A comparison of Hsp90a and Hsp90b interactionswith cochaperones and substrates

Aliakbar Taherian, Patrick H. Krone, and Nick Ovsenek

Abstract: Hsp90 chaperone complexes function in assembly, folding, and activation of numerous substrates. The 2 verte-brate homologues encoded by the genes hsp90a and hsp90b are differentially expressed in embryonic and adult tissues andduring stress; however, it is not known whether they possess identical functional activities in chaperone complexes. Thisquestion was addressed by examining potential differences between the Hsp90 isoforms with respect to both cochaperoneand substrate interactions. Epitope-tagged proteins were expressed in mammalian cells or Xenopus oocytes and subjectedto immunoprecipitation with an array of cochaperones. Both isoforms were shown to participate equally in multichaperonecomplexes, and no significant differences in cochaperone distribution were observed. The substrates Raf-1, HSF1, Cdc37,and MEK1 interacted with both Hsp90a and Hsp90b, and the relative patterns of these interactions were not affected byheat shock. The substrate kinases c-Src, CKIIB, A-raf, and Erk interacted with both isoforms; however, significantly moreHsp90a was recovered after heat shock. The data demonstrate that Hsp90a and Hsp90b exhibit similar interactions withcochaperones, but significantly different behaviors with respect to substrate interactions under stress conditions. These re-sults reveal both functional similarities and key functional differences in the individual members of this protein family.

Key words: Hsp90a, Hsp90b, heat shock, heterocomplex, chaperone.

Resume : Les complexes chaperons Hsp90 agissent dans l’assemblage, le repliement et l’activation de plusieurs substrats.Deux homologues chez les vertebres codes par les genes hsp90a et hsp90b sont exprimes de facon differentielle dans lestissus embryonnaires et adultes et durant le stress; cependant, on ignore si ceux-ci possedent des activites fonctionnellesidentiques dans les complexes chaperons. Cette question a ete traitee en examinant les differences potentielles qui existententre les isoformes de Hsp90 relativement aux interactions que ces deux cochaperons etablissent avec un substrat. Des pro-teines marquees par un epitope ont ete exprimees dans des cellules mammiferes et dans des ovocytes de Xenopus, et ellesont ete soumises a des immunoprecipitations a l’aide d’une variete de cochaperons. Les deux isoformes participaient egale-ment a la formation de complexes multi-chaperons et aucune difference significative dans la distribution des cochaperonsn’a ete observee. Les substrats Raf-1, HSF1, Cdc37 et MEK1 interagissaient tant avec Hsp90a qu’avec Hsp90b, et les pa-trons relatifs de ces interactions n’etaient pas affectes par le choc thermique. Les kinases substrats c-Src, CKIIB, A-raf etErk interagissaient avec les deux isoformes quoiqu’une quantite significativement plus grande de Hsp90a ait ete recupereeapres un choc thermique. Ces resultats demontrent que Hsp90a et Hsp90b interagissent de facon similaire avec des cocha-perons, mais qu’elles adoptent des comportements significativement differents lors des conditions de stress. Ces resultatsrevelent tant des similarites fonctionnelles que des differences fonctionnelles importantes au sein des membres individuelsde cette famille de proteines.

Mots-cles : Hsp90a, Hsp90b, choc thermique, complexes heterogenes, chaperon.

[Traduit par la Redaction]

Introduction

Hsp90 is highly abundant in unstressed eukaryotic cells(Lai et al. 1984; Welch and Feramisco 1982), and isessential for viability under all conditions in eukaryotes(Borkovich et al. 1989; Cutforth and Rubin 1994). Hsp90 islocalized predominantly in the cytoplasm, but very smallamounts are present in the nucleus (Langer et al. 2003). Itpurifies as a dimer and dimerization is required for its func-

tion in vivo (Minami et al. 1994). Hsp90 function is ATP-dependent and an intrinsic ATPase activity is required forthe operation of a functional chaperone cycle leading to thestabilization of substrate proteins (Pearl and Prodromou2000). General chaperone activity has been observed invitro, preventing aggregation of a variety of substrates(Craig 1985; Craig et al. 1994; Lindquist 1986; Mager a4ndFerreira 1993; Morimoto et al. 1992; Wu 1995).

