Regulation of client by a Cullin5-RING E3

Elana S. Ehrlicha, Tao Wanga, Kun Luoa, Zuoxiang Xiaoa, Anna Maria Niewiadomskaa, Tara Martineza, Wanping Xub, Len Neckersb, and Xiao-Fang Yua,1

aDepartment of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205; and bUrologic Oncology Branch, National Cancer Institute, Bethesda, MD 20892

Edited by William J. Muller, McGill University, Montreal, Canada, and accepted by the Editorial Board September 23, 2009 (received for review October 22, 2008) We report a link between Cullin5 (Cul5) E3 and the Here we demonstrate that Cul5 regulates Hsp90 clients. Our heat shock 90 (Hsp90) complex. Hsp90 partici- results indicate that Cul5 interacts with the Hsp90 chaperone pates in the folding of its client proteins into their functional complex and the Hsp90 client ErbB2 and demonstrate that Cul5 conformation. Many Hsp90 clients have been reported to be is recruited to the site of ErbB2 on the plasma membrane, aberrantly expressed in a number of cancers. We demonstrate Cul5 thereby inducing its polyubiquitination and - interaction with members of the Hsp90 chaperone complex as well mediated degradation. Other Hsp90 client proteins, such as as the Hsp90 client, ErbB2. We observed recruitment of Cul5 to the HIF1-␣, were also regulated by Cul5. We observed Cul5- site of ErbB2 at the plasma membrane and subsequent induction of mediated degradation of ErbB2 to occur in the absence of the polyubiquitination and proteasomal degradation. We also dem- traditional Cul5 adaptors ElonginB and ElonginC, suggesting onstrate Cul5 involvement in regulation of another Hsp90 client, that a component of the Hsp90 chaperone complex may serve Hif-1␣. We observed Cul5 degradation of ErbB2 to occur indepen- this function. This is an example of a link between Hsp90 and the dently of ElonginB-ElonginC function. The involvement of Cul5 in Cullin family of E3 ubiquitin . This is also a report of an Hsp90 client regulation has implications in the effectiveness of ElonginB-ElonginC-independent Cul5 E3 ubiquitin ligase. Hsp90 targeted chemotherapy, which is currently undergoing Hsp90 is a well-established target for chemotherapy (16). Our CELL BIOLOGY clinical trials. The link between Cul5 and Hsp90 client regulation data now provide an explanation for the decrease or loss of Cul5 may represent an avenue for cancer drug development. expression that is observed in a number of cancers (17). Our data also suggest that Cul5 levels could potentially influence suscep- chaperone ͉ erbb2 tibility to certain cancers and the effectiveness of anti-cancer treatment with geldanamycin or its derivatives, which are cur- rently in human clinical trials. he regulation of client proteins by 90 T(Hsp90) plays an important role in critical cellular processes Results such as control and . Dysregulation is linked to Cul5 Interacts with the Hsp90 Chaperone Complex. While the cellular cancer and neurological diseases (1, 2). Hsp90 is a molecular function of Cul5 is poorly understood, it is known to be hijacked chaperone responsible for the correct folding of proteins, allowing by the HIV type 1 (HIV-1) Vif protein to suppress the antiviral them to attain their proper functional conformations (3). Many protein Apobec3G (A3G) (18–20) and by adenovirus E4orf6 client proteins of Hsp90 are overexpressed oncogenes that are and E1B55K to degrade , Mre11, and DNA ligase IV (18, critical for the transformed phenotype observed in tumors (4–6). 21–25). To identify potential cellular partners of the Cul5 E3 Clients of Hsp90 are also regulated by the ubiquitin/proteasome ligase, we immunoprecipitated HA-tagged Cul5 and identified system (7). However, the identity of the cellular E3 ubiquitin proteins that specifically co-immunoprecipitated with Cul5-HA ligase(s) that regulate(s) Hsp90 client proteins is still elusive. but not with a cmyc-tagged Cul5 control protein. In repeated The Cullin family of RING E3 ubiquitin ligases are modular experiments, Cul5-HA, but not Cul5-myc, co-precipitated that act as a scaffolding to bring a specific substrate , as indicated by mass spectrometry analysis (Fig. 1A). within close proximity to the E2 ubiquitin conjugating , Interaction of Hsp70 with Cul5-HA but not Cul5-myc was thereby facilitating ubiquitination and subsequent proteasomal confirmed by Western blotting with an anti-Hsp70 antibody (Fig. degradation (8, 9). There are seven known human Cullin pro- 1B). We also observed an interaction between Hsp90 and teins, Cul1, 2, 3, 4a, 4b, 5, and 7, with diverse functions from cell Cul5-HA (Fig. 1B), suggesting that Cul5 may be involved in the cycle regulation, to DNA repair, to regulation of developmental regulation of Hsp90 client proteins. Hsp90 is a chaperone that is processes. Substrate specificity is determined by the combina- required for the maturation and function of a number of classes torial nature of E3 ligase assembly. In the case of Cul1, diversity of proteins, namely receptors, kinases, and transcription factors is achieved by the assembly of different F-box substrate receptor (7, 26). Hsp90 assembles with a number of co-chaperones, proteins with Cul1 through a single Skp1 adaptor protein (10). including Hsp70, and coordinates the maturation of its client Each F-box protein determines the specificity of Cul1 substrates. proteins. Client protein maturation occurs via a dynamic ATP- Cul3 is unique in having one protein with two domains, one dependent process (7). ATP hydrolysis or inhibition of Hsp90 functioning as an adaptor forming an interface with Cullin, and the other acting as a substrate receptor (9). Cul2 and 5 are interesting in that they both use ElonginB-C Author contributions: E.S.E. designed research; E.S.E., T.W., K.L., Z.X., and A.M.N. per- formed research; T.M., W.X., and L.N. contributed new reagents/analytic tools; E.S.E., L.N., adaptor proteins, through which they bind a SOCS box contain- and X.