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Development of Fluorescence Polarization and FRET Assays for Tyrosine and Serine

Outline. Development and optimization of a FRET assay for tyrosine kinase 1Reagent profilingComparison of FP and FRET assays for tyrosine kinase 2Sensitivity of FP, FRET and SPA assaysDevelopment and optimization of FP assays for Serine/threonine kinasesFP assay for PKCFP assay for Akt. Comp

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Development of Fluorescence Polarization and FRET Assays for Tyrosine and Serine

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    1. Development of Fluorescence Polarization and FRET Assays for Tyrosine and Serine/Threonine Kinases Dr. Jinzi J. Wu Novartis Institute for Biomedical Research Summit, New Jersey

    2. Outline Development and optimization of a FRET assay for tyrosine kinase 1 Reagent profiling Comparison of FP and FRET assays for tyrosine kinase 2 Sensitivity of FP, FRET and SPA assays Development and optimization of FP assays for Serine/threonine kinases FP assay for PKC FP assay for Akt I will discuss three topics today: I will spend half of my time discussing the last topic: Developing sensitive and robust FP assays for PKC and Akt.I will discuss three topics today: I will spend half of my time discussing the last topic: Developing sensitive and robust FP assays for PKC and Akt.

    3. Fluorescence polarization is another fluorescence technology which is different from time-resolved fluorescence. The principle of this assay is that when a fluorescence-labeled ligand binds to its acceptor molecule, resulting a slow rotation, polarized light source remains polarized. In the presence of a specific inhibitor, more labeled ligand molecules become unbound, resulting in a fast rotation of free ligand molecules, and light becomes depolarized. The advantages of this assay are nonradioactive and homogeneous. Fluorescence polarization is another fluorescence technology which is different from time-resolved fluorescence. The principle of this assay is that when a fluorescence-labeled ligand binds to its acceptor molecule, resulting a slow rotation, polarized light source remains polarized. In the presence of a specific inhibitor, more labeled ligand molecules become unbound, resulting in a fast rotation of free ligand molecules, and light becomes depolarized. The advantages of this assay are nonradioactive and homogeneous.

    4. FP and FRET: Assay diagrams The FP assays presented in my lecture are competitive FP assays.First, you shine polarized light through wells in the plate. In the absence of kinase, fluorescence-labeled phosphotracer binds to Ab, bound tracer rotates slowly and polarized light remains polarized. However, in the presence of kinase, subtrate gets phosphorylated and phosphosubtrate competes with fluorescent tracer for Ab. Unbound tracer rotates rapidly and polarized light becomes depolarized. In the presence of kinase inhibitors, less phosphosubstrate is generated, polarized light still remain polarized. The advatages of FP assays are 1)One component (here is the subtrate) must be labeled; 2) sensitive and robust; 3) less expensive. TR-FRET assay is TR- fluorescence resenance energy transfer assay. In the presence of kinase, Eu-labeled Ab and SA-APC are brought in close proximity through phosphotyrisine-anti-pY Ab and biotin-SA interactions, generating a time-resloved signal at 665 nm via energy transfer from Eu to APC. This assay is sensitive and robust. However, 2 or 3 components must labled.The FP assays presented in my lecture are competitive FP assays.First, you shine polarized light through wells in the plate. In the absence of kinase, fluorescence-labeled phosphotracer binds to Ab, bound tracer rotates slowly and polarized light remains polarized. However, in the presence of kinase, subtrate gets phosphorylated and phosphosubtrate competes with fluorescent tracer for Ab. Unbound tracer rotates rapidly and polarized light becomes depolarized. In the presence of kinase inhibitors, less phosphosubstrate is generated, polarized light still remain polarized. The advatages of FP assays are 1)One component (here is the subtrate) must be labeled; 2) sensitive and robust; 3) less expensive. TR-FRET assay is TR- fluorescence resenance energy transfer assay. In the presence of kinase, Eu-labeled Ab and SA-APC are brought in close proximity through phosphotyrisine-anti-pY Ab and biotin-SA interactions, generating a time-resloved signal at 665 nm via energy transfer from Eu to APC. This assay is sensitive and robust. However, 2 or 3 components must labled.

    5. FRET for TK1: Assay optimization Assays in my presentaion are developed in both 96- (70-200 ul) and 384-well (<70 ul) plates. This slide shows titration of kinase, substrate, SA-APC and Ab. In the opitimzed assay, the concentration of Ab is 0.4 nM and ratio of SA_APV to poly GAT is 1:1.Assays in my presentaion are developed in both 96- (70-200 ul) and 384-well (<70 ul) plates. This slide shows titration of kinase, substrate, SA-APC and Ab. In the opitimzed assay, the concentration of Ab is 0.4 nM and ratio of SA_APV to poly GAT is 1:1.