A unique feature of Hsp90 among cellular chaperones issubstrate specificity. The majority of substrate proteins aresignal transduction proteins involved in cell growth and pro-liferation (Isaacs et al. 2003; Maloney and Workman 2002)or transcription factors (Pratt et al. 2004; Pratt and Toft2003). The best-characterized Hsp90-dependent substratesare members of the steroid receptor family. Hsp90 chaper-one machinery is required for the steroid receptor to achievea conformation that enables binding and response to steroidhormones. Maturation of steroid receptor complexes, for ex-

Received 4 October 2007. Revision received 11 December 2007.Accepted 12 December 2007. Published on the NRC ResearchPress Web site at bcb.nrc.ca on 30 January 2008.

A. Taherian, P.H. Krone, and N. Ovsenek.1 Department ofAnatomy and Cell Biology, College of Medicine, University ofSaskatchewan, Health Science Building A308, 107 Wiggins Rd.,Saskatoon, SK S7N 5E5, Canada.

1Corresponding author (e-mail: nick.ovsenek@usask.ca).

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ample, involves a dynamic assembly pathway in which thereceptor is passed from intermediate complexes containingHsp90, Hsp70, FKBP51/52, Cyp40, p60 (Hop), and p48(Hip) (Craig and Jacobsen 1984; Halladay and Craig 1995;Werner-Washburne et al. 1987) to a mature complex con-taining Hsp90, low levels of Hsp70, p23, and 1 of 3 immu-nophilins (Halladay and Craig 1995). In addition to thesteroid receptors, Hsp90 is a known chaperone of HSF1, themajor activator of the stress response (Ali et al. 1998; Bhar-adwaj et al. 1999; Duina et al. 1998a; Guo et al. 2001;Marchler and Wu 2001; Nadeau et al. 1993; Zou et al.1998a).

A number of cell-signaling molecules are chaperoned bythe Hsp90 machinery, including the Raf family of proteins(Raf-1, A-Raf, and B-Raf) (Hagemann and Rapp 1999; Liet al. 2001; Stancato et al. 1993, 1997; Vojtek et al. 1993;Workman 2003), MEK1/2 (Piatelli et al. 2002; Stancato etal. 1997), Erk (Stancato et al. 1997), Src (Brugge 1986;Stancato et al. 1997; Xu and Lindquist 1993) Cdk4 (Stepa-nova et al. 1996; Vaughan et al. 2006), and casein kinaseCKII (Dougherty et al. 1987; Miyata et al. 1997; Miyataand Yahara 1992, 1995). Cdc37 has been identified in asso-ciation with a number of Hsp90 substrate proteins, such asRaf-1, Cdk4, and Src family kinases (Grammatikakis et al.1999; Hartson et al. 2000; Hunter and Poon 1997; Kise etal. 2006; Park et al. 2007; Siligardi et al. 2002).

Two higher vertebrate Hsp90 isoforms are encoded by thehsp90a (Hickey et al. 1989) and hsp90b genes (Rebbe et al.1989). The Hsp90a and Hsp90b homologs in vertebrate spe-cies show approximately 81% identity to each other (Gupta1995). Although both the isoforms are expressed at basallevels even under nonstress conditions, various forms of cel-lular stress conditions increase their expression to differentdegrees (Barnier et al. 1987; Sass et al. 1996, 1999). For ex-ample, the zebrafish hsp90a gene is strongly expressed fol-lowing heat shock, whereas the hsp90b is only weaklyupregulated under similar stress conditions (Krone and Sass1994). Whereas the Hsp90-based heterocomplex has beenextensively studied, a systematic comparison of the functionof Hsp90a and Hsp90b isoforms has not been reported. Inthe present study, epitope-tagged (hemagluttanin, HA) ze-brafish Hsp90a and Hsp90b were individually expressed inNIH3T3 cells and Xenopus oocytes. The use of fish hsp90in human cells and frog oocytes was a deliberate strategy todirectly compare the activities of the same Hsp90 proteins inthe 2 model systems. HA–Hsp90a and HA–Hsp90b interac-tions with cochaperones and selected substrate proteins werethen examined by co-immunoprecipitation and Western blotanalysis. The results show similarities between isoformswith respect to cochaperone interactions, and reveal impor-tant functional differences in the chaperoning of specificsubstrate proteins under heat shock conditions.

