Down-regulation of the Mixed-lineage Dual Leucine Zipper-bearing Kinase by Heat Shock Protein 70 and Its Co-chaperone CHIP

Down-regulation of the Mixed-lineage Dual LeucineZipper-bearing Kinase by Heat Shock Protein 70 and ItsCo-chaperone CHIP*

Received for publication, August 9, 2006 Published, JBC Papers in Press, August 24, 2006, DOI10.1074/jbc.M607612200

Alex Daviau‡, Roxanne Proulx‡, Karine Robitaille‡, Marco Di Fruscio‡, Robert M. Tanguay§, Jacques Landry¶,Cam Patterson�, Yves Durocher**, and Richard Blouin‡1

From the ‡Departement de Biologie, Faculte des Sciences, Universite de Sherbrooke, Sherbrooke, Quebec J1K 2R1, Canada,§Laboratoire de Genetique Cellulaire et Developpementale, Departement de Medecine, Universite Laval, Pavillon C. E. Marchand,Ste-Foy, Quebec G1K 7P4, Canada, ¶Centre de Recherche en Cancerologie de l’Universite Laval, l’Hotel-Dieu de Quebec, CentreHospitalier Universitaire de Quebec, Quebec G1R 2J6, Canada, �Carolina Cardiovascular Biology Center, University of NorthCarolina, Chapel Hill, North Carolina 27599, and **Animal Cell Technology and Downstream Processing Group,Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada

Dual leucine zipper-bearing kinase (DLK) is a mixed-lineagekinase family member that acts as an upstream activator of thec-Jun N-terminal kinases. As opposed to other components ofthis pathway, very little is currently known regarding the mech-anisms by which DLK is regulated in mammalian cells. Here weidentify the stress-inducible heat shock protein 70 (Hsp70) as anegative regulator of DLK expression and activity. Support forthis notion derives from data showing that Hsp70 induces theproteasomal degradation of DLK when both proteins are co-expressed in COS-7 cells. Hsp70-mediated degradation occurswith expression of wild-type DLK, which functions as a consti-tutively activated protein in these cells but not kinase-defectiveDLK. Interestingly, the Hsp70 co-chaperone CHIP, an E3 ubiq-uitin ligase, seems to be indispensable for this process sinceHsp70 failed to induceDLKdegradation inCOS-7 cells express-ing a CHIP mutant unable to catalyze ubiquitination or inimmortalized fibroblasts derived from CHIP knock-out mice.Consistentwith these data,wehave found that endogenousDLKbecomes sensitive to CHIP-dependent proteasomal degrada-tion when it is activated by okadaic acid and that down-regula-tion of Hsp70 levels with an Hsp70 antisense attenuates thissensitivity. Therefore, our studies suggest that Hsp70 contrib-utes to the regulation of activated DLK by promoting its CHIP-dependent proteasomal degradation.

Dual leucine zipper-bearing kinase (DLK)2 is a serine/threo-nine kinase that belongs to a family of mitogen-activated pro-tein kinase kinase kinases, known as mixed-lineage kinases(MLKs) (1). Members of this family, which also include MLK1,

MLK2, MLK3, MLK4, leucine zipper-bearing kinase, andleucine zipper and sterile �-motif kinase (1), are characterizedat the structural level by the presence of a catalytic domainbearing amino acid motifs found in serine/threonine and tyro-sine kinases and one or two leucine zipper motifs, which regu-late their activity by mediating protein dimerization or oli-gomerization (2–5). A number of other interesting motifs thatare likely important for protein binding have also been identi-fied in specific members of the MLK family. For instance,MLK2 and MLK3 contain a Src homology 3 (SH3) domain intheir N-terminal region that binds, respectively, the GTPasedynamin and the Ste20-related protein kinaseHPK1 (6, 7). BothMLK proteins also possess a functional Cdc42/Rac interactivebinding (CRIB)motif thatmediates associationwithCdc42 andRac1 in a GTP-dependent manner (8, 9).The importance of the MLKs as signaling molecules is high-

lighted by the fact that these proteins act as key regulators of thec-Jun N-terminal kinase (JNK) subgroup of mitogen-activatedprotein kinases (1). Specifically, all MLK family members regu-late the JNK pathway by phosphorylating and activating theJNK direct upstream activators MKK4 and MKK7 (10–14). Inaddition to their role in catalyzing JNK activation, MLKs arealso known to contribute to apoptosis in neuronal cells. Indeed,when dominant negative forms of MLKs were expressed inneuronal PC12 cells and sympathetic neurons, death caused bynerve growth factor deprivation was severely inhibited (15).Evidence supporting the involvement of theMLKs in apoptosisalso derives from studies with theMLK inhibitor CEP1347 (16),which provides neuroprotection against numerous death-in-ducing stimuli (17–19).As is the case for the majority of MLK family members, rel-

atively little is known about themechanisms responsible for theactivation and regulation of DLK in mammalian cells. Currentevidence suggests that the leucine zipper domain of DLK playsa role in the activation process, as deletion of this region pre-vents dimerization, autophosphorylation, and stimulation ofthe JNK pathway (20).Work from a number of laboratories hasalso established that the regulation of DLK involves heterolo-gous interactions with scaffolding and inhibitory proteins. Thebinding of DLK to these proteins, in particular JNK-interacting

* This work was supported by grants from the Canadian Institutes of HealthResearch (to R. B., M. D., and R. M. T.). The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed. Tel.: 819-821-8000 (ext.62062); Fax: 819-821-8049; E-mail: [email protected].