-F.Y. analyzed data; and E.S.E. wrote the paper. ing substrate receptor (8). Whether a substrate receptor recruits The authors declare no conflict of interest. Cul2 or Cul5 depends on an additional Cullin binding interface. This article is a PNAS Direct Submission. W.J.M. is a guest editor invited by the Editorial In the case of cellular substrate receptors, Cullin selection is Board. determined by the presence of a VHL or SOCS box motif (11), 1To whom correspondence should be addressed. E-mail: [email protected]. in the case of HIV and SIV, a zinc-stabilized helix mediates Cul5 This article contains supporting information online at www.pnas.org/cgi/content/full/ selection (12–15). 0810571106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810571106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 24, 2021 IP HA 293T cells U87 cells also confirmed in U87cells (Fig. 1C). Immunoprecipitation of A kD B C Cl5Cu - Cul5Cul5- endogenous Hsp70 also co-precipitated endogenous Cul5 (Fig. CuCl5 Hsp90- Hsp90- Hsp70 1D). Conversely, immunoprecipitation of endogenous Cul5 also 66 Hsp70- Hsp70- co-precipitated endogenous Hsp90 and Hsp70 (Fig. 1E). 121 2 121 2 Hsp90 has been shown to be expressed at high levels, along + - Cul5-HA + - -45 Cul5-HA with a number of its client proteins, in a variety of cancers and 1 2 Cul5-cmyc -+ Cul5-cmyc -+ has therefore become a promising target for intervention (7). Cul5-HA + - IP Cul5:Anti-HA IP Cul5:Anti-HA Cul5-cmyc -++ Treatment of cells with the benzoquinone ansamycin antibiotic Coomassie stain Cell lysate geldanamycin (GA) or its analogs results in the proteasomal EFCl5Cu - degradation of Hsp90 client proteins (16). GA is an ATPase D Hsp90- Ab α : IgG ErbB2 inhibitor that binds the nucleotide-binding pocket with an af- IP: Hsp70 IgG Hsp70- Erbb2- finity greater than that of ATP or ADP, shifting the chaperone Abα : IgG Cul5 Cul5- Cul5Cul5- complex into a conformation that favors client-protein degra- Hsp70- Cul5- 1 2 3 1 2 Hsp90- dation (27). ErbB2 is an Hsp90 client that has been extensively GA -+ - IP Endogenous Hsp70 Hsp70- characterized. This receptor tyrosine kinase is overexpressed in 1231 2 3 IP ErbB2 approximately 30% of breast cancers and is required for tumor GA -+ - cell proliferation. Treatment of ErbB2- overexpressing cells with IP Endogenous Cul5 GA or its analogs results in rapid proteasomal degradation of Fig. 1. Cul5 interacts with the Hsp90-Hsp70 chaperone complex. (A) Cul5-HA ErbB2 (28, 29). While the U-box-containing E3 ligase, CHIP, and Cul5-cmyc were immunoprecipitated with anti-HA-conjugated agarose has been implicated in the degradation of ErbB2 via Hsp/Hsc70, beads. The eluates were analyzed by SDS-PAGE and Coomassie staining, and GA-mediated ErbB2 degradation still occurs in CHIPϪ/Ϫ cells, bands were identified by mass spectrometry. (B and C) Cul5-HA and Cul5-cmyc suggesting that an additional E3 ligase is also involved in were immunoprecipitated with anti-HA-conjugated agarose beads in (B) 293T GA-mediated degradation of ErbB2 (29). and (C) U87 cells. Eluates were analyzed by SDS-PAGE and Western blotting with antibodies against HA, Hsp90, and Hsp70. (D) 293T cell lysates were To examine the possibility that Cul5, interacting with the incubated with antibody against Hsp70 or IgG and protein A/G agarose. Hsp90 chaperone complex, might serve as an E3 ligase to Protein complexes were immunoprecipitated and analyzed by SDS-PAGE and degrade Hsp90 client proteins, we characterized the interaction Western blotting with antibodies against Hsp70 and Cul5. (E) 293T cells were of Cul5 with ErbB2. We treated 293T cells transfected with treated with GA or DMSO as indicated. Protein complexes were immunopre- ErbB2 or empty vector with 2 ␮M GA or control DMSO and 5 cipitated with antibodies against Cul5 and protein A/G agarose. Eluates were ␮M MG132. Even though equal amounts of ErbB2 were immu- analyzed by SDS-PAGE and Western blotting with antibodies against Cul5, noprecipitated, Cul5 interaction was detected only in GA- Hsp90, and Hsp70. (F) 293T cells were transfected with the ErbB2 expression treated cells (Fig. 1F). These data demonstrate the endogenous vector and treated with GA or MG132 as indicated. Cell lysates were incubated with antibody against ErbB2 and with protein A/G agarose. Immunoprecipi- interaction of Cul5 with ErbB2 and the Hsp90 chaperone tates were analyzed by SDS-PAGE, followed by Western blotting with anti- complex, suggesting the involvement of Cul5 in the regulation of body against Cul5. Hsp90 client proteins.