Materials and methods

CloningFull-length zebra fish hsp90a and hsp90b were ampli-

fied from plasmid DNA (GenBank accession Nos. Q90474and AAC21566.1, respectively) with the following primers.hsp90a: forward, 5’-CGGGGGATCCATGCCTGAGGCT-CACGAGCAG-3’; reverse, 5’-GCCCGAATTCTTAGT-

CGACTTCCTCCATTCT-3’). hsp90b: forward, 5’-CGG-GGGATCCATGCCTGAAGAAATGCGCCCA-3’; reverse,5’-GCCCGAATTCTTAATCGACTTCCTCCATGC-3’. Forboth hsp90a and hsp90b, the 5’ primers contained a restric-tion digest site for BamH1 and the 3’ primers contained arestriction site for EcoR1. PCR products were digestedwith BamH1–EcoR1 and cloned into BamH1–EcoR1-di-gested HA3-CMV vector, a modified form of pACTAG2(Charest et al. 1995), yielding plasmids HA–Hsp90a andHA–Hsp90b, which express N-terminally tagged proteins.

Xenopus laevis oocytesFrogs were purchased from Xenopus I, Inc. (Dexter,

Mich., USA). All experiments were carried out according toThe Guide for the Care and Use of Laboratory Animals andwere approved by the Ethics Committee of the University ofSaskatchewan. Oocytes were surgically removed from adultfemales and defolliculated in calcium-free OR-2 medium(82.5 mmol/L NaCl, 2.5 mmol/L KCl, 1 mmol/L NaH2PO4,5 mmol/L HEPES (pH 7.8), 10 mg/L streptomycin sulfate,10 mg/L benzylpenicillin) containing 2 mg/mL of collage-nase (type II, Sigma; www.sigmaaldrich.com) for 2–3 hwith rocking at 18 8C (Wallace et al. 1973). Oocytes werewashed extensively and allowed to recover overnight inOR2 containing 1 mmol/L CaCl2 at 18 8C. Healthy stageVI oocytes were injected into nuclei with 0.5 ng of eitherCMV-Hsp90a or CMV-Hsp90b using a Narashige IM 300microinjector. After incubation overnight at 18 8C, oocyteswere either left at 18 8C (N, nonshock) or heat shocked(HS) 1 h in circulating water bath at 33 8C, immediatelyprior to preparations of protein extracts. Nuclei were ob-tained under OR2 by scoring animal hemispheres of stageVI oocytes with a needle and gently squeezing the equato-rial region with watchmaker’s forceps. Nuclei were washedgently by gentle pipetting to remove residual cytoplasm.Protein extracts were prepared as described previously(Bharadwaj et al. 1999) using either Buffer C (50 mmol/LTris–HCl (pH 7.9), 20% (v/v) glycerol, 50 mmol/L KCl,and 0.1 mmol/L EDTA (pH 8.0)) for SDS–PAGE analysis,or IP buffer (20 mmol/L (Tris pH 7.4), 5 mmol/L EDTA,5 mmol/L EGTA, 100 mmol/L NaCl, 50 mg/mL PMSF,1 mmol/L iodo acetamide, 1 mmol/L pepstatin, 1 mmol/L 1,10 phenanthroline, 50 nmol/L okadaic acid, 0.5 mmol/L so-dium orthovanadate, 50 mmol/L KFl, 4% Triton X-100,10 mg/mL aprotinin, 10 mg/mL leupeptin, and 2 mmol/LDTT) (Jing et al. 1999) for immunoprecipitation experi-ments. Oocytes were homogenized in 10 mL/oocyte bufferC or 15 mL/oocyte IP buffer and centrifuged (10 000g,10 min).