2 The abbreviations used are: DLK, dual leucine zipper-bearing kinase; MLK,mixed-lineage kinase; JNK, c-Jun N-terminal kinase; Hsp70, stress-induc-ible heat shock protein 70; CHIP, C terminus of Hsc70-interacting protein;DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum;PMSF, phenylmethylsulfonyl fluoride.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 42, pp. 31467–31477, October 20, 2006Printed in the U.S.A.

OCTOBER 20, 2006 • VOLUME 281 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 31467

at UN

IVE

RS

ITE

LAV

AL on O

ctober 16, 2006 w

ww

.jbc.orgD

ownloaded from

http://www.jbc.org

protein (JIP)-1 and MAPK upstream kinase (MUK)-bindinginhibitory protein (MBIP), is likely to play an important role inDLK regulation by preventing its dimerization (21–23).Another importantmechanismbywhichDLK is proposed to beregulated is through changes in its phosphorylation status.Experimental evidence supporting the involvement of phos-phorylation in DLK regulation derives from the observation thatoligomerization-dependent autophosphorylation is required foractivation ofDLK and stimulation of the JNK pathway (20). Fur-thermore, other studies have revealed that DLK exists in part asa phosphoprotein in vivo and that treatment of cells with oka-daic acid, an inhibitor of protein phosphatases 1 and 2A, resultsin accumulation of phosphorylated DLK (24). Finally, Nakataet al. (25) recently demonstrated that DLK protein levels inCaenorhabditis elegans are down-regulated by an E3 ubiquitinligase, termed RPM-1, which was found to stimulate DLKubiquitination.In the current study we report that activated DLK either

exogenously or endogenously expressed is down-regulated bythe stress-inducible heat shock protein 70 (Hsp70), amolecularchaperone that confers cytoprotection in response to variousstress stimuli (26–29). Our study also shows that Hsp70-de-pendent DLK down-regulation is mediated by a proteasome-dependent mechanism involving the CHIP ubiquitin ligase.Thus, our results support a role for Hsp70 and CHIP as impor-tant regulators of DLK protein levels.

EXPERIMENTAL PROCEDURES

Chemicals, Reagents, and Antibodies—The proteasome inhibi-tor lactacystin and the rabbit polyclonal antibody against actinwere purchased from Sigma-Aldrich. The mouse monoclonalantibody for the detection of phospho-JNK and the rabbit poly-clonal antibody insensitive to the phosphorylation state of JNKwere purchased from Cell Signaling Technology Inc. (Beverly,MA). Themousemonoclonal antibodies against the T7 and thehexahistidine tag sequences were from Novagen, Inc. (Madi-son, WI) and Invitrogen, respectively. The anti-Myc mono-clonal antibody (clone 9E10) and okadaic acid were obtainedfrom Calbiochem-Novabiochem. The rabbit polyclonal anti-bodies against DLK, Hsp70, Hsp27, Hsp40, Hsp60, Hsc70,Hsp90�, Hsp90�, and CHIP were described previously (30–33). The rabbit anti-ubiquitin antibody was purchased fromRockland Immunochemicals, Inc. (Gilbertsville, PA). Recombi-nant Hsp70 was purchased from Stressgen BiotechnologiesCorp. (Victoria, BC, Canada). Cell culture reagents were fromInvitrogen,HyClone Laboratories (Logan, UT), CambrexCorp.(East Rutherford, NJ), or BIOSOURCE International Inc.(Camarillo, CA).Cell Culture—COS-7 cells were cultured in Dulbecco’s mod-

ified Eagle’s medium (DMEM) supplemented with 10% (v/v)heat-inactivated fetal bovine serum (FBS), 100 units/ml peni-cillin, 100 �g/ml streptomycin, and 25 �g/ml amphotericin B.Transformed lung fibroblasts derived from CHIP�/� andCHIP�/� mice were also maintained in DMEM plus 10% FBS.Plasmids and Transfection—The T7-tagged DLK expression

vector was generously provided by Dr. S. Hirai (Yokohama CityUniversity School of Medicine). The expression vector forT7-tagged catalytically inactiveDLKwas produced by replacing

the invariant lysine at position 185 within kinase subdomain IIwith arginine using the Stratagene QuikChange site-directedmutagenesis kit (La Jolla, CA). The entire coding region of thecloned gene for human hsp70 (34) was inserted into a mamma-lian expression vector under the control of the cytomegaloviruspromoter (CMV5). A mutant form of Hsp70 lacking the ATPbinding domain (Hsp70�ABD) was constructed with theHsp70 expression vector by in-frame deletion of amino acidresidues 120–428 of the published human Hsp70 sequence(34). pCW8 (expressing Myc-tagged ubiquitin K48R) was a giftfrom Dr. R. Kopito (35). The pcDNA3 expression plasmidsencoding wild-type Myc-tagged CHIP (Myc-CHIP) and itsU-box deletion mutant (Myc-CHIP�E4) have been describedpreviously (36). The antisense hsp70 pcDNA3 plasmid, con-taining a 500-base pair fragment of the human inducible hsp70cDNA in the antisense orientation (974–475), was a kind gift ofDr. M. Jaattela (37). For gene transfer experiments, COS-7,CHIP�/�, andCHIP�/� cells in exponential growth phasewereseeded at 2.5 � 105 viable cells/60-mm dish and allowed torecover for 24 h. Thereafter, cells were transfected or cotrans-fected with the different expression vectors mentioned aboveusing FuGENE 6 transfection reagent (Roche Diagnostics).Cells were harvested and processed for immunoblot analysis48 h after transfection. When indicated, medium was removed48 h after transfection, and cells were treated with lactacystin(10�M) and/or okadaic acid (400 nM) in 3ml of DMEM supple-mented with 10% (v/v) heat-inactivated FBS, 100 units/ml pen-icillin, 100 �g/ml streptomycin, and 25 �g/ml amphotericinB/60 mm at 37 °C. Then cells were harvested and processed forfurther analyses.Preparation of Cell Lysates and Immunoblotting—Cells were

