Hsp90-binding Immunophilins Link p53 to Dynein During p53 Transport to Nucleus

Hsp90-binding Immunophilins Hsp90-binding Immunophilins Link p53 to Dynein During p53 Link p53 to Dynein During p53

Transport to NucleusTransport to Nucleus

M. Galigniana, J. M. Galigniana, J. Harrell, H. O’Hagen, M. Harrell, H. O’Hagen, M.

Ljungman, W. PrattLjungman, W. Pratt

OverviewOverview

•Important Terms

•Introduction/Background

•Results

•Discussion

•How this relates to cancer

Important TermsImportant Terms

MicrotubuleMicrotubuleDyneinDyneinDynactinDynactinDynamitinDynamitinp53p53Hsp90Hsp90ImmunophilinImmunophilinGlucocorticoid receptorGlucocorticoid receptor

Background-MicrotubuleBackground-Microtubule

Part of the cytoskeleton Part of the cytoskeleton

Serve as structural Serve as structural components of the cellcomponents of the cell

Involved in cellular Involved in cellular processes such as: processes such as:

MitosisMitosis

CytokinesisCytokinesis

Vesicle Transport Vesicle Transport

++ and and -- ends which are ends which are attributed to their polarityattributed to their polarity

The eukaryotic cytoskeleton. Microtubules in green, and the nuclei are in blue.

Microtubules, cont.Microtubules, cont.

Motor proteins, Motor proteins, such as dynein, such as dynein, move along move along microtubules in a microtubules in a highly regulated highly regulated mannermanner

Motor proteins bind Motor proteins bind to and transport to and transport cargocargo

Background-DyneinBackground-Dynein

•Molecular motor that moves proteins towards the nucleus

•Two identical heavy chains-contain the ATPase activity responsible for generating movement along the microtubule

•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

Background-DynactinBackground-Dynactin

Dynein activator complex Dynein activator complex that stimulates transportthat stimulates transport

Projecting sidearm that Projecting sidearm that interacts with dynein and interacts with dynein and an actin-like minifilament an actin-like minifilament backbone that is thought backbone that is thought to bind cargoto bind cargo

Contains dynamitin Contains dynamitin subunit that regulates subunit that regulates microtubule-dependent microtubule-dependent motor functionmotor function

Background-DynamitinBackground-Dynamitin

Dynamitin is a subunit Dynamitin is a subunit of the dynein-dynactin of the dynein-dynactin complexcomplex

Dynamitin Dynamitin overexpression blocks overexpression blocks dynein function by dynein function by dissociating dynein dissociating dynein from its cargo from its cargo

Background-p53Background-p53

Transcription factor that acts as a tumor Transcription factor that acts as a tumor suppressor suppressor

p53, cont.p53, cont.

Induces cell growth Induces cell growth arrest, apoptosis, cell arrest, apoptosis, cell differentiation, and DNA differentiation, and DNA repair in response to DNA repair in response to DNA damagedamage

Mutations in p53 Mutations in p53 contribute to ~2/3 of all contribute to ~2/3 of all human cancershuman cancers

Moves between Moves between cytoplasm and nucleuscytoplasm and nucleus

Background-Hsp90Background-Hsp90

Ubiquitous molecular Ubiquitous molecular chaperonechaperone

Involved in cell signaling, Involved in cell signaling, protein folding, and tumor protein folding, and tumor repressionrepression

Cytoplasmic, globular Cytoplasmic, globular proteinprotein

3 important binding 3 important binding regions: ATP binding, regions: ATP binding, protein binding, and protein binding, and dimerizing regionsdimerizing regions

protein

Background-ImmunophilinsBackground-ImmunophilinsReceptors for Receptors for immunosuppressive drugs (ex-immunosuppressive drugs (ex-FKBP and Cyclosporin A)FKBP and Cyclosporin A)

Contain tetratricopeptide repeat Contain tetratricopeptide repeat (TPR) domains that interact with (TPR) domains that interact with TPR site on hsp90TPR site on hsp90

Contain PPIase domain involved Contain PPIase domain involved in protein folding processin protein folding process

PPIase domain functions as PPIase domain functions as protein-protein binding domain to protein-protein binding domain to link GR-hsp90 complex to dyneinlink GR-hsp90 complex to dynein

TPR domainPPIase domain

IntroductionIntroduction

p53 is transported to the nucleus along p53 is transported to the nucleus along microtubules by dyneinmicrotubules by dyneinThe p53 protein shuttles between the The p53 protein shuttles between the cytoplasm and the nucleuscytoplasm and the nucleusExclusion of p53 from the nucleus may Exclusion of p53 from the nucleus may lead to its inactivationlead to its inactivationSome p53 mutants isolated to the Some p53 mutants isolated to the cytoplasm were found to complex with cytoplasm were found to complex with hsp90hsp90