    6. Filtration, SPA and FRET assays for TK1 In FRET, better S:N ratio was observed with ployGAT. This may result from higher binding affinity of Ab to phospho-polyGAT. In contrast,, polyGT seems to be better substrate in filtration and SPA assays. Sensitivity of FRET assay in terms of concentration of kinase is much better than either filtration or SPA assay.In FRET, better S:N ratio was observed with ployGAT. This may result from higher binding affinity of Ab to phospho-polyGAT. In contrast,, polyGT seems to be better substrate in filtration and SPA assays. Sensitivity of FRET assay in terms of concentration of kinase is much better than either filtration or SPA assay.

    7. FRET for TK1: Reagent profiling Take home message from this slide is that there is no significant difference between Packard, Wallac and Prozyme reagents. Triplicates 0.1% BSATake home message from this slide is that there is no significant difference between Packard, Wallac and Prozyme reagents. Triplicates 0.1% BSA

    8. Summary of the FRET assay of TK1 A high throughput, sensitive, homogeneous tyrosine kinase FRET assay has been developed in 384-well plates. This FRET assay is 30 to 60-fold more sensitive than the filtration and SPA assay. After assay optimization, the cost of reagents (Eu-Ab+SA-APC) has been reduced to 6 per well in 384-well plates with an assay volume of 70 ?l.

    9. FP for TK2: Sensitivity of the LJL Analyst In this experiment, we just mixed fluo-tracer with Ab. LJL Analyst can detect as low as 10 pM tracer. However, reproduciblity becomes an issue when tracer concentratio is less than 50pM.In this experiment, we just mixed fluo-tracer with Ab. LJL Analyst can detect as low as 10 pM tracer. However, reproduciblity becomes an issue when tracer concentratio is less than 50pM.

    10. FP, FRET and SPA for TK2: Enzyme titration Again, assays presesnted here are developed in both 96-and 384- well plates. This slide indicates that sensitivity of FRET and FP ssays sre much better than SPA assay.Again, assays presesnted here are developed in both 96-and 384- well plates. This slide indicates that sensitivity of FRET and FP ssays sre much better than SPA assay.

    11. IC50s in FP, FRET and SPA assays IC50s determined in FP, FRET and SPA assays are consistent despite that theconcentartion of kinase is significantly higher in SPA than in FP and FRET assays.IC50s determined in FP, FRET and SPA assays are consistent despite that theconcentartion of kinase is significantly higher in SPA than in FP and FRET assays.

    12. Summary of FP and FRET assays for TK2 The concentration of fluorescein tracers can be reduced to < 100 pM in development of FP assays with the LJL Analyst. Sensitivities of FRET (1 ng/well) and FP (4 ng/well) assays are much better than SPA assay (>200 ng/well). IC50s determined in all these assay are consistent.

    13. Development of FP assays for S/T kinases Selection of anti-phosphoserine/anti-phosphothreonine antibodies Selection of fluorescein-labeled tracers FP assay for PKC FP assay for Akt We have tested 15 anti-phosphoserine or anti-phosphothreonine antibodies from different sources. None of them worked in the FP assay except an anti-phosphoserine ab. I cant release the clone name and source of this ab now. We will this information in the near future. We have also tested a number of flurescein labeled tracer to select high binding affinity tracers and to reduce propellor effect. Under certain circusmutances, even though the fluorescein tracer binds to Ab, fluorescein moiety still rotates rapidly. This is called propellor effect. I will presnt FP assays for PKC and Akt.We have tested 15 anti-phosphoserine or anti-phosphothreonine antibodies from different sources. None of them worked in the FP assay except an anti-phosphoserine ab. I cant release the clone name and source of this ab now. We will this information in the near future. We have also tested a number of flurescein labeled tracer to select high binding affinity tracers and to reduce propellor effect. Under certain circusmutances, even though the fluorescein tracer binds to Ab, fluorescein moiety still rotates rapidly. This is called propellor effect. I will presnt FP assays for PKC and Akt.

    14. FP for PKC: Enzyme titration In this signal decrease assay, the substrate of PKC is 13-mer peptide with a Km of 0.2 uM. The tracer is the phosphorylated same peptide with fluorescein labeled at N-terminus. Ab used is a specific ab against the phosphpeptide. This slide indicates that 1) phosphotidylserine activates PKC kinase acitivty as observed in other assay formats published in the literature; 2) phosphatidylserine does not compete with the tracer for Ab. pPhosphotidylsereine is one of major conerns for developing FP assays for PKC since it may bind to anti-phosphoserine ab.In this signal decrease assay, the substrate of PKC is 13-mer peptide with a Km of 0.2 uM. The tracer is the phosphorylated same peptide with fluorescein labeled at N-terminus. Ab used is a specific ab against the phosphpeptide. This slide indicates that 1) phosphotidylserine activates PKC kinase acitivty as observed in other assay formats published in the literature; 2) phosphatidylserine does not compete with the tracer for Ab. pPhosphotidylsereine is one of major conerns for developing FP assays for PKC since it may bind to anti-phosphoserine ab.