lysed for 30 min at 4 °C in 15 mM Tris-HCl, pH 7.4, 1% TritonX-100, 0.5% sodium deoxycholate, 0.2% SDS, 150 mM NaCl, 1mM EGTA, 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride(PMSF), 1 �g/ml leupeptin, and 1 �g/ml aprotinin. Lysateswere clarified by centrifugation (13,000 rpm for 10min at 4 °C),and the concentration of total protein in the supernatant frac-tion was quantified by the modified Bradford protein assay(Bio-Rad). For immunoblotting, equal amounts of proteinswere fractionated on 10% reducing SDS-PAGE and transferredonto polyvinylidene difluoride membranes (Roche Diagnos-tics) using a semidry transfer apparatus (Hoefer ScientificInstruments). Membranes were blocked overnight at 4 °C in 20mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20 containing 5%skimmilk powder before incubation with the primary antibodyfor 1 h at room temperature. Immunoreactive bands weredetected by enhanced chemiluminescence using secondaryhorseradish peroxidase-conjugated antibodies (ECL PlusWestern blotting kit, Amersham Biosciences).Immunoprecipitation—Cells were lysed for 30 min at 4 °C in

50mMTris-HCl, pH 7.4, 150mMNaCl, 5 mM EDTA, 1% TritonX-100, 50 mM NaF, 0.2 mM Na3VO4, 1 mM PMSF, 1 �g/mlleupeptin, and 1�g/ml aprotinin. Lysates were clarified by cen-trifugation (13,000 rpm for 10 min at 4 °C), and the concentra-tion of total protein in the supernatant fraction was quantifiedusing themodified Bradford protein assay (Bio-Rad). Typically,500 �g of protein extracts were incubated for 4 h at 4 °C withconstant rotationwith the primary antibody andproteinA-aga-

Hsp70 and CHIP Act as Negative Regulators of DLK Protein Levels

31468 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 42 • OCTOBER 20, 2006

at UN

IVE

RS

ITE

LAV

AL on O

ctober 16, 2006 w

ww

.jbc.orgD

ownloaded from

http://www.jbc.org

rose beads. Immune complexes were collected by centrifuga-tion (13,000 rpm for 1 min) and washed three times with lysisbuffer. The resulting pellet was resuspended in 2� SDS-PAGE

sample buffer, boiled for 5min, frac-tionated on 10% SDS-PAGE, andprocessed for immunoblot analysisas described above.In Vitro Interaction Assay—The

full-length coding sequence of wild-type DLK was inserted in-framewith a His-tag sequence in thepTriEx-4 expression vector (Nova-gen) for production of recombinantproteins using the TNT� T7 Cou-pled Reticulocyte Lysate system(Promega Corp., Madison, WI).After nonradioactive translationHis-tagged wild-type DLK wasincubated for 2 h at 30 °C alone orin combination with recombinantHsp70 (1�g) in 50mMTris-HCl, pH7.4, 150 mM NaCl, 5 mM EDTA, 1%Triton X-100, 1 mM PMSF, 1 �g/mlleupeptin, and 1 �g/ml aprotinin.Proteins were then subjected toimmunoprecipitation with the anti-Hsp70 antibody, and the resultingimmunocomplexes were analyzedby immunoblotting with either theanti-Hsp70 or anti-His antibody.Pulse-ChaseAnalysis—COS-7cells

transiently expressing T7-taggedwild-type DLK in the presence orabsence of Hsp70 were starved inDMEMplus0.5%FBSwithoutmethi-onine and cysteine for 16 h and thenmetabolically labeled with [35S]me-thionine/cysteine (Easy TagTMEXPRESS,PerkinElmerLifeSciences)for 45 min. Subsequently, cells werechased in nonradioactivemedium forthe times indicated, lysed, and sub-jected to immunoprecipitation withthe anti-T7 antibody. Immunocom-plexes were resolved by SDS-PAGEand visualized by autoradiography.Immunocomplex Kinase Assay for

DLK—Cultures of CHIP�/� andCHIP�/� cells were incubated inthe absence or presence of okadaicacid (400 nM) and lactacystin (10�M) before homogenization in lysisbuffer (50mMTris-HCl, pH 7.4, 150mM NaCl, 5 mM EDTA, 1% TritonX-100, 50mMNaF, 0.2mMNa3VO4,1mM PMSF, 1 �g/ml leupeptin, and1�g/ml aprotinin). The lysateswereclarified by centrifugation, and the

concentration of total protein in the supernatant fraction wasquantified using themodifiedBradford protein assay (Bio-Rad).Typically, 600 �g of protein extract were incubated for 2 h at

FIGURE 1. Hsp70 down-regulates the expression of DLK. A, COS-7 cells were transiently transfected with anempty vector (3 �g) or with an expression construct (3 �g) encoding T7 epitope-tagged wild-type or kinase-defective (K185R) DLK. At 48 h after transfection, cells were lysed and processed for immunoblotting withantibodies to either the T7 epitope, Hsp70, Hsp27, Hsp40, Hsp60, Hsc70, Hsp90�, Hsp90�, or phosphorylatedJNK. The total level of JNK in cells was also analyzed by immunoblotting (WB) with a specific antibody. B, COS-7cells were transfected with an expression vector (0.5 �g) encoding T7 epitope-tagged wild-type or kinase-defective (K185R) DLK either alone or together with an expression construct for human Hsp70 (0.5 �g). At 48 hafter transfection, cells were lysed and processed for immunoblotting with antibodies to either the T7 tag orHsp70. As a control for protein loading, immunoblots were probed in parallel with an antibody targeting actin.Densitometry analysis was used to determine the relative levels of DLK expression in the absence or presenceof Hsp70. The total amount of plasmid was kept constant in each transfection condition by including an emptyvector. C, COS-7 cells were transiently transfected with a plasmid (0.5 �g) for T7-DLK either alone or togetherwith a vector (0.5 �g) for Hsp70 or Hsp70�ABD. At 48 h after transfection cells were lysed and processed forimmunoblotting with antibodies to either the T7 tag or Hsp70. As a control for protein loading, immunoblotswere probed in parallel with an anti-actin antibody. The total amount of plasmid was kept constant in eachtransfection condition by including an empty vector. The data in this figure and all the other figures arerepresentative of at least three independent experiments.