Complex formation between hsp90 and Complex formation between hsp90 and “client” proteins stabilizes the “client” “client” proteins stabilizes the “client” proteinprotein

This stabilization targets the “client” This stabilization targets the “client” protein for degradation by proteolysisprotein for degradation by proteolysis

Complex formation between hsp90 and Complex formation between hsp90 and immunophilins is also required for immunophilins is also required for movement of the GRmovement of the GR

Introduction Introduction

GR-hsp90 complexes with:GR-hsp90 complexes with:– Either one of four TPR and PPIase domain Either one of four TPR and PPIase domain

immunophilins (FKBP52, FKBP51, CyP-40, or PP5)immunophilins (FKBP52, FKBP51, CyP-40, or PP5)– DyneinDynein– hsp70hsp70– p23p23


GR-hsp90 heterocomplexes contain GR-hsp90 heterocomplexes contain cytoplasmic dyneincytoplasmic dynein

Previous work: GR links dynein motor, but Previous work: GR links dynein motor, but may/may not be dynein dependent. p53 may/may not be dynein dependent. p53 movement to nucleus is dynein movement to nucleus is dynein dependent, but how are the two dependent, but how are the two complexed together? complexed together?

PurposePurpose

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

How did they do it?How did they do it?

DLD-1 human colon cancer cells and DLD-1 human colon cancer cells and HT29-tsp53 cells that contained a HT29-tsp53 cells that contained a temperature sensitive p53 mutanttemperature sensitive p53 mutant

Utilized immunoadsorption assays and Utilized immunoadsorption assays and Western blots to visualize proteins Western blots to visualize proteins

Fluorescence microscopy to visualize Fluorescence microscopy to visualize movement of complexes into the nucleus movement of complexes into the nucleus through transfection and incubation through transfection and incubation

Immunoadsorption Immunoadsorption

Western Blot (Immunoblot) Western Blot (Immunoblot)

Method used to detect proteinsMethod used to detect proteins

Procedure:Procedure:– Obtain protein(s) of interestObtain protein(s) of interest– Use gel electrophoresis to separate Use gel electrophoresis to separate

denatured proteins by weight (smaller denatured proteins by weight (smaller proteins at the bottom)proteins at the bottom)

– Transfer to nitrocellulose membraneTransfer to nitrocellulose membrane– Probe/label proteins with specific antibody Probe/label proteins with specific antibody

ResultsResultsComposition of p53-hsp90 Heterocomplexes Composition of p53-hsp90 Heterocomplexes

in DLD-1 Cellsin DLD-1 Cells

DLD-1 cells have point mutation of Ser-DLD-1 cells have point mutation of Ser-241 of p53 to Phe241 of p53 to Phe

Mutant is localized in cytoplasm and Mutant is localized in cytoplasm and complexes with hsp90complexes with hsp90

Immunoadsorbed p53 and GR from DLD-1 Immunoadsorbed p53 and GR from DLD-1 cytosolcytosol

Identified proteins by immunoblottingIdentified proteins by immunoblotting

Galigniana, M. D. et al. J. Biol. Chem. 2004;279:22483-22489

Immunoadsorption of p53 and GR heterocomplexes

p53 and GR Immunoblotsp53 and GR Immunoblots

Conclusions: Conclusions: – Same complexes Same complexes

of proteins are co-of proteins are co-immunoadsorbed immunoadsorbed with p53 and GRwith p53 and GR

ResultsResultsImmunophilins Link the p53-hsp90 Complex Immunophilins Link the p53-hsp90 Complex

to Dyneinto Dynein

Reconstitution of p53-hsp90-immunophilin-Reconstitution of p53-hsp90-immunophilin-dynein complexes by rabbit reticulocyte lysate dynein complexes by rabbit reticulocyte lysate In vivoIn vivo, reticulocytes are highly specialized and , reticulocytes are highly specialized and synthesize hemoglobinsynthesize hemoglobinHemoglobin represents 90% of protein made by Hemoglobin represents 90% of protein made by reticulocytesreticulocytesImmature red blood cellsImmature red blood cells* * Reticulocyte lysate contains hsp90/hsp70 chaperone Reticulocyte lysate contains hsp90/hsp70 chaperone machinery that assembles client protein-hsp90 machinery that assembles client protein-hsp90 complexes, and GR-hsp90 complexes; it also contains complexes, and GR-hsp90 complexes; it also contains immunophilins and dynein*immunophilins and dynein*


