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JAK-STAT signalling

JAK-STAT signalling. type I cytokine receptor. extracellular. intracellular. Y Y Y. Y Y Y. Y. JAK. JAK. JAK kinase. Y. P. Y Y Y. Y Y Y. Y. JAK. JAK. Y. P. JAK-STAT signalling. cytokine. P. Y Y Y. Y Y Y. Y. JAK. JAK. Y. P. P. P. P. P. P. P.

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JAK-STAT signalling

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  1. JAK-STAT signalling type I cytokine receptor extracellular intracellular Y Y Y Y Y Y Y JAK JAK JAK kinase Y

  2. P Y Y Y Y Y Y Y JAK JAK Y P JAK-STAT signalling cytokine

  3. P Y Y Y Y Y Y Y JAK JAK Y P P P P P P P JAK-STAT signalling

  4. Y JAK JAK Y P P Y P P P P Y JAK-STAT signalling P Y Y Y Y Y Y P STAT

  5. Y JAK JAK Y P P Y Y P P P P P P Y Y P P JAK-STAT signalling P Y Y Y Y Y Y P cytoplasm nucleus STAT responsive promoter

  6. MAPPIT cytokine receptor F F F F F F Y JAK2 JAK2 Y leptin receptor (Y>F)

  7. P P MAPPIT ligand F F F F F F Y JAK2 JAK2 Y

  8. P P MAPPIT ligand F F F F F F Y JAK2 JAK2 Y

  9. P P MAPPIT ligand F F F F F F Y JAK2 JAK2 Y bait

  10. P P MAPPIT ligand F F F F F F Y JAK2 JAK2 Y Y Y Y Y gp130 prey

  11. ligand P F F F F F F Y JAK2 JAK2 Y P Y Y Y Y P P P P MAPPIT

  12. P P Y P P P Y P MAPPIT ligand F F F F F F Y JAK2 JAK2 Y Y Y Y Y STAT3

  13. P P P Y Y P P P Y Y P MAPPIT ligand F F F F F F Y JAK2 JAK2 Y P Y Y Y Y P P rPAP1 promoter reporter gene

  14. P P P Y Y P P P Y Y P P P MAPPIT ligand F F F F F F Y JAK2 JAK2 Y Y Y Y Y P rPAP1 promoter reporter gene

  15. MAPPIT assets • operates in intact human cells, a close to normal physiological context • works in different cell types (epithelial, haematopoietic, neuronal) • tight background control • interaction and effector zone are separated • ligand-inducible system • easy to perform, simple readout, can be automated

  16. MAPPIT constitutes a toolbox of related interaction mapping technologies • MAPPIT (2H): protein-protein interaction -> protein interaction mapping • ReverseMAPPIT (R2H): disruption of protein-protein interaction -> compound screening • MASPIT (3H): compound-protein interaction -> compound target profiling

  17. MAPPIT toolbox can be applied in a drug discovery pipeline target interaction lead compound optimised lead compound MAPPIT Reverse MAPPIT MASPIT protein

  18. P P MASPIT Epo EpoR F F F F F F Y JAK2 JAK2 Y LR-F3 DHFR

  19. P P MASPIT Epo F F F F F F Y JAK2 JAK2 Y compound PEG linker methotrexate

  20. P P Y Y Y Y MASPIT Epo F F F F F F Y JAK2 JAK2 Y Y Y Y Y

  21. P P P P Y Y P Y Y P P P P P P P Y Y P P P P MASPIT Epo F F F F F F Y JAK2 JAK2 Y Y Y Y Y Y Y Y Y rPAP1 promoter luciferase

  22. MASPIT

  23. Fold induction MFC concentration(µM) MASPIT

  24. Fold induction MFC concentration(µM) Fold induction Time (hours) Fold induction pMG-CDK2 plasmid (mg) gp-130-CDK2 MASPIT

  25. MFC: RGB-286649 Target : DHFR MFC: RGB-286649 MFC: RGB-286649 Target : Abl Target : Abl Epo % Control % Control % Control P F F F F F F Y JAK2 JAK2 P [Methotrexate] µM Y [Imatinib] µM [PD-173955] µM Y Y Y Y Abl-prey PD173955 methotrexate EC50 0.07µM EC50 0.03µM MASPIT competition assays affinity determination in intact cells >> MASPIT EC50 better reflects reality than IC50 obtained with in vitro assays (e.g. compound uptake, interference of other proteins with binding)

  26. P P MASPIT screening with PD173955 • all identified preys are kinases • known (Abl, Lyn, Fyn, FGFR1) and novel (Eph, GAK) • validated with in vitro kinase activity assays Epo F F F F F F Y JAK2 JAK2 Y PD173955 methotrexate

  27. MAPPIT validation of Y2H protein network maps

  28. empirical confidence score from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009

  29. empirical confidence score from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009

  30. mapping the human interactome • 3 year NIH grant • Y2H: 16.000 x 16.000 full lenght human ORFs (~ 50% of total matrix of 22.000 x 22.000) • interaction toolkit re-test: ~25-30.000 interactions (~10.000/year; ~20% of the map)

  31. benchmarking binary interaction mapping methods >> MAPPIT performance is similar to that of the other tested methods from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009

  32. benchmarking binary interaction mapping methods >> the interaction mapping methods are highly complementary from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009

  33. hIL5Rα hIL5Rα Y Y CMV 5’LTR CD90 gp130 prey CMV LR-F3 bait rPAP1 hIL5RαΔcyt MAPPIT cDNA library screening MACS enrichment anti-PE magnetobead FACS sort anti-mIgG-PE Y Y retroviral prey cDNA library Y Y anti-hIL5Rα mEcoR

  34. towards an efficient screening format: reverse transfection

  35. ArrayMAPPIT screening prey (+reporter) plasmid human ORFeome collection transfection reagent reverse transfection mix -/+ ligand MAPPIT prey collection MAPPIT bait cell line luciferase read-out MAPPIT prey array (stable for months !)

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