NIH 3T3 cellsNIH 3T3 cells were maintained in Dulbecco’s modified

Eagle’s medium (GIBCO, catalogue No. 41500-034) supple-mented with 10% fetal bovine serum (Summit Biotechnol-ogy, catalogue No. FP-100-05), and 100 IU/mL eachpenicillin and streptomycin at 37 8C in a humidified cham-ber supplemented with 5% CO2. Cells were grown to*50%–80% confluency before transfection and then grownto 90%–100% confluency before protein extraction. NIH3T3 cells were transfected using LipofectAMINE and PlusReagents (Invitrogen Life Technologies, www.invitrogen.

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com) with HA–Hsp90a, HA–Hsp90b, or pCMV vectors.Cells were either left at 37 8C (N) or heat shocked (HS) at42 8C for 1 h immediately prior to protein extraction. Pro-tein extraction was performed by the method of Andersonand Ismail (1998). Cells were rinsed 3 times with cold PBSand then mixed with 800 mL of ice-cold RIPA buffer(50 mmol/L Tris (pH 8), 150 mmol/L NaCl, 0.1% (w/v)SDS, 0.5% sodium deoxycholic acid, 1% Nonidet P-40,10 mg/mL aprotinin, and 10 mg/mL leupeptin) on ice for20 min. Cells were recovered by scraping with a rubber cellscraper and homogenized with a 25 gauge 5/8 inch needle.Homogenates were centrifuged (13 000g, 30 min) and thesupernatants were stored at –80 8C.

Immunoprecipitation (IP)IP was performed according to the method of Anderson

and Ismail (1998). All steps of IP were performed on ice orat 4 8C. For each reaction, 800 mg of total protein of cell oroocyte lysate (assayed with a TP0300 Lowry kit, Sigma)was precleared by adding 20 mL of protein G suspension(Sigma, catalogue No. p-3296) and 3 mg normal mouse IgG(sc-2343, Santa Cruz, www.scbt.com) followed by incuba-tion for 2 h at 4 8C with gentle rotation. Samples were cen-trifuged at 13 000g for 20 min and supernatants weresubjected to IP. For each reaction, 100 mL preswollen pro-tein G – sepharose was washed with 1 mL of RIPA bufferand mixed with 5 mg of antibody and 800 mL of RIPA buf-fer for 4 h with rocking. Samples were centrifuged at 1000gfor 2 min and washed with 500 mL RIPA buffer. Antibody-coupled protein G was mixed with precleared cell lysate andincubated overnight with rotation. Samples were centrifuged(1000g for 2 min). The pellets were washed 3 times with1 mL RIPA buffer and mixed with 40 mL Laemmli buffer.For both oocytes and NIH3T3 cells, all experiments were re-peated at least 3 times, and representative Western blots arepresented in the Results. Antibodies used for IP were as fol-lows (with catalogue numbers given in parentheses): caseinkinase IIb (sc-12739), c-Src (sc-5266), IkB-a (sc-847),PCNA (sc-56), and Erk (sc-94) from Santa Cruz; MEK1(9122), Cyp-40 (PA3-023), FKBP51 (PA-020), HSF1 (PA3-017), hsp70 (MA3-006), P23 (MA3-414), and Cdc37 (MA3-029) from Affinity BioReagents; and Hip (SPA-766), p60(SRA-1500, FKBP52 (SRA-1400), and HSF1 (SPA-901)from Stressgen.

Western blot analysisProtein lysates (10 mg) were fractionated by SDS–PAGE,

transferred to nitrocellulose membranes (Laemmli et al.1976; Towbin and Gordon 1984), and analyzed by Westernblotting, as described previously (Bharadwaj et al. 1999).The SPA830 antibody used to detect Hsp90 was fromStressgen.