to Dyneinto DyneinProcedure: Immnuonadsorbed p53 with Procedure: Immnuonadsorbed p53 with an associated antibody, precipitated it, an associated antibody, precipitated it, incubated different fractions in varying incubated different fractions in varying combinations of rabbit reticulocyte lysate, combinations of rabbit reticulocyte lysate, differentiated proteins associated with differentiated proteins associated with p53-hsp90 heterocomplex, and analyzed p53-hsp90 heterocomplex, and analyzed results using electrophoresis and results using electrophoresis and immunoblottingimmunoblotting


Effects of geldanamycin and competition with TPR and PPIase domain peptides on p53 heterocomplexes assembled by reticulocyte lysate


to Dyneinto DyneinConclusionsConclusions::– p53 complexes with hsp90, p53 complexes with hsp90,

dynein, PP5, FKBP52 and dynein, PP5, FKBP52 and CyP-40 CyP-40 in vitroin vitro (Lane 5) (Lane 5)

– In presence of hsp90 In presence of hsp90 inhibitor, no immunophilins inhibitor, no immunophilins or dynein associated with or dynein associated with p53 (Lane 6)p53 (Lane 6)

– TPR fragments of TPR fragments of immunophilins compete for immunophilins compete for binding to p53-hsp90; p53-binding to p53-hsp90; p53-hsp90 complexes form that hsp90 complexes form that lack dynein and lack dynein and immunophilins (Lane 7)immunophilins (Lane 7)


to Dyneinto DyneinConclusions, cont.Conclusions, cont.– When PPIase domain fragments of immunophilins When PPIase domain fragments of immunophilins

are present, p53-hsp90-immunophilin complexes are are present, p53-hsp90-immunophilin complexes are formed that lack dynein (Lane 8)formed that lack dynein (Lane 8)

– Thus immunophilins link p53-hsp90 complex to Thus immunophilins link p53-hsp90 complex to dynein dynein in vitroin vitro

ResultsResults: : Radicicol (RAD) Inhibits p53 Transport to Radicicol (RAD) Inhibits p53 Transport to

the nucleusthe nucleusUsed colon carcinoma cells HT29-tsp53 that Used colon carcinoma cells HT29-tsp53 that express a temperature sensitive mutant of p53express a temperature sensitive mutant of p53

Mutant is active at 32Mutant is active at 32oo C, inactive at 39 C, inactive at 39o o C and is C and is isolated in the cytoplasmisolated in the cytoplasm

Mutant switches from mutant form to wt under Mutant switches from mutant form to wt under permissive temperature conditionspermissive temperature conditions

Radicicol=hsp90 inhibitorRadicicol=hsp90 inhibitor*RAD was used instead of geldanamycin because *RAD was used instead of geldanamycin because geldanamycin produces hydrogen peroxide in vivo which geldanamycin produces hydrogen peroxide in vivo which stimulates nuclear translocation*stimulates nuclear translocation*


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

TransfectionTransfection

The introduction of foreign material into a The introduction of foreign material into a eukaryotic celleukaryotic cell


p53 translocation to the nucleus is impeded by radicicol


the nucleusthe nucleusTake home messages:Take home messages:– In absence of RAD, all of In absence of RAD, all of

p53 mutant has moved to p53 mutant has moved to nucleus after 60 min at nucleus after 60 min at permissive temp.permissive temp.

– The presence of RAD The presence of RAD inhibits, but does not inhibits, but does not block movement of p53 block movement of p53 into the nucleusinto the nucleus

*Some studies show that 20uM of RAD will inhibit nuclear translocation, but *Some studies show that 20uM of RAD will inhibit nuclear translocation, but HT29-tsp53 cells do not respond to concentrations that high.*HT29-tsp53 cells do not respond to concentrations that high.*

Results:Results: Inhibition of p53 Transport by Expression of Inhibition of p53 Transport by Expression of

a PPIase Domain Fragmenta PPIase Domain Fragment

Procedure: HT29-tsp53 cells were Procedure: HT29-tsp53 cells were incubated at 32incubated at 32oo C on a coverslip, they C on a coverslip, they were fixed, permeabilized and stained with were fixed, permeabilized and stained with antibodies either against p53 or antibodies either against p53 or mycmyc (which they used as a tag for dynamitin) (which they used as a tag for dynamitin) and nuclear translocation was monitored and nuclear translocation was monitored by fluorescence microscopy by fluorescence microscopy