at UN

IVE

RS

ITE

LAV

AL on O

ctober 16, 2006 w

ww

.jbc.orgD

ownloaded from

http://www.jbc.org

4 °C with constant rotation using a polyclonal antibody againstDLK (30) and protein A-Sepharose beads. After incubation, theimmunocomplexes were washed 3 times with lysis buffer and 3times with kinase buffer (10 mM Tris-HCl, pH 7.4, 150 mMNaCl, 10 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mM PMSF, 0.2mM sodium orthovanadate, 1 �g/ml leupeptin, and 1 �g/mlaprotinin). Immunocomplex kinase assays were performed byincubating the immune complexes in 40 �l of kinase buffercontaining 2.5 �Ci of [�-32P]ATP (Amersham Biosciences), 25�MATP, and 1�g ofmyelin basic protein as a substrate. After a20-min incubation at 30 °C, the reaction was stopped by addingan appropriate volume of 6� SDS-PAGE sample buffer andboiling for 5 min. Phosphorylated proteins were visualized byautoradiography after fractionation by SDS-PAGE.

RESULTS

Down-regulation of DLK by Hsp70—During the course of astudy recently conducted in our laboratory, we noticed thatoverexpression of wild-type DLK inMCF-7 human breast can-cer cells resulted in the accumulation of a protein with amolec-ular weight (Mr) of �70,000 (p70), as revealed by CoomassieBlue staining of cell extracts fractionated on reducing SDS-PAGE. This protein bandwas excised from the gel and analyzedby tandemmass spectrometry at the EasternQuebec ProteomicCenter (Universite Laval, Quebec, Canada). A correlativesearch of the NCBI Protein Data base with the peptide production spectra generated by tandem mass spectrometry identifiedp70 as the stress-inducible protein Hsp70, a molecular chaper-one that has been shown to protect cells against the potentiallyfatal consequences of diverse physiological and environmentalinsults (26, 27). This result was subsequently confirmed byimmunoblot analysis of total proteins isolated from MCF-7cells overexpressing DLK with an antibody specific for theinducibleHsp70 protein (31). To determinewhetherDLKover-expression could elicit a similar induction of Hsp70 in anothercell system, we transfected COS-7 cells with an expression vec-tor encoding a T7 epitope-tagged form of wild-type DLK. At48 h after transfection cells were lysed and processed for immu-noblot analyses using either anti-T7 or anti-Hsp70 antibodies.The data presented in Fig. 1A demonstrate that overexpressedDLK, which functions as a constitutively activated protein ableto induce phosphorylation of endogenous JNK, substantiallyincreased the levels of endogenousHsp70when comparedwithcells transfected with an empty vector. Interestingly, this effectis specific for Hsp70, because expression of the other Hspsexamined, including Hsp27, Hsp40, Hsp60, Hsc70, Hsp90�,and Hsp90�, was not changed in DLK-transfected cells. More-over, although it was expressed at levels comparable with wild-type DLK, a catalytically inactive T7-tagged DLK mutant didnot influence the expression of any Hsps in transfected cells.Thus, these results confirm that the ectopic expression of DLKin COS-7 cells is sufficient to up-regulate in a specific mannerHsp70 protein levels and suggest that DLK kinase activity isrequired for Hsp70 induction.To investigate the physiological relevance of Hsp70 induc-

tion inDLK-transfected cells, we next assayed the effects of thismolecular chaperone on the steady-state levels of wild-typeand kinase-defective T7-DLK in COS-7 cells co-transfected

with an expression plasmid for humanHsp70. After transfection,cells were lysed and processed for immunoblot analysis with an-ti-T7 and anti-Hsp70 antibodies. Results shown in Fig. 1B revealsthat Hsp70 co-expression, as demonstrated by the immunoblotdata, reduces by �80% the abundance of wild-type T7-DLK intransfected cells. In contrast, the levels of the catalytically inactiveformof T7-DLK remained constant in response toHsp70 overex-pression. These data indicate that wild-type T7-DLK undergoesdown-regulation mediated at least in part by its own activity incells which overexpressed Hsp70.Because Hsp70 is composed of two functionally distinct

domains, an N-terminal ATPase domain responsible for itsprotein folding function and a C-terminal peptide-bindingdomain that specifically binds both unfolded and folded sub-strates (28, 38), we asked whether the ATPase activity of Hsp70could play a role inDLKdown-regulation. To this endwe trans-fected COS-7 cells with the T7-tagged DLK expression con-struct either alone or together with a plasmid encoding aHsp70deletion mutant that lacks the ATPase domain (amino acids120–428, Hsp70�ABD) and measured the protein levels ofDLK in these cells byWestern blot analysis (Fig. 1C). Aswas thecase for wild-type Hsp70, overexpression of Hsp70�ABD alsoresults in a dramatic decrease in ectopic DLK protein levels,indicating that the ATPase activity of Hsp70 is not essential topromote the down-regulation of DLK.Hsp70 Associates with Wild-type DLK in Intact Cells but Not

in Vitro—As an approach to explore the mechanism by whichHsp70 down-regulates DLK protein levels, we first examinedwhether these proteins could associate physically in intact cells.For this purpose, COS-7 cells transiently transfected with T7-tagged DLKwere subjected to immunoprecipitation with anti-T7antibody, and the resultant immunocomplexes were examined byWesternblot analysisusing theanti-Hsp70antibody (Fig. 2A).Theimmunoblot data reveals that endogenous Hsp70 associates withwild-type T7-DLK in COS-7 cells. Interestingly, this association

