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 PerkinElmer Life and Analytical Sciences, 940 Winter Street, Waltham, MA USA (800) 762-4000 (+1) 203 925-4602 www.perkinelmer.com