Novel Substrates for Fluorescence-based Protein Tyrosine Kinase Assays

Novel Substrates for Fluorescence-based Protein Tyrosine Kinase Assays
Julie Blouin, Mireille Caron, Anja Rodenbrock, Anne Labonté, Mireille Legault, Christian Fafard, Véronique Brechler, Philippe Roby, Lucille Beaudet and Francesco Lipari
PerkinElmer Life and Analytical Sciences, Montreal (QC), Canada H3J 1R4
3
LANCE® Ultra is a TR-FRET technology
for kinase assays. In these kinase assays,
the binding of a Eu-labeled anti-phospho-Tyr
antibody to the phosphorylated ULight™labeled substrate brings donor and acceptor
molecules into close proximity. After
irradiation of the kinase reaction at 320 or
340 nm, the energy from the Eu donor is
transferred to the ULight acceptor dye which,
in turn, generates light at 665 nm. The
intensity of the light emission is proportional
to
the
level
of
ULight-substrate
phosphorylation.
ULight-poly GT (4:1) & ULight-poly GAT (1:1:1)
Polypeptide sequences : polymers of amino acids (20-50 kDa) in which the ratios of amino acids
are as indicated (G = glutamic acid, T = tyrosine, A = alanine)
Origin: these synthetic polypeptides were originally described by Braun et al 1984
ULight-IRS-1 (Tyr983)
Peptide sequence : CKKSRGDYMTMQIG
Origin: peptide derived from residues 979-989 of mouse Insulin receptor substrate 1 (IRS-1).
The peptide contains the YMxM motif shown to be a consensus site for PTK phosphorylation
(Shoelson et al 1992, Garcia et al 1993)
ULight-JAK-1 (Tyr1023)
Peptide sequence : CAGAGAIETDKEYYTVKD
Origin: peptide derived from amino acids 1015-1027 of human Janus kinase 1 (JAK-1). The
peptide is a substrate for JAK3 (Wang et al 2003)
ULight-TK
Peptide sequence and origin : patent pending
4
Materials - ULight-poly GT (4:1), ULight-poly GAT (1:1:1), ULight-IRS-1 (Tyr983),
ULight-JAK-1 (Tyr1023), ULight-CDK1 (Tyr15), ULight-TK, ULight-Streptavidin, and
Europium-anti-phospho-Tyr (PT66) Antibody were from PerkinElmer LAS, Inc (PKI). All
kinases were recombinant (human origin) and purchased from Carna Biosciences.
General assay procedure - The assays were performed in White OptiPlate™-384
microplates from PKI. All assay incubations were performed at room temperature and
the microplates were sealed with TopSeal-A™ (PKI) during that time. The fluorescence
signals were read using an excitation filter of 320 nm and an emission filter of 665 nm
on an EnVision® Multilabel Reader (PKI). The final assay volume was 20 µL.
Determination of kinase concentration – 0 to 30 nM of each kinase was incubated with
50 nM of biotinylated phospho-Tyr-peptide in the presence of 200 µM ATP for 2 h. The
reaction was stopped by adding 10 mM EDTA and 50 nM ULight-Streptavidin for 5 min.
2 nM Europium-anti-phospho-Tyr (PT66) Antibody was added and incubated for
another 1 h before reading the fluorescence.
Kinase profiling – The kinase was incubated with 50 (CDK1, JAK-1 and TK) or 100 nM
(IRS-1, poly GT and poly GAT) substrate in the presence or absence of 200 µM ATP.
The enzyme reaction proceeded for 2 h before stopping with 10 mM EDTA for 5 min. 2
nM Europium-anti-phospho-Tyr (PT66) Antibody was added and incubated for another
1 h before reading the fluorescence. The signal to background (S/B) ratio was
calculated by dividing the fluorescence signal of the reaction with ATP by the signal
from the reaction without ATP.
