1 / 35

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.

penn
Download Presentation

JAK-STAT signalling

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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 !)

More Related