Results

Expression and subcellular localization of HA–Hsp90aand HA–Hsp90b

The first aim of this study was to test for potential differ-ences in the behavior of Hsp90 isoforms with respect totheir interactions with known Hsp90 cochaperones. Re-combinant HA-tagged Hsp90a or Hsp90b proteins were in-

dividually expressed in oocytes and NIH3T3 cells anddetected by Western blotting with anti-HA antibody(Fig. 1A). In controls, no signal was detected in nontrans-fected cells or uninjected oocytes. Expression conditionswere modified to provide equal expression of HA–Hsp90aand HA–Hsp90b in either model system, and IP from cellextracts specifically precipitated HA–Hsp90a and HA–Hsp90b (Fig. 1B). We currently do not know whetherexpressed Hsp90 proteins form dimmers with endogenousproteins in vivo.

Following expression, the subcellular location of HA–Hsp90a or HA–Hsp90b was examined in oocytes undernonstress control and heat-shock conditions. The expressedproteins were detected primarily in the cytoplasm, with aminor portion appearing in the nuclear fractions. No signifi-cant difference was observed in the relative subcellular dis-

Fig. 1. Expression of HA-tagged Hsp90a or Hsp90b in oocyte andNIH 3T3 cells. Cells were transfected or microinjected with eitherHA–Hsp90a or HA–Hsp90b expression plasmids. (A) Total protein(10 mg) was analyzed by Western blotting with anti-HA antibody.(B) Untransfected or uninjected (C, control) samples were subjectedto IP with HA antibody and analyzed by Western blotting forHsp90. (C) Uninjected oocytes (control) or oocytes expressingHA–Hsp90a or HA–Hsp90b were incubated at a nonshock (N)temperature (18 8C) or a heat-shock (HS) temperature (33 8C) for1 h and analyzed by Western blotting for total Hsp90 (top panel) orHA–Hsp90 (bottom panel).

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tribution of these proteins in control and HS cells, althougha slight decline in Hsp90a was observed in some experi-ments (Fig. 2). A similar pattern of distribution was seen byimmunohistochemical analysis of NIH3T3 cells (data notshown). Western blot data also confirmed that expression ofHA-tagged proteins did not significantly increase the totalamount of cellular Hsp90 (Fig. 1C).

Interaction of Hsp90a or Hsp90b with cochaperonesWe next tested for potential differences between the inter-

actions of Hsp90a and Hsp90b with a panel of cochaperoneproteins: Hsp70, p60, FKBP52, FKBP51, Cyp-40, Hsp40,and p23. The experimental strategy was to immunoprecipi-tate individual cochaperones and test comparatively foreach of the HA-tagged Hsp90 proteins expressed in parallelcell or oocyte cultures under both control and heat-shockconditions (Fig. 3). The results showed no significant differ-ences in the amount of either Hsp90 isoform in IP experi-ments; each of the different cochaperones pulled downalmost equal amounts of HA-tagged Hsp90a or Hsp90b. Mi-nor differences in the relative levels of Hsp90a and Hsp90bwere detected in immunoprecipitated material using thepanel of cochaperone antibodies (Fig. 3). Particularly inNIH3T3 cells, there appeared to be a difference in cochaper-one interactions with Hsp90b after heat shock. However, itis clear that both isoforms strongly interact with all cocha-perones. For Hsp90a, no significant differences were consis-tently observed in the quantity of tagged proteins recoveredin IP experiments either before or after heat shock. Further-more, similar results were obtained with both NIH 3T3 cellsand Xenopus oocytes (Fig. 3). These results demonstrate thatnascent HA-tagged Hsps expressed in either cell type assem-ble into chaperone complexes, but more importantly, theyreveal an equal ability of either Hsp90 isoform to interactwith the key heterocomplex components. The pattern ofHsp90–cochaperone interactions for either isoform was notaltered by heat shock.