Cul5 Co-Localizes with ErbB2 at the Plasma Membrane. Since ErbB2 function through drug treatment results in the reorganization of is a receptor tyrosine kinase, we hypothesized that Cul5 might be the chaperone complex and subsequent destabilization of the acting on ErbB2 at the plasma membrane. We therefore trans- client protein (7). Interaction of Cul5 with Hsp90 and Hsp70 was fected 293T cells with either the ErbB2 expression vector or

ABCTIF -C 5luC 2BbrE-enimadohR M egre

Control

DEF

ErbB2

Fig. 2. Cul5 is recruited to ErbB2 at the plasma membrane. (A–F) 293T cells were transfected with the ErbB2 expression vector (D–F) or empty vector control (A–C). Twenty-four hours after transfection, the cells were plated on glass coverslips and incubated for 16 h. Cells were fixed, permeablized, and stained with antibodies against endogenous Cul5 and ErbB2. Slides were visualized using a Zeiss Meta 510 confocal microscope and viewed with LSM software. Images are equivalent slices of a z-stack.

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810571106 Ehrlich et al. Downloaded by guest on September 24, 2021 A B ErbB2-

ErbB2- Ribosomal p19- CHIP- 12341 2 3 4 Ribosomal p19- Control vector + + - - 1 2 3 4 Cul5 Nedd8 - - + + Erbb2 + + + + GA + - + - Control siRNA + + - - D IP: anti-ErbB2 CHIP siRNA - - + + 170- GA + - + - 130- 95- -Anti-Ub

C SKBR3 Ces ll ErbB2 Cul5 β -actin -ErbB2 12341 2 3 4 170- 130- Cul5 shRNA - - + + 1 2 3 4 5 GFP shRNA + + - - -Cul5ΔNedd8 Fig. 4. Cul5 is required for ErbB2 and Hif-1␣ degradation. (A and B) Cul5 but GA -+ -+ ErbB2 - + + + + not Cul2 is required for GA-mediated ErbB2 degradation. 293T cells were Cul5 ΔNedd8 ---+ + transfected with the ErbB2 expression vector and Cul5, Cul2, or control siRNA GA - + - + - as indicated. Forty-eight hours post transfection, the cells were treated with GA or DMSO for 16 h. ErbB2 stability was assessed by Western blotting with MG132 + + + + +