FIGURE 2. Hsp70 associates with DLK in intact cells but not in vitro. A,COS-7 cells were transiently transfected with empty vector (1 �g) or expres-sion plasmids for either T7-tagged wild-type DLK (1 �g) or T7-tagged kinase-defective (K185R) DLK (1 �g). At 48 h after transfection cells were lysed andimmunoprecipitated (IP) with anti-T7 antibody. Immune complexes werethen subjected to Western blotting (WB) with antibodies to Hsp70 or the T7tag. B, His-tagged DLK was incubated either alone or in combination withrecombinant Hsp70 for 2 h at 30 °C. After incubation proteins were subjectedto immunoprecipitation with the anti-Hsp70 antibody, and the resultingimmunocomplexes were analyzed by Western blotting using an antibodyagainst Hsp70 or the His tag. The presence of His-DLK and/or Hsp70 in inputmaterial (5%) was verified by Western blotting with the same antibodies.



at UN

IVE

RS

ITE

LAV

AL on O

ctober 16, 2006 w

ww

.jbc.orgD

ownloaded from

http://www.jbc.org

appears to be regulated by DLK kinase activity, since the catalyti-cally inactive T7-tagged DLKmutant did not co-immunoprecipi-tate endogenous Hsp70 (Fig. 2A).As a logical extension to thesedata,wenext sought to analyze in

vitro whether Hsp70 could interact directly with DLK. To do sorecombinant His-tagged DLK, produced by in vitro translation,was incubated either alone or in combination with recombinantHsp70. After incubation, proteins were subjected to immunopre-cipitationanalyseswith the anti-Hsp70antibody, and the resultingcomplexes were probed with either the anti-Hsp70 or anti-Hisantibody. The results shown in Fig. 2B indicates thatHis-DLKwasnot found in Hsp70 immunoprecipitates under these conditions.Taken together, these results suggest that the interactionobservedbetweenHsp70 andwild-typeDLK inCOS-7 cells is weak or indi-rect andpossibly involves theparticipationof at least anothermol-ecule that could bridge or stabilize this interaction.Hsp70 Reduces the Half-life of Nascent DLK—Given the ob-

servation that the steady-state levels of wild-type DLK were re-duced in cells co-expressingHsp70,wenext askedwhetherHsp70could affect the turnover ofDLK.To test this hypothesiswe deter-mined the half-life of DLK in the presence or absence ofHsp70 byusing pulse-chase experiments. Cells transfected with T7-taggedDLK either alone or together with Hsp70 were metabolically la-beled for 45 min with a mixture of [35S]methionine and [35S]cys-teine and then chased with unlabeled medium for various timeperiods. Subsequently,T7-DLKwas immunoprecipitatedwith the

anti-T7 antibody, resolved in SDS-PAGE, and visualized by autoradiog-raphy. As shown in Fig. 3, similarquantities of newly synthesized T7-DLK were detected at chasing time 0in COS-7 cells transiently co-trans-fected with or without Hsp70. How-ever, in the presence of Hsp70 thehalf-life of T7-DLK was reduced bygreater than50%.Thus, our data indi-cate that overexpressed Hsp70 dra-matically enhances thedegradationofDLK in COS-7 cells.Wild-type but Not Kinase-defective

DLK Is an Hsp90 Client Protein—Alarge number of signaling proteins inmammalian cells is known to associ-ate with a multichaperone complexconsisting of Hsp70, Hsp90, and dif-ferent co-factors (39).This chaperonesystem plays a major role not only inthe folding and maturation of clientproteins but also in their degradationdepending on the set of co-factors(39). Because wild-type T7-taggedDLK was found associated withHsp70, we examined whether endog-enous Hsp90 also forms a complexwith this protein in transiently trans-fected COS-7 cells. By analogy to thedata presented above, we found thatendogenousHsp90 could be detected

FIGURE 3. Hsp70 reduces the half-life of DLK. COS-7 cells were transfected withan expression vector (0.5 �g) encoding T7 epitope-tagged wild-type DLK eitheralone or together with an expression construct for human Hsp70 (0.5 �g). At 48 hafter transfection cells were serum-starved for 16 h, pulsed for 45 min with 25�Ci/ml [35S]methionine/cysteine, and then chased for the indicated time periodsin DMEM containing 10% FBS. 35S-Labeled T7-DLK was immunoprecipitated (IP)with the anti-T7 antibody, fractionated by SDS-PAGE, and visualized by autora-diography. The relative amount of newly synthesized T7-DLK was determined byliquid scintillation counting of the labeled T7-DLK protein band cut from the gel.The total amount of plasmid was kept constant in each transfection condition byincluding an empty vector.