Comparative interactions of Hsp90a or Hsp90b withchaperone substrates in unshocked and heat-shockedcells

We used essentially the same experimental protocol asthat described in the previous section to examine potentialdifferences in the interaction of chaperone heterocomplexes,containing Hsp90a and Hsp90b, with a panel of substrateprotein signaling molecules, including A-Raf, Raf-1, c-Src,CKIIB, Cdc37, MEK1, and Erk, as well the heat-inducibletranscription factor HSF1. All of these proteins are knownto require interactions with the Hsp90-based machinery fornormal function. The results presented in Fig. 4 showedequal recovery of either isoform, with 4 of these proteins,i.e., HSF1, Raf-1, Cdc37 and MEK1. The interactions witheither the Hsp90a or Hsp90b isoform were not affected byheat shock.

Both HA–Hsp90a and HA–Hsp90b were seen to interactwith A-Raf, c-Src, CKIIB, and Erk. The interactions withHA–Hsp90b were similar in unstressed and heat-shockedcells. In contrast, we consistently observed enhanced inter-actions between HA–Hsp90a and c-Src, CKIIB, and Erkafter heat shock (Fig. 4). This pattern was observed in oo-cytes, and more so, in NIH3T3 cells. A-Raf interaction wasenhanced after heat shock in NIH3T3 cells, but reduced inoocytes. These findings suggest isoform-specific functionalroles for Hsp90a-containing complexes with respect to chap-eroning selected kinases in heat-shocked cells.

DiscussionTo our knowledge, this is the first systematic comparison

of the Hsp90 isoforms with respect to cochaperone and mul-tiple substrate interactions. Our experimental strategy was toexpress small amounts of epitope-tagged hsp90 isoformsHsp90a and Hsp90b, in 2 distinct cellular models, and com-pare their interactions with a panel of cochaperones andchaperone substrates in both nonstress and heat-shocked

Fig. 2. Nucleocytoplasmic distribution of HA-tagged Hsp90a and Hsp90b. Oocytes were microinjected with HA–Hsp90a or HA–Hsp90b(indicated at top), and following expression for 24 h, were subjected to nonshock (N; 18 8C) or heat-shock (HS; 33 8C for 1 h) conditions(indicated at left). Whole cell, cytoplasmic, and nuclear fractions were analyzed by Western blotting with HA, PCNA (nuclear marker), andIkB (cytoplasmic marker) antibodies.

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conditions. Coimmunoprecipitation of endogenous proteinswas performed in parallel to compare interactions with ei-ther isoform. This protocol was used to circumvent potentialcross-reactivity between existing Hsp90 antibodies with the2 highly conserved isoforms. Expression conditions werecontrolled to provide equal expression of either isoform inboth NIH3T3 cells and in Xenopus oocytes. Both recombi-nant proteins were distributed primarily in the cytoplasmwith a minor component of nuclear localization, and we didnot detect any significant change in the localization oftagged Hsp90 after heat shock. The nascent tagged proteinstherefore adopted a similar pattern of subcellular localizationas endogenous Hsp90s, and none of the results could be at-

tributed to nucleocytoplasmic redistribution of native ortagged proteins under heat-shock conditions. It is also im-portant to note that, in the majority of experiments, similarresults were obtained in the 2 distinct cell models.

The first major conclusion is that Hsp90a and Hsp90b in-teract similarly with the major cochaperone components ofthe Hsp90 chaperone machine. Immunopreciptiation experi-ments showed that each of Hsp70, p60, FKBP52, FKBP51,Cyp-40, Hsp40, or p23 brought down almost equal amountsof either Hsp90a or Hsp90b (Figs. 3A and 3B). Another im-portant observation was that heat shock did not alter the ap-parent interactions with any of the cochaperones. Therefore,stress conditions do not appear to alter the dynamics of the

Fig. 3. Interaction of Hsp90 Hsp90a and Hsp90b with cochaperones.(A) Oocyte protein extracts were subjected to immunoprecipitation (IP)with the indicated antibodies, and samples were analyzed by Western blotting for HA-tagged proteins. Starting cell extracts were analyzedfor HA-tagged protein expression (right panel) to ensure equal levels in all samples. (B) NIH 3T3 cells were subjected to IP and analyzedby Western blot analysis, as in A.