20000
Concentration
chosen for assay
(1 nM)
10000
0
-∞ -13 -12 -11 -10
-9
-8
-7
-6
Log [Kinase] (M)
Several kinases contain phospho-Tyr residues that can bind to the antibody in the assay and
compete with the phosphorylated substrate; therefore, the kinase concentration has to be chosen so
that it does not interfere in the signal generation. Preliminary tests with the kinase and a phosphopeptide were performed to determine the concentration of kinase to use in the assay. Enough ATP
was added to allow for potential protein autophosphorylation to occur. The fluorescence signal was
plotted versus the concentration of enzyme and the highest concentration of enzyme providing
minimal or no interference of signal was chosen for determination of activity versus the various
ULight-substrates. The competition curve for JAK2 kinase is illustrated.
5
Kinase Profiling
LANCE Signal (665 nm)
Materials and Methods
ABL [T315I] (ABL1)
- ATP
+ ATP
150000
125000
100000
75000
50000
25000
0
Kinases
Kinase
EphA3
EphA4
EphA5
EphA6
EphA7
EphA8
EphB1
EphB2
EphB3
EphB4
FAK
FER
FES
FGFR2
FGFR3
FGFR4
S/B ratio
22.4
31.5
35.5
5.4
17.2
32.3
29.9
28.8
24.7
29.8
1.7
31.0
24.9
27.1
24.4
17.7
A panel of 16 kinases were tested with the substrate ULight-TK. The results clearly demonstrate that
this substrate can be used to assay numerous kinases.
ACK
ARG
SRC
BLK
TEC
BMX
SRC
BRK
TEC
BTK
CSK
CSK
CSK
CTK
FER
FAK
FER
FER
FES
SRC
FGR
SRC
FRK
SRC
FYN
SRC
HCK
TEC
ITK
JAKA
JAK1
JAKA
JAK2
JAKA
JAK3
SRC
SRC
LCK
LYNa
SRC
LYNb
FAK
PYK2
SRC
SRC
SRC
SRM
SYK
SYK
TEC
TEC
ACK
TNK1
TEC
TXK
JAKA
TYK2
SRC
YES
SYK
ZAP70
ALK
30000
hA
Ep 3
h A (0 .
4 5
E p (0 n M
)
.
Ep h A 5 n
h A 5 ( M)
Ep 6 1 n
h (0 M
E p A 7 .5 n )
h A (0 M )
.
Ep 8 ( 5 n
h 25 M
Ep B1 0 p )
h B (0 M )
.5
2
Ep ( n
h B 25 M )
Ep 3 0 p
h B (0 . M )
4 1n
(0 M
F A .5 )
FE K nM
R (3 n )
(2 M
5
F
FG E 0 p )
F S M
F G R2 ( 1 n )
F R (2 5 M )
3 0p
F G (2 M
FR 50 )
4 pM
(3 )
nM
)
2
40000
Ep
The results demonstrated that poly GT, poly GAT, and the TK peptide are universal
substrates, whereas the other peptide substrates work only with certain kinases. Due to its
small size, the TK peptide may offer unique advantages in assay development compared
to the large poly GT and poly GAT substrates in regard to assay reproducibility and signal
stability. To our knowledge, this is the first time such extensive analysis of human PTK
activity versus different substrates has been undertaken.
ABL
ABL
FAK
50000
Kinase
ABL
ABL
Determination of Kinase Concentration
Six ULight-labeled PTK substrates were developed. The substrates are poly GT (4:1),
poly GAT(1:1:1), three peptides of 14-18 amino acids in length derived from known
phosphorylated targets: CDK1 (Tyr15), IRS-1 (Tyr983), and JAK-1 (Tyr1023), and a fourth
peptide containing a novel sequence phosphorylated by most tyrosine kinases (TK peptide;
patent pending). These peptide targets were initially validated using PTKs cited in the
literature. For example, the JAK-1 peptide was assayed with the enzyme JAK3 (Wang et al
2003). Our objective was to further evaluate these substrates with a large number of
human PTKs in order to determine the applicability of the substrates to assay other PTKs.