FIGURE 4. Wild-type but not kinase-defective DLK is an Hsp90 client protein. A, COS-7 cells weretransiently transfected with empty vector (1 �g) or expression constructs for either T7-tagged wild-typeDLK (0.5 �g) or T7-tagged kinase-defective (K185R) DLK (0.5 �g) in the presence or absence of Hsp70 (0.5�g). At 48 h after transfection, cells were lysed and processed for immunoprecipitation (IP) with theanti-T7 antibody. The immunocomplexes were then subjected to Western blotting (WB) with anti-Hsp90�antibody. DLK and Hsp70 protein levels were determined by probing the lysates with the anti-T7 andanti-Hsp70 antibodies, respectively. As a control for protein loading, immunoblots were probed in parallelwith an anti-actin antibody. The total amount of plasmid was kept constant in each transfection conditionby including an empty vector. B, COS-7 cells transfected with either T7-tagged wild-type (1 �g) or kinase-defective (K185R) DLK (1 �g) were exposed for the indicated time periods to geldanamycin (GA) and thenprocessed for Western blotting with anti-T7 antibody. As a control for protein loading, immunoblots wereprobed in parallel with an anti-actin antibody.



at UN

IVE

RS

ITE

LAV

AL on O

ctober 16, 2006 w

ww

.jbc.orgD

ownloaded from

http://www.jbc.org

in immunoprecipitates of T7-tagged wild-type but not kinase-de-fectiveDLK (Fig. 4A). Becausebinding toHsp90 is required for thestability and/or activity of certain protein kinases (39), we won-dered whether Hsp90 association with wild-type DLK could bealtered by co-expression of Hsp70. Results shown in Fig. 4A dem-onstrated that overexpressedHsp70 enhances rather than inhibitsthe interactionofwild-typeDLKwithendogenousHsp90. In theseconditions,Hsp70was also able to induce the formation of a com-plex between the kinase-defective DLK variant and endogenousHsp90. Thus, it appears that down-regulation of wild-type DLKcaused by Hsp70 is not attributable to disruption of its binding toHsp90.To further examine the influence of Hsp90 on the stability of

DLK, we monitored the steady-state levels of both wild-typeand kinase-defective T7-DLK in COS-7 cells exposed for vari-ous times to the ansamycin antibiotic geldanamycin, whichspecifically binds to Hsp90 and usually promotes the degrada-tion of its client proteins (40). Treatment of transfected cellswith 5�M geldanamycin for 18 h led to the complete disappear-ance of wild-type DLK, whereas no significant effect on theexpression of its kinase-defective counterpart was observedunder the same conditions (Fig. 4B). Immunoblots processed inparallel with an antibody against actin ensured that this trendwas not attributed to variations in the loading of protein sam-ples. These data are, therefore, consistent with the idea thatwild-type but not kinase-defective DLK requires Hsp90 for sta-bility in COS-7 cells.DLK Is Subject to Proteasome-dependent Degradation in

Hsp70-transfected Cells—To further characterize the mecha-nism underlying DLK down-regulation in cells overexpressingHsp70, we asked whether DLK could serve as a substrate of theproteasomal degradationmachinery, which is linked to the heatshock protein network (36, 41–44). To test this we transfectedCOS-7 cells with a vector expressing T7-tagged DLK alone ortogether with an expression plasmid for Hsp70. At 48 h aftertransfection, cells were incubated with and without the protea-some inhibitor lactacystin before being processed for immuno-blotting with anti-T7 and anti-Hsp70 antibodies. As shown inFig. 5A, lactacystin treatment substantially suppresses the dis-appearance of T7-DLK in Hsp70-transfected cells, therebysuggesting that the 26 S proteasome might be responsible atleast in part for the down-regulation of DLK in response toHsp70 overexpression.Because the 26 S proteasome generally degrades intracellular

proteins in a ubiquitin-dependent manner (45), we next under-took experiments to determine the role of ubiquitination inHsp70-mediated degradation of T7-DLK. For this purpose weexamined by immunoblot analyses the steady-state level ofexogenously expressed T7-DLK in COS-7 cells that were co-transfected with Hsp70 and increasing amounts of a Myc-tagged ubiquitin mutant (K48R), which prevents the synthesisof polyubiquitin chains and inhibits degradation by the protea-some (35). As depicted in Fig. 5B, transfection of the K48Rmutant ubiquitin suppresses the Hsp70-induced down-regula-tion of T7-DLK in a dose-dependent manner. This resultimplies that Hsp70 promotes the proteasomal degradation ofDLK by a mechanism involving polyubiquitination.

CHIP Is Required for Hsp70-mediated Degradation of DLK—The ability of Hsp70 to promote proteasomal degradation ismediated at least in part by its interaction with the co-chaper-one CHIP, an E3 ubiquitin ligase of the U-box protein family(33, 46). To test the hypothesis thatCHIP is required forHsp70-mediated degradation of DLK, we first examined by coimmu-noprecipitation assay whether endogenous CHIP associateswith wild-type DLK transiently transfected in COS-7 cells.These studies showed that CHIP is present in immunoprecipi-tates of T7-tagged wild-type DLK (Fig. 6A). Interestingly, CHIP

FIGURE 5. Proteasome inhibition prevents the down-regulation of DLK inHsp70-transfected cells. A, COS-7 cells were transfected with empty vector(1 �g) or expression plasmids for either T7-tagged DLK (0.5 �g), Hsp70 (0.5�g) or T7-tagged DLK (0.5 �g) and Hps70 (0.5 �g). At 48 h after transfectioncells were left untreated or were treated with 10 �M lactacystin for 3 h. Afterincubation cells were lysed and processed for immunoblotting with anti-T7 oranti-Hsp70 antibodies. As a control for protein loading, immunoblots (WB)were probed in parallel with an anti-actin antibody. B, COS-7 cells were tran-siently transfected with a plasmid (0.5 �g) for T7-DLK either alone or togetherwith a vector (0.5 �g) for Hsp70 in the absence or presence of increasingamounts (0.5, 1, 2, and 4 �g) of an expression construct for a Myc-taggedubiquitin mutant (K48R). At 48 h after transfection, cells were lysed and pro-cessed for immunoblotting with anti-T7 or anti-Hsp70 antibodies. As a con-trol for protein loading, immunoblots were probed in parallel with an anti-actin antibody. The total amount of plasmid was kept constant in eachtransfection condition by including an empty vector.