antibodies against ErbB2, Cul2, Cul5, and ribosomal p19. (C and D) Cul5 is CELL BIOLOGY Cell lysate required for GA-mediated degradation of HIF-1␣. 293T cells were cotrans- ␣ Fig. 3. Cul5 is required for polyubiquitination and proteasomal degradation fected with HIF-1 -HA expression vector and Cul5 or control siRNA or ⌬ of ErbB2. (A) CHIP knockdown does not completely stabilize ErbB2. 293T cells Cul5 Nedd8 as indicated. At 48 h after transfection, the cells were treated ␣ were transfected with ErbB2 and either CHIP or control siRNA as indicated. At with GA or DMSO for 16 h. HIF-1 stability was assessed by Western blotting 48 h after transfection, the cells were treated with GA or DMSO for 16 h. ErbB2 with antibodies against HA, Cul5, and ribosomal p19. stability was assessed by Western blotting with antibodies against ErbB2, CHIP, and ribosomal p19. (B) ErbB2 degradation is inhibited by the Cul5 dominant-negative mutant. 293T cells were transfected with ErbB2 and suggesting that CHIP is necessary but not sufficient for GA- Cul5⌬Nedd8-cmyc where indicated. At 48 h after transfection, the cells were mediated degradation of ErbB2 (29). In fact, we observed the treated with GA or DMSO for 16 h as indicated. ErbB2 stability was assessed by same phenotype in our system. Transfection with CHIP siRNA Western blotting with antibodies against ErbB2, cmyc, and ribosomal p19. (C) resulted in efficient CHIP knockdown; however, treatment with Cul5 shRNA inhibits GA-mediated degradation of ErbB2 in SKBR3 cells. SKBR3 GA still resulted in ErbB2 degradation, supporting the hypoth- cells were infected with lentiviruses containing GFP targeting or Cul5 target- ing shRNA. Twenty-four hours post infection, cells were selected with puro- esis that an additional mechanism of GA-induced degradation is mycin for 1 week. Cells were treated with GA or equivalent volume of DMSO at work in this system (Fig. 3A). for 2 h. Cells were harvested and analyzed by Western blot against ErbB2, Cul5, To determine whether Cul5 is involved in ErbB2 degradation, or ␤-actin where indicated. (D) A Cul5 dominant-negative mutant inhibits we used a dominant-negative mutant, Cul5⌬Nedd8, to assess the ErbB2 polyubiquitination. 293T cells were transfected with ErbB2 and requirement of Cul5 for ErbB2 degradation (Fig. 3B). In the Cul5⌬Nedd8-cmyc as indicated. At 48 h after transfection, the cells were absence of Cul5⌬Nedd8, treatment of ErbB2-transfected 293T ␮ ␮ treated with 5 M MG132 and 3 M GA or DMSO for4hasindicated. Cell cells with GA resulted in ErbB2 degradation (Fig. 3B, compare lysates were incubated with anti-ErbB2 and protein G-conjugated agarose. ⌬ Immunoprecipitates were then analyzed by Western blotting with antibodies lanes 1 and 2). However, after the addition of Cul5 Nedd8, against ErbB2, myc, and ubiquitin. ErbB2 was stabilized even in the presence of GA (Fig. 3B, compare lanes 3 and 1). We have also demonstrated that GA induced degradation of endogenous ErbB2 in the SKBR3 breast empty vector control for 48 h, then stained for ErbB2 and cancer cell line was inhibited when Cul5 was efficiently knocked- endogenous Cul5. In the absence of ErbB2, Cul5 was evenly down. We observed the stabilization of ErbB2 in the SKBR3 distributed throughout the cell (Fig. 2A). However, in the cells even in the presence of GA when Cul5 expression was presence of ErbB2, Cul5 expression appeared to be concentrated reduced by the shRNA strategy via a lentiviral delivery system at the plasma membrane (Fig. 2 D and E), suggesting that the (Fig. 3C, lane 4), compared to SKBR3 cells treated with control presence of substrate results in the recruitment of Cul5 to the shRNA targeting GFP (Fig. 3C, lane 2). GA treatment induced plasma membrane. This interaction was specific to Cul5, as Cul2 polyubiquitination of ErbB2 (Fig. 3D, lane 2), when compared did not co-localize with ErbB2 (Fig. S1). Cul5 appears to to the control treated cells (lane 3). Cul5⌬Nedd8 also inhibited co-localize with ErbB2 at the plasma membrane as seen in the polyubiquitination of ErbB2 induced by GA (Fig. 3D, multiple (2-␮m) frames of a zstack (Fig. S2). ErbB2 expression compare lanes 2 and 4). did not affect cell integrity as seen by DAPI and phase contrast To further examine the role of Cul5 in GA-induced degrada- (Fig. S3). tion of Hsp90 clients, we used siRNA against the Cul5 coding region to knock down Cul5 expression. Addition of Cul5-specific Cul5 Is Required for Polyubiquitination and Proteasomal Degradation siRNA (Fig. 4A, lanes 3 and 4), but not control siRNA (Fig. 4A, of ErbB2. Treatment of ErbB2-expressing cells with GA is known lanes 1 and 2), resulted in efficient Cul5 knockdown. GA induced to result in rapid proteasomal degradation of the receptor; efficient degradation of ErbB2 in control siRNA-treated cells however, degradation of ErbB2 still occurs in CHIPϪ/Ϫ cells, (Fig. 4A, compare lanes 1 and 2), but Cul5 knockdown served to

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E2 A ErbB2- B A3G-HA- C Rbx ElonginB- ElonginB- Cullin ElonginC- ElonginC- lamosobiR p 91 - iR ob som la p -91 B C 1 2 3 4 5 6 Φ ElonginCΔ4 ElonginB siRNA Vif - + + --+ SOCS box Control siRNA + + - ElonginB siRNA - - + Substrate receptor GA + - + Control siRNA + + -

D E A3G-HA- ErbB2- Vif- ElonginCΔ4-HA- Ribosomal p19- ElonginC 4-HA- 1 2 3 4 Β-Tubulin-Tubulin ErbB2 + + + + 1 2 3 4 Empty vector + + - - A3G-HA + + + + ElonginCΔ4 - - + + Empty vector + + - - GA + - + - ElonginCΔ4 - - + + Vif -+ -+