S/B ratios
ACK
ULight-CDK1 (Tyr15)
Peptide sequence: CAGAGKIGEGTYGVVYK
Origin: peptide derived from amino acids 9-20 of human cyclin-dependent kinase 1 (CDK1). The
peptide is a substrate for several Src family kinases (Cheng et al 1992)
7
Profiling Data
Family
Cytoplasmic Tyrosine Kinases
Protein tyrosine kinases (PTK) are
involved in the regulation of many aspects of
the cell cycle, including proliferation,
differentiation, secretion and apoptosis.
Aberrant protein kinase expression or
function is a cause or consequence of many
human
diseases
like
cancer
and
atherosclerosis. As a result, kinases became
very important targets in drug discovery. This
has prompted the development of many
kinase assay technologies such as timeresolved fluorescence energy transfer (TRFRET).
6
The Substrates
ALK
ALK
AXL
DDR
DDR1
DDR
DDR2
EGFR
EGFR
EGFR
EGFR [T790M]
EPH
EphA1
EPH
EphA2
EPH
EphA3
EPH
EphA4
EPH
EphA5
EPH
EphA6
EPH
EphA7
EPH
EphA8
EPH
EphB1
EPH
EphB2
EPH
Receptor Tyrosine Kinases
Introduction
LANCE Signal (665nm)
1
EphB3
EPH
EphB4
FGFR
FGFR1
FGFR
FGFR2
FGFR
FGFR3
FGFR
FGFR4
VEGFR
FLT1
PDGFR
FLT3
VEGFR
FLT4
PDGFR
FMS (CSFR)
EGFR
HER2
EGFR
HER4 (ERBB4)
INSR
IGF1R
INSR
INSR
INSR
IRR
VEGFR
KDR
PDGFR
KIT
ALK
LTK
AXL
MER
MET
MET
MUSK
MUSK
PDGFR
PDGFRa
PDGFR
PDGFRβ
RET
RET
MET
RON
ROR
ROR1
ROR
SEV
ROR2
ROS
TIE
TIE2
TRK
TRKA (NTRK1)
TRK
TRKB (NTRK2)
TRK
TRKC (NTRK3)
AXL
TYRO3
Kinase
concentration
(nM)
ULight -poly GT
ULight -poly GAT
ULight -CDK-1
ULight -IRS-1
ULight -JAK-1
ULight -TK
0.25
0.5
20
0.5
0.5
0.5
3
0.25
20
20
3
0.25
1
0.1
0.5
0.25
0.25
0.5
7
1
0.5
0.5
0.5
1
0.25
0.5
0.5
0.5
0.5
1
3
4
0.25
4
4
4
0.5
0.5
0.5
0.5
0.5
0.25
0.5
0.5
1
0.5
0.5
0.25
0.5
0.25
0.1
0.5
0.5
0.25
0.25
3
0.25
0.1
0.25
0.25
10
0.1
0.25
20
0.5
0.25
2
1
0.25
0.5
1
0.5
0.25
0.5
0.25
20
20
0.25
0.5
0.5
0.5
0.5
0.25
13.1
6.6
27.3
13.2
16.5
13.2
17.4
11.8
10.4
12.6
20.0
12.9
17.1
25.1
20.8
12.8
21.3
9.8
17.6
8.9
6.7
10.4
15.6
18.6
15.5
15.1
18.3
2.3
25.4
9.3
11.0
6.2
15.3
11.1
9.8
9.8
2.0
4.1
1.7
18.1
24.1
12.3
17.4
12.6
18.0
14.2
22.7
16.4
25.1
27.9
29.0
13.7