at UN

IVE

RS

ITE

LAV

AL on O

ctober 16, 2006 w

ww

.jbc.orgD

ownloaded from

http://www.jbc.org

association with DLK seemed to depend onDLK kinase activitybecauseCHIPwas not co-immunoprecipitatedwith the kinase-defective DLK mutant even though this variant was expressedat levels similar to that of its wild-type counterpart.In the next series of experiments wemeasured the steady-state

levels of T7-DLK inCOS-7 cells that were transfectedwithHsp70

or co-transfected with Hsp70 and amutant form of CHIP lacking ubiq-uitin ligase activity (Myc-CHIP�E4).This mutant has been previouslyshown to act in a dominant-negativemanner to block the ubiquitinationand degradation of CHIP substrates,such as the glucocorticoid receptor(36) and the cystic-fibrosis trans-membrane conductance regulator(47).Asexpected fromtheabovedata,expression of Hsp70 greatly reducesthe steady-state levels of T7-DLK intransfected cells (Fig. 6B). However,inhibition of endogenous CHIPactivity by overexpression of itsU-box mutant almost completelyantagonizes the effects of Hsp70 onT7-DLK, suggesting that Hsp70inducesDLKdegradation inaCHIP-dependent manner.To further confirm a role for CHIP

in Hsp70-mediated degradation ofDLK, we compared the steady-statelevels of T7-tagged DLK in CHIP�/�

and CHIP�/� cells that have beentransiently transfected with Hsp70.Consistent with the results obtainedin COS-7 cells, the level of ectopicallyexpressedwild-typeDLKwas consid-erably reduced in CHIP�/� cells co-expressing Hsp70, whereas that of ki-nase-defective DLK remainedconstant (Fig. 6C). Importantly, nei-ther wild-type nor kinase-defectiveDLK steady-state levels were affectedby expression of Hsp70 in CHIP�/�

cells. However, reintroduction ofCHIP in CHIP�/� cells led to a dra-matic decrease in the steady-state lev-els of T7-tagged wild-type DLK, andthis was further enhanced when theHsp70 expression construct wasadded to the transfection mixture(Fig. 6D). As expected, down-regula-tion of DLK protein expression inthese cells was accompanied by a re-duction in phosphorylation but notprotein level of JNK. Thus, these datasuggest thatCHIP is essential in regu-lating the Hsp70-induced degrada-tion of active DLK.

In Its Active Form, Endogenous DLK Undergoes CHIP-dependent Ubiquitination and Degradation—After havingobserved in preliminary experiments that both CHIP�/� andCHIP�/� cells express endogenous levels of DLK, we pro-ceeded to investigate whether endogenous DLK could be sub-ject to CHIP-dependent ubiquitination and degradation. To do

FIGURE 6. CHIP is required for Hsp70-mediated DLK degradation. A, COS-7 cells were transiently trans-fected with empty vector (1 �g) or expression plasmids for either T7-tagged wild-type DLK (1 �g) or T7-taggedkinase-defective (K185R) DLK (1 �g). At 48 h after transfection, cells were lysed and immunoprecipitated (IP)with anti-T7 antibody. Immune complexes were then subjected to Western blotting (WB) with antibodies toCHIP. DLK protein levels were determined by probing the lysates with the anti-T7 antibody. As a control forprotein loading, immunoblots were probed in parallel with an anti-actin antibody. B, COS-7 cells were tran-siently transfected with a plasmid (0.5 �g) for T7-DLK either alone or together with a vector (0.5 �g) for Hsp70in the absence or presence of an expression construct (1 �g) for an Myc-tagged CHIP mutant lacking ubiquitinligase activity (Myc-CHIP�E4). At 48 h after transfection cells were lysed and processed for immunoblottingwith anti-T7, anti-Hsp70, or anti-Myc antibodies. As a control for protein loading, immunoblots were probed inparallel with an anti-actin antibody. The total amount of plasmid was kept constant in each transfectioncondition by including an empty vector. C, CHIP�/� and CHIP�/� cells were transiently transfected with expres-sion constructs for either T7-tagged wild-type DLK (0.5 �g) or T7-tagged kinase-defective (K185R) DLK (0.5 �g)in the presence or absence of Hsp70 (0.5 �g). At 48 h after transfection, cells were lysed and processed forimmunoblotting with anti-T7, anti-Hsp70, or anti-CHIP antibodies. As a control for protein loading immunob-lots were probed in parallel with an anti-actin antibody. The total amount of plasmid was kept constant in eachtransfection condition by including an empty vector. D, CHIP�/� cells were transiently transfected with aplasmid (0.5 �g) for T7-DLK either alone or together with a vector (0.5 �g) for Hsp70 and an expressionconstruct (2 �g) encoding a Myc-tagged wild-type form of CHIP. At 48 h after transfection cells were lysed andprocessed for immunoblotting with anti-T7, anti-Hsp70, or anti-Myc antibodies. The level of phosphorylated(p) and total JNK in these cells was also analyzed by immunoblotting with specific antibodies. As a control forprotein loading, immunoblots were probed in parallel with an anti-actin antibody. The total amount of plasmidwas kept constant in each transfection condition by including an empty vector.