Fig. 5. Cul5-mediated degradation of ErbB2 occurs via an ElonginB-ElonginC-independent mechanism. (A) ElonginB and ElonginC are not required for GA-mediated degradation of ErbB2. 293T cells were cotransfected with ErbB2 expression vector and ElonginB or control siRNA as indicated. At 48 h after transfection, the cells were treated with GA or DMSO for 16 h. ErbB2 stability was assessed by Western blotting with antibodies against ErbB2, ElonginB, ElonginC, and ribosomal p19. (B) ElonginB and ElonginC are required for HIV Vif-mediated degradation of APOBEC3G (A3G). 293T cells were cotransfected with A3G and Vif expression vectors and ElonginB or control siRNA where indicated. At 48 h after transfection, the cells were harvested, and A3G stability was assessed by Western blotting with antibodies against A3G-HA, ElonginB, ElonginC, and ribosomal p19. (C) The ElonginC dominant negative mutant can no longer interact with the SOCS box containing substrate receptor, yet retains interaction with Cul5. (D) ElonginC is not required for GA-mediated degradation of ErbB2. 293T cells were cotransfected with ErbB2 expression vector and ElonginC⌬4 or control empty vector as indicated. At 48 h after transfection, the cells were treated with GA or DMSO for 16 h. ErbB2 stability was assessed by Western blotting with antibodies against ErbB2, HA-tagged ElonginC ⌬4, and ribosomal p19. (E) ElonginC is required for Vif-mediated degradation of A3G. 293T cells were cotransfected with A3G and Vif expression vectors and ElonginC⌬4 or control empty vector where indicated. Forty-eight hours post transfection A3G stability was assessed by Western blotting with antibodies against HA-A3G, HIV Vif, HA-tagged ElonginC ⌬4, and ribosomal p19.

stabilize ErbB2, even in the presence of GA (Fig. 4A, compare Hsp90 client protein HIF1-␣, suggesting that Cul5 may regulate lanes 3 and 1). In contrast, knockdown of another Cullin E3 multiple Hsp90 client proteins. ligase (Cul2) had little effect on GA-induced ErbB2 degradation (Fig. 4B). Taken together, these data suggest that Cul5 is Cul5-Induced Degradation of ErbB2 Occurs Independent of ElonginB required for GA-induced polyubiquitination and degradation of and ElonginC. Cul5 classically requires the adaptor proteins the Hsp90 client protein ErbB2. ElonginB and ElonginC to efficiently induce the proteasomal

Cul5 Is Required for Degradation of HIF1-␣, Another Hsp90 Client. Ub These findings led us to ask whether Cul5 also participates in the E1 regulation of other Hsp90 client proteins. HIF1-␣ is a transcrip-

tion factor that is involved in the regulation of angiogenesis and Ub GA E2 metabolism (30). Under normoxic conditions, the Cul2- Ub VHL E3 ubiquitin ligase induces the degradation of HIF1-␣, but ␣ Hsp70 under hypoxic conditions, HIF1- is stabilized (31). Stabilization Hsp70 Ub of HIF1-␣ under normoxic conditions has been observed in Erbb2 Erbb2 Erbb2 Cul5 Cul5 E2 tumors and VHL-null or mutant cell lines (32). Constitutive ␣ expression of HIF1- is important for vascularization, adapta- Hsp90 Hsp90 GA GA Hsp90 tion to hypoxia, and overall tumor survival. HIF1-␣ has also been Ub Ub Ub shown to associate with Hsp90 and is sensitive to GA-induced Ub

degradation via a mechanism that is independent of oxygen and Proteasome VHL-Cul2 (7, 33). We examined the potential involvement of Cul5 in GA-mediated, oxygen-independent degradation of Erbb2 HIF1-␣. In the presence of control siRNA, treatment with 3 ␮M GA for 16 h induced HIF1-␣ degradation (Fig. 4C, lane 1), when compared to control cells that were not subjected to GA (Fig. 4C, lane 2). However, when Cul5 expression was reduced by siRNA against Cul5, GA-induced degradation of HIF1-␣ was inhibited Fig. 6. A proposed model for Cul5-mediated regulation of Hsp90 client proteins. GA treatment or ATP hydrolysis induces remodeling of the Hsp90 (Fig. 4C, compare lane 3 to lane 1). We observed a similar effect chaperone complex, resulting in recruitment of the Cul5-E3 ubiquitin ligase, with the Cul5 dominant negative mutant, Cul5⌬Nedd8 (Fig. 4D). followed by polyubiquitination and proteasomal degradation of ErbB2 or Thus, Cul5 apparently also plays a role in the regulation of the other Hsp90 client proteins.