2.9
7.8
16.0
9.2
22.2
20.8
6.9
14.6
9.3
25.2
21.8
18.3
21.6
8.6
13.0
21.3
29.2
17.0
2.5
17.8
11.2
1.4
27.7
3.4
4.2
23.8
19.3
16.3
22.9
34.1
21.5
11.1
6.4
27.2
12.7
7.8
9.0
3.0
19.6
20.3
18.8
13.0
17.1
19.0
17.7
22.9
10.4
13.2
8.5
16.4
7.9
6.9
16.5
8.9
13.2
7.7
18.4
20.1
4.1
15.4
15.2
18.9
5.3
14.0
15.3
7.2
5.6
3.0
5.1
5.0
10.8
11.4
7.1
8.0
11.3
21.9
15.5
11.1
7.8
7.9
13.2
11.2
4.0
5.0
11.6
10.7
5.6
15.4
13.2
6.5
6.2
8.4
14.4
14.6
25.4
9.1
4.3
21.7
25.1
15.9
13.2
10.6
10.6
12.5
3.2
25.7
1.5
1.4
9.0
9.4
17.5
26.3
22.3
9.1
1.3
1.3
4.8
1.4
9.6
2.2
1.4
3.1
1.7
1.0
1.0
16.1
10.3
4.1
1.3
19.7
12.8
2.5
1.3
7.2
1.3
6.1
14.3
24.0
2.3
16.6
8.0
1.2
2.2
2.4
3.2
1.3
12.8
1.1
2.6
1.5
1.0
1.0
1.8
1.1
3.1
2.1
4.4
6.7
19.2
1.2
1.4
3.1
14.1
13.1
8.3
12.6
1.6
1.6
1.4
1.2
1.1
3.6
1.8
1.2
1.1
1.1
3.1
1.1
2.4
1.3
1.0
2.4
9.7
4.1
1.8
1.3
1.6
1.5
1.3
0.9
1.0
4.4
1.6
5.5
7.0
1.1
4.7
1.1
1.2
1.5
1.2
1.1
1.1
1.3
1.0
1.3
1.0
1.0
9.1
7.2
1.1
1.1
1.0
1.2
1.6
3.7
21.4
6.0
1.0
1.1
1.2
1.1
1.1
1.3
1.8
1.0
1.6
1.2
7.3
1.0
1.1
16.8
1.6
1.1
4.9
2.5
1.2
1.2
1.8
3.5
2.4
7.6
1.1
1.3
1.6
8.4
3.8
2.3
4.8
13.5
15.5
8.1
3.4
1.9
4.5
11.4
1.4
1.1
1.1
15.2
3.2
15.6
5.1
1.0
3.9
5.6
4.5
1.1
2.4
2.0
6.9
4.4
1.0
0.9
20.5
2.1
11.8
11.1
1.7
4.8
1.2
1.3
2.0
1.4
2.6
1.2
1.1
1.5
3.9
1.0
1.0
5.8
4.0
3.2
2.2
2.3
5.1
1.6
19.5
41.8
37.3
1.7
7.4
18.3
1.3
5.9
2.0
20.0
1.8
1.2
1.3
15.2
3.0
6.7
3.9
1.2
1.0
1.0
19.0
3.9
1.2
3.9
3.8
6.4
21.2
1.3
1.5
5.6
10.6
5.7
4.0
12.0
30.8
33.2
27.6
20.8
10.0
25.0
36.9
6.3
1.4
1.5
12.8
1.6
12.7
30.8
3.3
2.7
3.2
19.7
3.9
9.0
3.0
23.6
4.9
1.0
1.0
7.7
5.4
15.1
17.0
1.4
2.1
4.1
4.8
17.3
10.6
31.0
7.1
2.4
6.8
27.2
2.5
1.7
31.0
24.9
29.3
9.7
25.9
29.7
8.4
13.7
35.5
16.1
12.3
32.5
33.1
2.9
31.7
32.5
24.6
18.6
6.8
5.9
7.3
28.0
7.6
8.8
1.8
1.0
1.0
10.0
3.3
10.8
26.9
22.4
31.5
35.5
5.4
17.2
32.3
29.9
28.8
24.7
29.8
26.4
27.1
24.4
17.7
4.1
25.9
20.0
3.0
1.4
3.1
25.1
4.0
19.2
16.9
1.5
13.7
12.9
24.2
3.1
8.0
4.7
20.3
3.5
1.1
1.0
24.5
17.7
23.2
28.0
6.4
6.1
The average S/B ratio was calculated, for each enzyme:substrate pair, from
two independent experiments.
