
their ability to block the proliferation of Ba/F3 cells stably expressing BCRꢀABL was
determined with a cell viability assay (Figure 5A). Ba/F3 cells are dependent on the kinase
activity of BCRꢀABL for survival when grown in the absence of cytokines. Probes 3 and 4 are
potent inhibitors of ABL kinase activity in vitro (Figure 1C), and should be able to block the
growth of BCRꢀABLꢀexpressing Ba/F3 cells if they possess sufficient cell permeability. Indeed,
probes 3 (EC50 = 18 nM) and 4 (EC50 = 11 nM) are able to block the proliferation of BCRꢀABLꢀ
expressing Ba/F3 cells, indicating that they are able to access intraꢀcellular protein kinase targets
(Figure 5A).
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Figure 5. Probes 3 and 4 are cell permeable. (A) The abilities of inhibitors 1ꢀ4 (1ꢀ10,000 nM) to
block the growth of BCRꢀABLꢀdependent Ba/F3 cells were determined with a cell viability
assay. The number of viable cells was normalized relative to a DMSO control. (B) In situ
labeling of A431 cells with probe 3. A431 cells were incubated with probe 3 (1 ꢁM) in the
presence (lane 2) or absence (lane 1) of an active site competitor (10 ꢁM) for 30 minutes and
then irradiated with UV light for 7 minutes. Lysate obtained form these cells was tagged with
rhodamineꢀazide, resolved by SDSꢀPAGE, and labeled proteins were detected with inꢀgel
fluorescence scanning. Lane 3 shows the results of an in situ labeling experiment performed
without UV irradiation. A coommassieꢀblue stained gel showing all of the proteins loaded is
shown on the right.
We next determined the abilities of probes 3 and 4 to label protein kinases in situ. To do
this, cultured mammalian cells were incubated with probes 3 or 4 for 30 minutes, irradiated with
UV light for seven minutes, washed with PBS, and lysed. Labeled proteins in the cell lysate were
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then conjugated to rhodamineꢀazide, separated by SDSꢀPAGE, and visualized using a fluorescent
gel scanner. Fluorescent gels for photoꢀcrosslinking experiments performed with probe 3 in
A431 cells and K562 cells are shown in Figure 5B and Figure S4, respectively. Consistent with
probe 3 possessing the ability to robustly label multiple kinases in situ, a large number of
fluorescent bands are observed. This labeling appears to be specific as most of these bands are
absent when photoꢀcrosslinking is performed in the presence of competitor 1 (10 ꢁM) or the
absence of UV light. Probe 3 was found to consistently provide more specific labeling than probe
4 in situ (data not shown) and was selected for further characterization.
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To initially characterize the intraꢀcellular targets of our photoꢀcrosslinkers, an in situ
photoꢀcrosslinking experiment was performed with probe 3 (1 ꢁM) in A431 cells metabolically
labeled with heavy Lys (Figure 6A). A parallel experiment was carried out with probe 3 (1 ꢁM)
in the presence of competitor 1 (10 ꢁM) with A431 cells grown in light Lys. After photoꢀ
crosslinking and cell lysis, equal amounts of heavy and light sample protein were mixed,
conjugated to biotinꢀazide, and enriched with streptavidinꢀconjugated beads. The beads were
subjected to extensive washing, followed by onꢀbead digestion with LysC, and the eluted
peptides were identified using tandem mass spectrometry. In total, 24 proteins were specifically
enriched (H/L ratio > 2) by probe 3, with eight of these being protein kinases (Table S3). Notable
in situ targets of probe 3 are kinases that have previously been characterized to adopt the DFGꢀ
out conformation, p38α, SRC, and YES, and the receptor tyrosine kinases EPHA2, EPHB2, and
EPHB3. Interestingly, the EPH receptor tyrosine kinases were labeled with particularly high
efficiency and specificity (H/L ratio between 9 and 67), which demonstrates that probe 3 is able
to label transmembrane proteins within live cells. To further confirm the specific labeling of
EPH receptor tyrosine kinases in situ, cells that have low endogenous levels of these kinases,
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COSꢀ7, were transiently transfected with a plasmid containing V5ꢀtagged EPHA2. Transfected
cells were then subjected to our standard in situ photoꢀcrosslinking conditions. COSꢀ7 cells that
were not transfected with V5ꢀtagged EPHA2 were photoꢀcrosslinked using the same conditions.