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heterocomplex population in cells, at least with respect tothe distribution of Hsp90 isoforms. Most importantly, the re-sults presented here support a model in which both Hsp90aor Hsp90b participate in the normal activity of the Hsp90-based machinery, with no apparent specificity for Hsp70,Hsp40, Hop, or the any of the immunophilins. The findingssuggest a nonselective distribution of endogenous Hsp90isoforms in the cellular chaperone machinery.

The second major conclusion is that, although bothHsp90a or Hsp90b interact with a number of known chaper-one substrates under nonstress conditions, Hsp90a displayedenhanced substrate preference under heat shock conditions.This was a general observation for a subset of substrates,namely c-Src, A-Raf, Erk, and CKIIb, with the exception ofa decline in A-Raf interaction in oocytes. The specificity ofthis observation is underlined by the fact that there were noapparent differences in the relative interaction of either iso-form with many of the chaperone substrates, namely HSF1,MEK1, Cdc37, or Raf-1 (Fig. 4). Moreover, some of the IPexperiments were repeated in oocytes and the observationswere largely consistent between the 2 very different modelsystems. It is important to note that this result was not at-tributable to enhanced expression of Hsp90a under heat-shock conditions because the HA-tagged protein was notunder the control of a stress-inducible promoter. The results,however, highlight an inherent functional difference betweenthe inducible (Hsp90a) and noninducible (Hsp90b) isoform.The data would suggest that these closely related isoforms,

which are rarely differentiated in the biochemical literature,may indeed carry out disparate roles in the cell. The preciseimplications of this finding and the nature of the functionalconsequences will require further analysis.

The mechanism underlying the enhanced interaction ofHsp90a with c-Src, A-Raf, Erk, and CKIIb is not yetknown. The observations reveal potential differences in thefunction, regulation, or chaperone requirement for thesemolecules during episodes of cell stress. The interaction be-tween Raf-1 and B-Raf proteins with Hsp90 has been re-ported previously (Stancato et al. 1993, 1997; Workman2003; Zhang and Burrows 2004). This interaction allowsRaf activity to be sensitive to geldanamycin (Hagemannand Rapp 1999; Li et al. 2001; Stancato et al. 1997; Vojteket al. 1993). Interaction between Erk and Hsp90 has beendemonstrated previously with coimmunoprecipitation experi-ments (Stancato et al. 1997), and both v-Src and c-Src havebeen previously shown to bind to Hsp90, affecting activityand membrane recruitment (Brugge 1986; Stancato et al.1997; Xu and Lindquist 1993). Our results extend the cur-rent data, in that they reveal an enhanced interaction of c-Src with Hsp90a after heat shock (Fig. 4). It is not yet clearwhether Hsp90a binding to c-Src after heat shock affectskinase activity, or heat-shock activation of c-Src affectsbinding to Hsp90a.

CKII, a member of a family of serine–threonine proteinkinases (Litchfield and Luscher 1993; Tuazon and Traugh1991), is a tetrameric complex consisting of either a a2b2

Fig. 4. Interaction of Hsp90 Hsp90a and Hsp90b with cochaperones. Oocyte and NIH3T3 cell extracts were subjected to immunoprecipita-tion using a panel of antibodies (indicated at left). Samples were analyzed by Western blotting for HA-tagged proteins. Starting cell extractswere analyzed for HA-tagged protein expression (right panels) to ensure equal levels in all samples.

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or an aa’b2 structure. Hsp90 binds to CKII, thereby prevent-ing self-aggregation and enhancing its kinase activity(Dougherty et al. 1987; Miyata and Yahara 1992, 1995). Itis possible that Hsp90a may aid in CKIIb phosphorylation,or alternatively, there is an enhanced requirement for chap-eroning under heat-shock conditions.

Overall, our results extend the current understanding ofHsp90–protein interactions by demonstrating an equal inter-action of Hsp90 isoforms with the general chaperone machi-nery, as well as a distinct interaction of a subset of substrateproteins with the Hsp90a following heat shock.

AcknowledgementsThis work was supported by Natural Sciences and Engi-

neering Research Council of Canada Discovery Grants toN.O. and P.K.

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