1. Hsp90-binding Immunophilins Link p53 to Dynein During p53 Transport to Nucleus M. Galigniana, J. Harrell, H. O’Hagen, M. Ljungman, W. Pratt

2. Overview Important Terms Introduction/Background Results Discussion How this relates to cancer

3. Important Terms MicrotubuleDyneinDynactinDynamitinp53Hsp90Immunophilin Glucocorticoid receptor

4. Background-Microtubule Part of the cytoskeleton Serve as structural components of the cell Involved in cellular processes such as: MitosisCytokinesis Vesicle Transport + and - ends which are attributed to their polarity The eukaryotic cytoskeleton. Microtubules in green, and the nuclei are in blue.

5. Microtubules, cont. Motor proteins, such as dynein, move along microtubules in a highly regulated manner Motor proteins bind to and transport cargo

6. Background-Dynein Molecular motor that moves proteins towards the nucleus Two identical heavy chains- contain the ATPase activity responsible for generating movement along the microtubuleATPase Two intermediate chains which to anchor the dynein to its cargo Several light chains Moves processively along microtubules, with one stalk always in contact with the microtubule

7. Background-Dynactin Dynein activator complex that stimulates transport Projecting sidearm that interacts with dynein and an actin-like minifilament backbone that is thought to bind cargo Contains dynamitin subunit that regulates microtubule-dependent motor function

8. Background-Dynamitin Dynamitin is a subunit of the dynein-dynactin complex Dynamitin overexpression blocks dynein function by dissociating dynein from its cargo

9. Background-p53 Transcription factor that acts as a tumor suppressor

10. p53, cont. Induces cell growth arrest, apoptosis, cell differentiation, and DNA repair in response to DNA damage Mutations in p53 contribute to ~2/3 of all human cancers Moves between cytoplasm and nucleus

11. Background-Hsp90 Ubiquitous molecular chaperone Involved in cell signaling, protein folding, and tumor repression Cytoplasmic, globular protein 3 important binding regions: ATP binding, protein binding, and dimerizing regions protein

12. Background-Immunophilins Receptors for immunosuppressive drugs (ex- FKBP and Cyclosporin A) Contain tetratricopeptide repeat (TPR) domains that interact with TPR site on hsp90 Contain PPIase domain involved in protein folding process PPIase domain functions as protein-protein binding domain to link GR-hsp90 complex to dynein TPR domain PPIase domain

13. Introduction p53 is transported to the nucleus along microtubules by dynein The p53 protein shuttles between the cytoplasm and the nucleus Exclusion of p53 from the nucleus may lead to its inactivation Some p53 mutants isolated to the cytoplasm were found to complex with hsp90

14. Introduction Complex formation between hsp90 and “client” proteins stabilizes the “client” protein This stabilization targets the “client” protein for degradation by proteolysis Complex formation between hsp90 and immunophilins is also required for movement of the GR

15. Introduction GR-hsp90 complexes with: –Either one of four TPR and PPIase domain immunophilins (FKBP52, FKBP51, CyP-40, or PP5) –Dynein –hsp70 –p23

16. Introduction GR-hsp90 heterocomplexes contain cytoplasmic dynein Previous work: GR links dynein motor, but may/may not be dynein dependent. p53 movement to nucleus is dynein dependent, but how are the two complexed together?

18. Purpose Using GR-hsp90 complex as model, is it possible to determine method of linkage of p53 to dynein-dependent transport to nucleus?

19. How did they do it? DLD-1 human colon cancer cells and HT29-tsp53 cells that contained a temperature sensitive p53 mutant Utilized immunoadsorption assays and Western blots to visualize proteins Fluorescence microscopy to visualize movement of complexes into the nucleus through transfection and incubation

20. Immunoadsorption

21. Western Blot (Immunoblot) Method used to detect proteins Procedure: –Obtain protein(s) of interest –Use gel electrophoresis to separate denatured proteins by weight (smaller proteins at the bottom) –Transfer to nitrocellulose membrane –Probe/label proteins with specific antibody

23. Results Composition of p53-hsp90 Heterocomplexes in DLD-1 Cells DLD-1 cells have point mutation of Ser- 241 of p53 to Phe Mutant is localized in cytoplasm and complexes with hsp90 Immunoadsorbed p53 and GR from DLD-1 cytosol Identified proteins by immunoblotting

24. Galigniana, M. D. et al. J. Biol. Chem. 2004;279:22483-22489 Immunoadsorption of p53 and GR heterocomplexes

25. p53 and GR Immunoblots Conclusions: –Same complexes of proteins are co- immunoadsorbed with p53 and GR

26. Results Immunophilins Link the p53-hsp90 Complex to Dynein Reconstitution of p53-hsp90-immunophilin- dynein complexes by rabbit reticulocyte lysate In vivo, reticulocytes are highly specialized and synthesize hemoglobin Hemoglobin represents 90% of protein made by reticulocytes Immature red blood cells * Reticulocyte lysate contains hsp90/hsp70 chaperone machinery that assembles client protein-hsp90 complexes, and GR-hsp90 complexes; it also contains immunophilins and dynein*

27. Results Immunophilins Link the p53-hsp90 Complex to Dynein Procedure: Immnuonadsorbed p53 with an associated antibody, precipitated it, incubated different fractions in varying combinations of rabbit reticulocyte lysate, differentiated proteins associated with p53-hsp90 heterocomplex, and analyzed results using electrophoresis and immunoblotting

28. Galigniana, M. D. et al. J. Biol. Chem. 2004;279:22483-22489 Effects of geldanamycin and competition with TPR and PPIase domain peptides on p53 heterocomplexes assembled by reticulocyte lysate