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810571106 Ehrlich et al. Downloaded by guest on September 24, 2021 degradation of its substrate. To determine whether ElonginB a number of cancers, suggesting that Cul5 suppression may be and ElonginC are required for ErbB2 degradation we used beneficial for tumor development or maintenance (35). Our data siRNA against ElonginB. Interestingly we observed that knock- also suggest that Cul5 can influence the effectiveness of anti- down of ElonginB resulted in a significant decrease in ElonginC cancer treatment with GA or its derivatives, and its potential (Fig. 5 A and B, lanes 3 and 6). This data supports the hypothesis effects should be considered when evaluating the clinical efficacy that ElonginB may be involved in stabilization of ElonginC (34). of these treatments. Thus, the relationship between oncogenesis In the presence of siRNA targeted against ElonginB, we did not and Cul5-mediated Hsp90 client regulation may represent an observe a defect in GA-induced degradation of ErbB2 when avenue for cancer drug development. compared to nontargeting control siRNA (Fig. 5A, compare lanes 1 and 3). However, we did observe a defect in HIV Methods Vif-mediated degradation of APOBEC3G, suggesting an effi- Cells, Plasmids, Transfection, Reagents, and Antibodies. 293T cells [AIDS Re- cient decrease in ElonginB and ElonginC levels (Fig. 5B, com- search Reagents Program, Division of AIDS, National Institute of Allergy pare lanes 2 and 3). To further evaluate the role of the and Infectious Diseases (NIAID), National Institutes of Health (NIH), cat no. 3522] were maintained in DMEM (Invitrogen) with 10% FBS and gentamy- ElonginB-C adaptor module in Cul5-mediated ErbB2 degrada- cin (5 ␮g/mL; D-10 medium) and passaged upon confluence. Plasmids tion, we used an ElonginC dominant negative mutant. This VR1012, pElonginC⌬4, pCul5-HA, pCul5-myc, and pCul5⌬Nedd8 have been mutant can bind Cul5 but can no longer bind the SOCS box in described (18, 19). pErbB2 and pCMV-HIF1-␣ HA were described previously the substrate receptor (Fig. 5C). This has been observed for both (29, 33). Cells were transfected with Mirus LT1 transfection reagent or HIV-1 Vif and adenovirus E4orf6 (19, 22). Transfection of the Lipofectamine2000 (Invitrogen) according to manufacturers’ instructions. ElonginC dominant negative mutant that can no longer interact The following antibodies used in this study have been described previously with the SOCS box had no effect on GA-mediated ErbB2 (18): anti-HA antibody-agarose conjugate, anti-Elongin B, anti-Elongin C, anti-myc, anti-HA, and anti-human ribosomal P antigens. In addition, the degradation (Fig. 5D, compare lane 1 with lane 3) but did effect following antibodies were used: anti-ErbB2 (Oncogene), anti-Cul5 (Santa- Vif-mediated A3G degradation (Fig. 5E, compare lanes 2 and 4). Cruz Biotechnology), anti-Cul2 (Zymed), anti-ubiquitin (Covance), anti- Since this mutant can no longer interact with the SOCS box, this Hsp70, and anti-Hsp90 (Stressgen). Geldanamycin (Invivogen) was stored as data suggests that Cul5-mediated degradation of ErbB2 occurs a 1 mM stock solution in DMSO and used at the indicated concentrations. in the absence of a SOCS box containing substrate receptor. In MG132 (EMD Biosciences) was stored as a 10 mM stock solution in DMSO addition, this data suggests Cul5-mediated ErbB2 regulation and used at 5 ␮M unless otherwise indicated. occurs via an ElonginB-ElonginC-independent, E3 ubiquitin ligase. Immunoprecipitation and Western Blotting. Immunoprecipitation was per- CELL BIOLOGY formed as described previously (18). The eluted materials were then analyzed Discussion by SDS-PAGE and Western blotting as described (18).