    15. FP for PKC: Inhibition of kinase activity Inhibition of PKC kinase activity by staurosporin. IC50 is similar to reporte in the lirature?!Inhibition of PKC kinase activity by staurosporin. IC50 is similar to reporte in the lirature?!

    16. FP for PKC: Conclusions A high affinity anti-phosphoserine Ab has been discovered for development of FP assay of PKC. Phosphatidylserine did not interfere with FP assays of PKC. No significant propeller effect was observed. This antibody may recognize a specific phosphopeptide only. However, the FP assay developed with the antibody may be adapted to other serine/threonine kinases since many of they share similar recognition motifs.

    17. Recognition motifs for S/T kinases Substrate recognition moifs for S/T kinases including PKA, PKC, Akt, CaM kinase II. All substrate recognition motifs underlined in this slide contain several positivly charged amino acids, suggesting that these S/T kinase may have some kind of similarity in substrate recognition. One specific peptide substrate can be a very efficient substrate for one kinase and a poor substrate for another kinase. We have exmained if we are able to apply the same peptide, tracer and Ab used in the PKC FP assay to development of the FP assay for other kinases such as Akt. This peptide is good substrat for PKC and a poor substrate for Akt.Substrate recognition moifs for S/T kinases including PKA, PKC, Akt, CaM kinase II. All substrate recognition motifs underlined in this slide contain several positivly charged amino acids, suggesting that these S/T kinase may have some kind of similarity in substrate recognition. One specific peptide substrate can be a very efficient substrate for one kinase and a poor substrate for another kinase. We have exmained if we are able to apply the same peptide, tracer and Ab used in the PKC FP assay to development of the FP assay for other kinases such as Akt. This peptide is good substrat for PKC and a poor substrate for Akt.

    18. FP for Akt: Tracer and antibody titration A few things I want to pint out:1) signal in mP is independent of tracer concentrastion up 250 pM; 2) signal increases as Ab concentration of antibody increases. At 250 pM tracer and 400 pM Ab, difference bwteewn plus and minus Ab is120 mP, suggesting that this is a high affinity Ab since in many FP assays, a signal of 140 mP often requires 10-fold excess of antibody or receptors.A few things I want to pint out:1) signal in mP is independent of tracer concentrastion up 250 pM; 2) signal increases as Ab concentration of antibody increases. At 250 pM tracer and 400 pM Ab, difference bwteewn plus and minus Ab is120 mP, suggesting that this is a high affinity Ab since in many FP assays, a signal of 140 mP often requires 10-fold excess of antibody or receptors.

    19. FP for Akt: Specificity of antibody

    20. FP for Akt: Substrate and enzyme titration Next question we asked is whether IC50s of known inhbiotrs of Akt determinde in FP assay is similar to tthose determinde in other assay format since the substrate used in FP assay of Akt is a poor substrate. Next question we asked is whether IC50s of known inhbiotrs of Akt determinde in FP assay is similar to tthose determinde in other assay format since the substrate used in FP assay of Akt is a poor substrate.

    21. FP and SPA for Akt: Inhibition curves Subtrate use in FP assay is poor substrate for Akt; substrate used in SPA assay, called Crosstide, is quite good substrate for Akt with a Km of 4 uM. IC50s obtained with both peptide substrates are consistent. Though Crosstide is much better substrate for Akt, the amount of kinase needed for the FP assay is still 40-fold less than that for SPA assay, suggesting that FP assay is more sensitive than SPA assay.Subtrate use in FP assay is poor substrate for Akt; substrate used in SPA assay, called Crosstide, is quite good substrate for Akt with a Km of 4 uM. IC50s obtained with both peptide substrates are consistent. Though Crosstide is much better substrate for Akt, the amount of kinase needed for the FP assay is still 40-fold less than that for SPA assay, suggesting that FP assay is more sensitive than SPA assay.

    22. Summary of FP assay for Akt FP assay for Akt has been developed in 384-well plates using the same peptide substrate and anti-phosphoserine antibody as those used for PKC. Concentration of the substrate is critical in development of FP assays due to specificity of the antibody. Sensitivity of FP assay is >40-fold higher than that of SPA assay. IC50s of staurosporine determined in both FP and SPA assays are consistent. Cost of reagents in the FP assay (10 per well in 384-well plates with a volume of 70 ?l) is less than that in the SPA assay.

    23. Acknowledgments Donna Yarwood Quynhchi Pham Michael Chin Dr. Robert Schweitzer Dr. Matthew Sills

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