29. Results Immunophilins Link the p53-hsp90 Complex to Dynein Conclusions: –p53 complexes with hsp90, dynein, PP5, FKBP52 and CyP-40 in vitro (Lane 5) –In presence of hsp90 inhibitor, no immunophilins or dynein associated with p53 (Lane 6) –TPR fragments of immunophilins compete for binding to p53-hsp90; p53-hsp90 complexes form that lack dynein and immunophilins (Lane 7)

30. Results Immunophilins Link the p53-hsp90 Complex to Dynein Conclusions, cont. –When PPIase domain fragments of immunophilins are present, p53-hsp90-immunophilin complexes are formed that lack dynein (Lane 8) –Thus immunophilins link p53-hsp90 complex to dynein in vitro

31. Results : Radicicol (RAD) Inhibits p53 Transport to the nucleus Used colon carcinoma cells HT29-tsp53 that express a temperature sensitive mutant of p53 Mutant is active at 32 o C, inactive at 39 o C and is isolated in the cytoplasm Mutant switches from mutant form to wt under permissive temperature conditions Radicicol=hsp90 inhibitor *RAD was used instead of geldanamycin because geldanamycin produces hydrogen peroxide in vivo which stimulates nuclear translocation*

32. Results : Radicicol (RAD) Inhibits p53 Transport to the nucleus Procedure: Transfected cells with temperature sensitive mutant and monitored movement of p53 into the nucleus at different time intervals in the presence and absence of a hsp90 inhibitor (moved from inactive temp. to permissive temp.)

33. Transfection The introduction of foreign material into a eukaryotic cell

34. Galigniana, M. D. et al. J. Biol. Chem. 2004;279:22483-22489 p53 translocation to the nucleus is impeded by radicicol

35. Results : Radicicol (RAD) Inhibits p53 Transport to the nucleus Take home messages: –In absence of RAD, all of p53 mutant has moved to nucleus after 60 min at permissive temp. –The presence of RAD inhibits, but does not block movement of p53 into the nucleus *Some studies show that 20uM of RAD will inhibit nuclear translocation, but HT29-tsp53 cells do not respond to concentrations that high.*

36. Results: Inhibition of p53 Transport by Expression of a PPIase Domain Fragment Procedure: HT29-tsp53 cells were incubated at 32 o C on a coverslip, they were fixed, permeabilized and stained with antibodies either against p53 or myc (which they used as a tag for dynamitin) and nuclear translocation was monitored by fluorescence microscopy

37. Galigniana, M. D. et al. J. Biol. Chem. 2004;279:22483-22489 Overexpression of dynamitin inhibits p53 movement to the nucleus

38. Galigniana, M. D. et al. J. Biol. Chem. 2004;279:22483-22489 Overexpression of the PPIase domain I fragment of FKBP52 inhibits p53 movement to the nucleus

39. Results: Inhibition of p53 Transport by Expression of a PPIase domain Fragment Take home messages: –Overexpression of dynamitin and the PPIase domain fragment of FKBP52 inhibited p53 movement to the nucleus –The PPIase domain fragment competes for binding of p53- hsp90-immunophilin complex to dynein and its overexpression inhibits p53 movement to the nucleus Overexpression of dynamitinOverexpression of the PPIase domain

40. Results: Immunophilin Interaction with Dynamitin Test to see if dynamitin binds directly to the immunophilin PPIase domain-if it does, it should be present in immunoadsorbed p53 heterocomplexes

41. Results: Immunophilin Interaction with Dynamitin Procedure: transfected DLD-1 cells with myc-dynamitin, immunoadsorbed p53 complexes, then probed Western blots with an anti-myc antibody to detect dynamitin

42. Galigniana, M. D. et al. J. Biol. Chem. 2004;279:22483-22489 Dynamitin binding to immunophilins

43. Results: Immunophilin Interaction with Dynamitin Conclusions: –Dynamitin gets co-immunoadsorbed with p53 and its presence is eliminated by competition with PPIase domain fragment of FKBP52 –Stripped dynamitin binds hsp90 binding immunophilins, but immunophilins do not bind in presence of competitor PPIase domains –Stripped dynamitin binds FKBP52, but binding is in competition with the PPIase domain suggesting a direct interaction between PPIase domains and dynamitin

44. Discussion Major Points argued: –Immunophilins link p53-hsp90 complex to dynein in vitro through TPR and PPIase domains TPR domainPPIase domain

45. Discussion, cont. –Prohibiting that interaction by overexpression of PPIase domain fragments inhibits p53 movement in vivo PPIase expression

46. Galigniana, M. D. et al. J. Biol. Chem. 2004;279:22483-22489 TPR domain immunophilins link the p53{middle dot}hsp90 heterocomplex to dynein for retrograde movement along microtubules

47. How does this relate to cancer progression? p53 is a transcription factor that acts as a tumor suppressor Translocation into the nucleus is imperative for subsequent p53 interactions

48. Cancer Progression, cont. p53 is activated upon DNA damage p53 is activated upon DNA damage DNA damage stimulates either a cell cycle arrest or apoptosis

49. Cancer Progression, cont. If p53 is not transported to the nucleus by dynein, it remains in the cytoplasm and can not perform its function as a transcription factor and tumor suppressor If DNA damage occurs, p53 exclusion from the nucleus would not be able to regulate/correct it

50. Cancer Progression, cont. Cell cycle progression would continue, damaged DNA would become replicated, and cell/tumor proliferation would occur p53

51. Questions?