The lysates obtained from both in situ photoꢀcrosslinking experiments were conjugated to
rhodamineꢀazide and separated with SDSꢀPAGE. A fluorescent band that corresponds to the
molecular weight of EPHA2 was observed in COSꢀ7 cells transiently expressing EPHA2, which
was absent in the nonꢀtransfected cells (Figure 6B and S5 (SI)). The high efficiency in which
probes 3 and 4 label receptor tyrosine kinases in situ will facilitate the interrogation of these
multiꢀdomain proteins in their native environment. Currently, we are using probe 3 to profile the
active site conformational changes that Eph receptor tyrosine kinases undergo upon ligand
stimulation.
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A
LCꢀMS/MS
13C6ꢀ Lys (Lys6)
C6ꢀ Lys (Lys0)
Identification and
quantification
+ Probe 3
and competitor 1
+ Probe 3
Irradiate with UV light
LysC digestion
Streptavidin
enrichment
Lyse Cells
+ Biotinꢀazide
TBTA, TCEP
Mix labeled
proteomes
Biotinꢀtagged
proteome
CuSO
4
B
Probe 3
+
+
+
ꢀ
+
+
+
ꢀ
EPHA2ꢀV5
Inꢀgel fluorescence
αꢀV5
Figure 6. Identification of in situ targets of probe 3. (A) A schematic representation of the in situ
photoꢀcrosslinking experiments performed with probe 3 in A431 cells. A table listing kinases
that were specifically enriched is in the Supporting Information (Table S3, SI). (B) (left panel)
EPHA2ꢀV5ꢀtransfected and nonꢀtransfected COSꢀ7 cells were photoꢀcrosslinked with probe 3 (1
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ꢁM). Lysate obtained from both samples was conjugated to rhodamineꢀazide, resolved by SDSꢀ
PAGE, and labeled proteins were detected with inꢀgel fluorescence scanning. (right panel)
Western blot analysis of EPHA2ꢀV5ꢀtransfected and nonꢀtransfected COSꢀ7 cells (antiꢀV5
antibody).
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Conclusions
Despite widespread interest in protein phosphorylation and the enzymes that catalyze this
postꢀtranslational modification, many aspects of protein kinase function remain unexplored due
to a lack of general profiling tools. Kinaseꢀdirected reagents can be used to perform inhibitor
selectivity screens and to globally profile the functional states of protein kinase active sites
during specific signaling events. Here, we describe two probes, 3 and 4, that are able to label the
ATPꢀbinding sites of protein kinases in cell lysates and live cells. To allow covalent modification
of protein kinase active sites and visualization/enrichment of labeled proteins, these reagents
contain a photoꢀactivatable trifluoromethylphenyl diazirine group and an alkyne tag,
respectively. The kinaseꢀtargeting elements of probes 3 and 4 are general type II inhibitors that
are selective for the DFGꢀout inactive conformation of protein kinases. Conversion of type II
inhibitors into photoꢀprobes does not affect their abilities to interact with kinases that adopt the
DFGꢀout conformation. Furthermore, probes 3 and 4 are able to efficiently photoꢀlabel the active
sites of the protein kinases that they are inhibitors of. Probes 3 and 4 were used to profile protein
kinases in A431 cell lysate. Using these probes in combination with a quantitative mass
spectrometry technique, SILAC, allowed the identification of a diverse set of protein kinase
targets that were selectively enriched. These targets include several protein kinases that have
previously been characterized to adopt the DFGꢀout conformation including, SRC, p38α, Pyk2,
and CSK. Furthermore, a number of protein kinases, particularly in the STE group, that have not
been previously characterized to adopt the DFGꢀout conformation were identified. We obtained a
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crystal structure of one of these kinases, LOK, bound to a type II inhibitor and found that its
ATPꢀbinding site is indeed in the DFGꢀout conformation. While selective labeling of a particular
kinase by one of our probes is not proof that its ATPꢀbinding site can adopt the DFGꢀout
conformation, the assembled list of kinases targets that have been identified are interesting
candidates for further characterization. We are in the process of performing structural studies
with several of the other novel kinases that are selectively labeled by probes 3 or 4. Beyond
lysate profiling experiments, we found that both probes 3 and 4 are cell permeable and able to
label a number of protein targets in A431 and K562 cells. Probe 3 was found to selectively label
a number of kinases, including p38α, SRC, YES, EPHA2, EPHB2, and EPHB3, in a preliminary
quantitative proteomic analysis of its intracellular targets. Current efforts are underway to obtain
a full in situ labeling profile of probes 3 and 4. This will not only allow a greater understanding
of the conformational state of protein kinase active sites but will also provide information on
which kinases can effectively be targeted with type II inhibitors. In addition, by comparing the
cell lysate and in situ labeling profiles of probes 3 and 4, a greater understanding of how the
local environment of a protein kinase affects its active site conformation will be obtained.