Data Analysis
Kinase representation:
Number of families
Number of kinases
Assay performance:
Receptor
16
49
Cytoplasmic
9
34
Total
25
83
Substrate
Highest S/B ratio obtained
Kinase(s)
ULight -polyGT
ULight -polyGAT
ULight -CDK1
ULight -IRS-1
ULight -JAK-1
ULight -TK
34
27
24
21
42
36
TRKC
ACK
LYNb
JAK2, ROS
JAK2
JAK2, EphA5
Selectivity of the substrates
ULight-poly GT (4:1) & ULight-poly GAT (1:1:1) & ULight-TK
77 kinases had an S/B ratio > 3 for ULight-poly GT.
81 kinases had an S/B ratio > 3 for ULight-poly GAT.
72 kinases had an S/B ratio > 3 for ULight-TK.
ULight-CDK1 (Tyr15)
33 kinases, from 12 families, had an S/B ratio > 3.
10/33 kinases are from the SRC family (cytoplasmic). Only 2/12 kinases (BRK and FRK) from
the SRC family did not phosphorylate the ULight-CDK1 substrate.
9/33 kinases are from the EPH family (receptor).
ULight-IRS-1 (Tyr983)
32 kinases, from 14 families, had an S/B ratio > 3. Only 2 of these families (6 kinases) are of the
cytoplasmic type: JAKA and FER.
ULight-JAK-1 (Tyr1023)
50 kinases, from 19 families, had an S/B ratio > 3.
8
Conclusions
83 human PTKs were assayed with six substrates using the TR-FRET technology. The
general conclusions are:
• It was possible to find at least one substrate for every kinase tested.
• Poly GT, poly GAT, and TK are general substrates for PTKs. They worked with over
87% of PTK enzymes.
• The CDK1 peptide was a selective substrate, as mostly kinases of the SRC and
EPH families phosphorylated this peptide.
• The IRS-1 peptide was also a selective peptide, as mostly kinases of the receptor
type were active with this peptide.
• On the other hand, the JAK-1 peptide was quite generic in that 50 kinases from 19
different families phosphorylated this substrate.
• Kinase concentration should be optimized carefully as
phosphorylated on tyrosines and could interfere in the assays.
9
many PTKs are
References
Braun, S., Raymond, W.E., and Racker, E. (1984) Synthetic tyrosine polymers as
substrates and inhibitors of tyrosine-specific protein kinases. J Biol Chem. 259, 2051-4
Cheng, H.C., Nishio, H., Hatase, O., Ralph, S., and Wang, J.H. (1992) A synthetic
peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine
kinases. J Biol Chem. 267, 9248-56
Garcia, P., Shoelson, S.E., George, S.T., Hinds, D.A., Goldberg, A.R., Miller, W.T.
(1993) Phosphorylation of synthetic peptides containing Tyr-Met-X-Met motifs by
nonreceptor tyrosine kinases in vitro. J Biol Chem. 268, 25146-51
Shoelson, S.E., Chatterjee, S., Chaudhuri, M., White, M.F. (1992) YMXM motifs of
IRS-1 define substrate specificity of the insulin receptor kinase. Proc Natl Acad Sci
USA. 89, 2027-31
S/B ratio > 10
S/B ratio > 5 and < 10
S/B ratio > 3 and < 5
Wang, R., Griffin, P.R., Small, E.C., and Thompson, J.E. (2003) Mechanism of Janus
kinase 3-catalyzed phosphorylation of a Janus kinase 1 activation loop peptide. Arch
Biochem Biophys. 410, 7-15
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