The data we present here suggests that Cul5 is recruited by the RNA Interference. siRNA duplexes (Dharmacon) were obtained as a smartpool. Hsp90 complex and serves to regulate Hsp90 client proteins Duplexes were transfected according to Dharmacon’s instructions using Lipo- via an ElonginB-ElonginC-independent mechanism. This is an fectamine 2000 (Invitrogen). Cells were transfected at 50% confluence and example of a Cullin-RING E3 ubiquitin ligase being involved harvested 72 h later. in chaperone-mediated protein regulation. We demonstrate Cul5 interaction with Hsp90, Hsp70, and ErbB2. Cul5 specif- shRNA Lentivirus Production and Transduction. shRNA and lentivirus packag- ically co-localizes with ErbB2 at the plasma membrane. Cul5 ing system were obtained from The RNAi Consortium collection available but not Cul2 is required for proteasomal degradation of through the Hit center at Johns Hopkins University as a gift from Jef Boeke. Lentivuruses were prepared, and cells were transduced following the ErbB2. protocol generated by the The RNAi Consortium at the Broad Institute. We used lentivirus delivered shRNA against Cul5 to eval- Briefly, 293T cells were plated in a 10-cm plate at a concentration of uate ErbB2 degradation in SKBR3 breast cancer cells and 1.3–1.5 ϫ 105 cells/mL and transfected at a confluence of approximately observed stabilization of ErbB2 in the presence of GA. We also 70% with the following plasmids: Cul5 or GFP shRNA pLKO.1 (20 ␮g), observed increased levels of ErbB2 in DMSO controls, sug- pMD2.G (6 ␮g), pRSV-Rev (5 ␮g), and pMDLg/pRRE (10 ␮g). Eighteen hours gesting that Cul5 plays a role in regulation of ErbB2 in both post transfection media was changed to high BSA virus production media. 293T cells ectopically expressing ErbB2 and ErbB2 positive Virus was harvested 48 h post transfection. Supernatant was centrifuged to SKBR3 breast cancer cells in the absence of GA. In addition, remove any cell debris. SKBR3 cells were infected by adding 10% of the ␣ harvested virus plus polybreene at a concentration of 8 ␮g/mL. Twenty-four Cul5 serves to regulate another Hsp90 client Hif1- , suggest- hours post infection, media was replaced with fresh media containing ing a role for Cul5 in Hsp90 client regulation. Interestingly, puromycin at a concentration of 2 ␮g/mL. Cells were selected for 1 week inhibition of the ElonginB-C module via siRNA and dominant before being used for the GA-induced degradation assay. negative mutant suggests that Cul5 may be functioning inde- pendently of the traditional adaptor proteins. The ElonginC In Vivo Ubiquitination Assays. 293T cells were cotransfected with ErbB2, dominant negative mutant can no longer interact with the Cul5⌬Nedd8, and control vector as indicated. At 48 h post transfection, the SOCS box in both HIV Vif and adenovirus E4orf6, suggesting cells were treated with 5 ␮M MG132 for 18 h. ErbB2 was immunoprecipitated that it can no longer interact with cellular SOCS box contain- as described previously. Immunoprecipitates were washed with a 500 ␮M NaCl ing substrate receptors. This data suggests that a Cul5 E3 ligase wash buffer. Samples were then analyzed by Western blot against ErbB2, ubiquitin, myc, and ␤-actin. is at work here. It is possible that Cul5 may be interacting directly with the Hsp90/Hsp70 chaperone complex, however Immunofluorescent Staining. 293T cells were transfected with the indicated this has been difficult to determine due to the dynamic nature plasmids as described above. At 24 h post transfection, the cells were of the complex. trypsinized and plated on glass coverslips at a dilution of one-eighth. Hsp90 client proteins play important roles in numerous cel- Sixteen hours post-plating, cells were fixed in 4% parafomaldehyde, per- lular processes, including signal transduction, regulation, meablized in 0.3% Triton X-100, then stained with the indicated antibod- cell cycle control, and apoptosis, and elevated expression of ies. Cells were visualized with a Zeiss Meta 510 confocal microscope and some Hsp90 client proteins has been implicated in the mainte- viewed with LSM software or a Nikon 90i and viewed with Velocity soft- nance and progression of a number of cancers. Our data raise the ware as indicated. possibility that Cul5 dysregulation may play a role in the ACKNOWLEDGMENTS. We thank R. Cohen, P.T. Sarkis, J. Romano, L. Tan, and overexpression of Hsp90 client proteins in certain tumors. Z. Huh for advice and technical assistance. This work was supported by Interestingly, Cul5 expression has been shown to be decreased in National Institutes of Health Grant AI062644 (to X.F.Y.).