Finally, the conformationꢀselective properties of probes 3 and 4 are being used to monitor the
active site conformational changes that specific kinases undergo during signaling events. This
will provide a greater understanding of the intraꢀcellular regulation of this important enzyme
family.
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ASSOCIATED CONTENT
Supporting Information Supplementary figures, supplementary tables, experimental methods,
and characterization of compounds 1ꢀ4. This material is available free of charge via the Internet
at http://pubs.acs.org.
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AUTHOR INFORMATION
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Corresponding Author
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Eꢀmail: maly@chem.washington.edu
Author Contributions
# P.R. and B.G.K.P contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This research was supported by the NIH: R01GM086858 (D.J.M) and R01AI089441 (E.A.M.);
and the Alfred P. Sloan Foundation (D.J.M.)
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A
Hinge Region
O
NH
Glu 310
R
1
2
3
4
5
6
7
8
NH
O
N
HN
R
HO
R
O
N
O
O
HN
H
O
H
DFG-out
Pocket
N
HN
N
N
CF
3
H
O
9
N
N
O
10
11
12
13
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H
N
O
Asp 404
Adenine
Pocket
O
HN
O
DFG-motif
Phe 405
Photo-crosslinking
Group
B
O
O
N
N
CF
3
N
N
HN
N
H
N
N
HN
N
H
CF3
HN
N
N
HN
O
N
O
N
O
N
1
3
O
NH
Alkyne Tag
O
O
N
N
CF3
N
N
H
N
N
H
CF3
HN
O
N
HN
O
2
4
NH2
NH
C
IC50 (nM)
Kinase
ABL
CSK
EPHA3
N/T
< 10
HCK
2.2 ± 0.2
< 0.5
LCK
< 2
< 2
34 ± 10
< 2
PKA
p38α
6.1 ± 0.5
< 10
22 ± 3
< 2
SRC
< 2
< 2
25 ± 2
< 2
1
2
3
4
1.5 ± 0.1 22 ± 1
8.4 ± 0.2
< 2
20 ± 5
< 2
36 ± 3 120 ± 10 10 ± 2
14 ± 1 2.7 ± 0.1
PAK5
6.7 ± 0.9
Kinase
IRAK4 MAP3K5 PAK4
p38δ
N/T
N/T
1
2
3
4
> 10,000 > 10,000 > 10,000 > 10,000 > 10,000
N/T
> 10,000
> 10,000 > 10,000 > 10,000
> 10,000 > 10,000 > 10,000 > 10,000 > 10,000 > 10,000
N/T > 10,000 > 10,000 > 10,000 > 10,000 > 10,000
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A
Probe 4
+
+
+
Probe 3
1
2
3
4
5
6
+
+
+
Competitor 2
-
+
+
-
-
Competitor 1
-
+
+
-
-
hν
+
hν
+
EPHA3
EPHA3
α-His6
7
8
α-His6
9
10
11
12
13
14
15
16
17
18
19
20
SRC
SRC
α-His6
α-His6
p38α
p38α
α-His6
α-His6
B
21
22
23
24
25
26
27
1.5
1.5
1.0
0.5
0.0
Probe 3
Probe 4
1.0
0.5
0.0
28
29
30
0
5
10
15
20
0
5
10
15
20
Irradiation Time (minutes)
Irradiation Time (minutes)
C
31
32
33
34
35
36
37
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60
2.5
2.0
1.5
1.0
0.5
0.0
= inactive HCK
= activated HCK
HCK (50 nm)
HCK (100 nm)
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A
B
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Biotin-tagged
proteome
Streptavidin
enrichment
13C6- Lys (Lys6) /
13C6- Arg (Arg6)
lysate
C6- Lys (Lys0) /
C6- Arg (Arg0)
lysate
35
18
55
Tryspin
Digestion
+ Biotin-azide
TCEP, TBTA,
CuSO4
+ Probe 3 or 4
and competitor
1 or 2
LC-MS/MS
Probe 3
Probe 4
+ Probe 3
or 4
C
Irradiate with UV Light
Identification and
quantification
21
15
28
Mix labeled proteomes
Probe 3
Probe 4
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C
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Probe 4
+
+
+
Competitor 2
-
9
LOK
10
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α-His6
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