Overexpression of dynamitin inhibits p53 movement to the nucleus


Overexpression of the PPIase domain I fragment of FKBP52 inhibits p53 movement to the nucleus

Results:Results: Inhibition of p53 Transport by Expression of Inhibition of p53 Transport by Expression of

a PPIase domain Fragmenta PPIase domain FragmentTake home messages:Take home messages:– Overexpression of dynamitin and the PPIase domain Overexpression of dynamitin and the PPIase domain

fragment of FKBP52 inhibited p53 movement to the nucleusfragment of FKBP52 inhibited p53 movement to the nucleus

– The PPIase domain fragment competes for binding of p53-The PPIase domain fragment competes for binding of p53-hsp90-immunophilin complex to dynein and its hsp90-immunophilin complex to dynein and its overexpression inhibits p53 movement to the nucleusoverexpression inhibits p53 movement to the nucleus

Overexpression of dynamitinOverexpression of the PPIase domain

Results:Results: Immunophilin Interaction with DynamitinImmunophilin Interaction with Dynamitin

Test to see if dynamitin binds directly to Test to see if dynamitin binds directly to the immunophilin PPIase domain-if it does, the immunophilin PPIase domain-if it does, it should be present in immunoadsorbed it should be present in immunoadsorbed p53 heterocomplexesp53 heterocomplexes


Procedure: transfected DLD-1 cells with Procedure: transfected DLD-1 cells with mycmyc-dynamitin, immunoadsorbed p53 -dynamitin, immunoadsorbed p53 complexes, then probed Western blots complexes, then probed Western blots with an anti-with an anti-myc myc antibody to detect antibody to detect dynamitindynamitin


Dynamitin binding to immunophilins


Conclusions: Conclusions: – Dynamitin gets co-immunoadsorbed with p53 Dynamitin gets co-immunoadsorbed with p53

and its presence is eliminated by competition and its presence is eliminated by competition with PPIase domain fragment of FKBP52with PPIase domain fragment of FKBP52

– Stripped dynamitin binds hsp90 binding Stripped dynamitin binds hsp90 binding immunophilins, but immunophilins do not bind immunophilins, but immunophilins do not bind in presence of competitor PPIase domainsin presence of competitor PPIase domains

– Stripped dynamitin binds FKBP52, but binding Stripped dynamitin binds FKBP52, but binding is in competition with the PPIase domain is in competition with the PPIase domain suggesting a direct interaction between suggesting a direct interaction between PPIase domains and dynamitin PPIase domains and dynamitin

DiscussionDiscussionMajor Points argued:Major Points argued:– Immunophilins link Immunophilins link

p53-hsp90 complex to p53-hsp90 complex to dynein dynein in vitro in vitro through through TPR and PPIase TPR and PPIase domainsdomains

TPR domain PPIase domain

Discussion, cont.Discussion, cont.

– Prohibiting that interaction by overexpression of Prohibiting that interaction by overexpression of PPIase domain fragments inhibits p53 movementPPIase domain fragments inhibits p53 movement in in vivovivo

PPIase expression


TPR domain immunophilins link the p53{middle dot}hsp90 heterocomplex to dynein for retrograde movement along microtubules

How does this relate to cancer How does this relate to cancer progression?progression?

p53 is a transcription factor that acts as a p53 is a transcription factor that acts as a tumor suppressor tumor suppressor

Translocation into the nucleus is imperative Translocation into the nucleus is imperative for subsequent p53 interactionsfor subsequent p53 interactions

Cancer Progression, cont.Cancer Progression, cont.

p53 is activated upon p53 is activated upon DNA damageDNA damage

DNA damage DNA damage stimulates either a stimulates either a cell cycle arrest or cell cycle arrest or apoptosisapoptosis


If p53 is not transported to the nucleus by If p53 is not transported to the nucleus by dynein, it remains in the cytoplasm and dynein, it remains in the cytoplasm and can not perform its function as a can not perform its function as a transcription factor and tumor suppressortranscription factor and tumor suppressor

If DNA damage occurs, p53 exclusion If DNA damage occurs, p53 exclusion from the nucleus would not be able to from the nucleus would not be able to regulate/correct itregulate/correct it


Cell cycle progression would continue, Cell cycle progression would continue, damaged DNA would become replicated, and damaged DNA would become replicated, and cell/tumor proliferation would occurcell/tumor proliferation would occur

p53

Questions?Questions?

Documents

Hsp90-binding Immunophilins Link p53 to Dynein During p53 Transport to Nucleus