Ehrlich et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 24, 2021 1. Dai C, Whitesell L, Rogers AB, Lindquist S (2007) Heat shock factor 1 is a powerful 20. Mehle A, Goncalves J, Santa-Marta M, McPike M, Gabuzda D (2004) Phosphorylation multifaceted modifier of carcinogenesis. Cell 130:1005–1018. of a novel SOCS-box regulates assembly of the HIV-1 Vif-Cul5 complex that promotes 2. Solit DB, Rosen N (2006) Hsp90: A novel target for cancer therapy. Curr Top Med Chem APOBEC3G degradation. Dev 18:2861–2866. 6:1205–1214. 21. Stracker TH, Carson CT, Weitzman MD (2002) Adenovirus oncoproteins inactivate the 3. Wegele H, Muller L, Buchner J (2004) Hsp70 and Hsp90—a relay team for protein Mre11-Rad50-NBS1 DNA repair complex. Nature 418:348–352. folding. Rev Physiol Biochem Pharmacol 151:1–44. 22. Luo K, et al. (2007) Adenovirus E4orf6 assembles with Cullin5-ElonginBElonginC E3 ubiq- 4. Zhang H, Burrows F (2004) Targeting multiple signal transduction pathways through uitin ligase through an HIV/SIV Vif-like BC-box to regulate p53. Faseb J 21:1742–1750. inhibition of Hsp90. J Mol Med 82:488–499. 23. Harada JN, Shevchenko A, Shevchenko A, Pallas DC, Berk AJ (2002) Analysis of the 5. Waterston RH, et al. (2002) Initial sequencing and comparative analysis of the mouse adenovirus E1B–55K-anchored proteome reveals its link to ubiquitination machinery. genome. Nature 420:520–562. J Virol 76:9194–9206. 6. Citri A, Kochupurakkal BS, Yarden Y (2004) The achilles heel of ErbB-2/HER2: Regula- 24. Querido E, et al. (2001) Degradation of p53 by adenovirus E4orf6 and E1B55K proteins tion by the Hsp90 chaperone machine and potential for pharmacological intervention. occurs via a novel mechanism involving a Cullin-containing complex. Genes Dev Cell Cycle 3:51–60. 7. Neckers L (2002) Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends 15:3104–3117. Mol Med 8(4 Suppl):S55–S61. 25. Baker A, Rohleder KJ, Hanakahi LA, Ketner G (2007) Adenovirus E4 34k and E1b 55k 8. Petroski MD, Deshaies RJ (2005) Function and regulation of cullin-RING ubiquitin oncoproteins target host DNA ligase IV for proteasomal degradation. J Virol 81:7034– ligases. Nat Rev Mol Cell Biol 6:9–20. 7040. 9. Pintard L, Willems A, Peter M (2004) Cullin-based ubiquitin ligases: Cul3-BTB complexes 26. Young JC, Agashe VR, Siegers K, Hartl FU (2004) Pathways of chaperone mediated join the family. EMBO J 23:1681–1687. in the cytosol. Nat Rev Mol Cell Biol 5:781–791. 10. Deshaies RJ (1999) SCF and Cullin/Ring H2-based ubiquitin ligases. Annu Rev Cell Dev 27. Prodromou C, et al. (1997) Identification and structural characterization of the ATP/ Biol 15:435–467. ADP-binding site in the Hsp90 molecular chaperone. Cell 90:65–75. 11. Kamura T, et al. (2004) VHL-box and SOCS-box domains determine binding specificity 28. Zhou P, et al. (2003) ErbB2 degradation mediated by the co-chaperone protein CHIP. for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases. Genes Dev 18:3055–3065. J Biol Chem 278:13829–13837. 12. Mehle A, Thomas ER, Rajendran KS, Gabuzda D (2006) A zinc-binding region in Vif 29. Xu W, et al. (2002) Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degra- binds Cul5 and determines Cullin selection. J Biol Chem 281:17259–17265. dative pathway for c-ErbB2/Neu. Proc Natl Acad Sci USA 99:12847–12852. 13. Xiao Z, Ehrlich E, Luo K, Xiong Y, Yu XF (2007) Zinc chelation inhibits HIV Vif activity and 30. Safran M, Kaelin WG, Jr (2003) HIF hydroxylation and the mammalian oxygen-sensing liberates antiviral function of the cytidine deaminase APOBEC3G. Faseb J 21:217–222. pathway. J Clin Invest 111:779–783. 14. Xiao Z, et al. (2007) Characterization of a novel Cullin5 binding domain in HIV-1 Vif. J 31. Lonergan KM, et al. (1998) Regulation of hypoxia-inducible mRNAs by the von Hippel- Mol Biol 373:541–550. Lindau tumor suppressor protein requires binding to complexes containing elongins 15. Xiao Z, et al. (2006) Assembly of HIV-1 Vif-Cul5 E3 ubiquitin ligase through a novel B/C and Cul2. Mol Cell Biol 18:732–741. zinc-binding domain-stabilized hydrophobic interface in Vif. Virology 349:290–299. 32. Maxwell PH, et al. (1999) The tumour suppressor protein VHL targets hypoxia inducible 16. Blagosklonny MV (2002) Hsp-90-associated oncoproteins: Multiple targets of geldana- factors for oxygen-dependent . Nature 399:271–275. mycin and its analogs. Leukemia 16:455–462. 17. Fay MJ, et al. (2003) Analysis of CUL-5 expression in breast epithelial cells, breast cancer 33. Isaacs JS, Jung YJ, Neckers L (2004) Aryl hydrocarbon nuclear translocator (ARNT) cell lines, normal tissues and tumor tissues. Mol Cancer 2:40. promotes oxygen-independent stabilization of hypoxia-inducible factor-1alpha by 18. Yu X, et al. (2003) Induction of APOBEC3G ubiquitination and degradation by an HIV-1 modulating an Hsp90-dependent regulatory pathway. J Biol Chem 279:16128–16135. Vif-Cul5-SCF complex. Science 302:1056–1060. 34. Stebbins CE, Kaelin WG, Jr, Pavletich NP (1999) Structure of the VHLElonginC-ElonginB 19. Yu Y, Xiao Z, Ehrlich ES, Yu X, Yu XF (2004) Selective assembly of HIV-1 Vif-Cul5- complex: Implications for VHL tumor suppressor function. Science 284:455–461. ElonginB-ElonginC E3 ubiquitin ligase complex through a novel SOCS box and up- 35. Chen XP, et al. (2002) Pim serine/threonine kinases regulate the stability of Socs- 1 stream cysteines. Genes Dev 18:2867–2872. protein. Proc Natl Acad Sci USA 99:2175–2180.

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