at UN

IVE

RS

ITE

LAV

AL on O

ctober 16, 2006 w

ww

.jbc.orgD

ownloaded from

http://www.jbc.org

so CHIP�/� and CHIP�/� cells were incubated with and with-out the proteasome inhibitor lactacystin to prevent degradationof ubiquitinated proteins. Lysates prepared from these cellswere then subjected to immunoprecipitation analyses with ananti-DLK antibody (30), and the resulting immunocomplexeswere probed with an anti-ubiquitin antibody. Because DLKkinase activity seems to be required for degradation, as sug-gested by our results with ectopically expressedDLK, cells werealso exposed to the serine/threonine phosphatase inhibitorokadaic acid before being processed for immunoprecipitationto favor activation of endogenous DLK. Okadaic acid was cho-sen as a potential activator of DLK based on the findings ofMata et al. (24), who have observed accumulation of phospho-rylated DLK in aggregating neural-glial cultures after exposureto this compound. Results shown in Fig. 7 indicate that endog-enous DLK was neither ubiquitinated nor degraded inCHIP�/� cells cultured in the presence of okadaic acid at a doseinducing its activation, as can be seen by theDLK immunocom-plex assay. By contrast, in CHIP�/� cells a reduction of endog-enous DLK levels was observed after activation by okadaic acid.Treatment of these cells with lactacystin before supplementingthe culturemediumwith okadaic acid was sufficient to stabilizeendogenous DLK and promote the accumulation of immuno-reactive bands that migrate as a high molecular mass smear,likely corresponding to DLK molecules associated with ubiq-uitin chains of different lengths. Thus, these results support thenotion that endogenous DLK undergoes ubiquitination and

degradation in CHIP�/� cells when it is activated by okadaicacid.Down-regulation of Endogenous Hsp70 Impairs the CHIP-de-

pendent Proteasomal Degradation of Active DLK—To investi-gate the role of Hsp70 in CHIP-dependent degradation ofendogenous DLK, we examined whether inhibition of Hsp70expression could result in DLK stabilization. Accordingly,CHIP�/� and CHIP�/� cells were transiently transfected witheither an empty vector or anHsp70 antisense construct capableof inhibiting selectively the expression of Hsp70 (37), and thenthe cellswere incubatedwith okadaic acid for 2 h to induceDLKactivation. In some cases, transfected cells were also heat-shocked at 43 °C for 2 h and then recovered at 37 °C for 24 hbefore being processed for immunoblotting analyses. Such heatshock caused induction of Hsp70 in control but not antisenseHsp70 transfectants, indicating the effectiveness of the anti-sense Hsp70 construct (Fig. 8A). Our results also revealed thatheat shock alone neither influenced expression (Fig. 8A) noractivity (Fig. 8B) of endogenous DLK in both CHIP�/� andCHIP�/� cells. As expected from the above findings, a reduc-tion of endogenous DLK levels was only detected in CHIP�/�

cells after treatment with okadaic acid (Figs. 8, A and B). How-ever, when these cells were transfected with the antisense con-struct (Fig. 8A) or exposed to lactacystin (Fig. 8B) before incu-bationwith okadaic acid, no noticeable decrease in endogenousDLK levels was observed. Therefore, Hsp70 appears essentialfor CHIP-dependent proteasomal degradation of endogenousDLK once it is activated by okadaic acid.

DISCUSSION

Like all members of the MLK family, DLK acts as a key com-ponent of the JNK signaling pathway (1, 48). Although signifi-cant progress has been achieved in the past few years in ourunderstanding of how DLK regulates the JNK pathway, verylittle is known about the identity of its physiological agonistsand the mechanisms responsible for its own regulation. In thisstudy we report that the active form of DLK is targeted to theproteasome for degradation by the stress-inducible proteinHsp70 and its associated co-chaperoneCHIP.Hsp70 belongs toa subfamily of Hsps that have been recognized as molecularchaperones due to their ability to facilitate the folding andmat-uration of key regulatory proteins, such as nuclear receptors,kinases, and transcription factors (49). By analogy to other Hspfamilymembers, Hsp70 is induced in response to a wide varietyof chemical and physiological stresses, including those involvedin JNK activation, and its expression provides protectionagainst cell death (26, 27, 29). Themechanisms that account forthe cytoprotective activity of Hsp70 within the cell appear to behighly dependent on its ability to prevent protein aggregationand to induce refolding of damaged proteins (50). In addition,recent evidence has accumulated to show that Hsp70 caninhibit cell death by directly regulating the activity of proteinsthat are involved in various aspects of apoptosis. For instance, ithas been reported that Hsp70 blocks both caspase-dependentand caspase-independent cell death through interactions withkey apoptotic effector molecules, including apoptotic activat-ing factor-1 (Apaf1) (51–53) and apoptosis-inducing factor(AIF) (54). Interactions betweenHsp70 and kinases of signaling

FIGURE 7. Endogenous DLK undergoes CHIP-dependent ubiquitinationand degradation in its active form. CHIP�/� and CHIP�/� cells were incu-bated in the presence or absence of lactacystin (10 �M) for 3 h followed byconcurrent incubation for 2 h with 400 nM okadaic acid. Cells were subse-quently lysed and subjected to immunoprecipitation (IP) with the DLK anti-serum. Immune complexes were subjected to Western blotting (WB) with anantibody to ubiquitin (Ub). DLK protein levels were determined by probingthe lysates with the DLK antiserum. DLK activity was determined in the lysatesfrom these cells by an immunocomplex kinase assay using myelin basic pro-tein (MBP) as a substrate. As a control for protein loading, immunoblots wereprobed in parallel with an anti-actin antibody.



at UN

IVE

RS

ITE

LAV

AL on O

ctober 16, 2006 w

ww

.jbc.orgD

ownloaded from

http://www.jbc.org

pathways that transduce stress signals have also been observed.Park et al. (55) have found that Hsp70 can inhibit JNK activa-tion in cells exposed to UV irradiation and that this inhibitoryfunction is linked to the Hsp70 ability to directly bind JNK andsuppress its association with and phosphorylation by MKK4.The anti-apoptotic activity of Hsp70 is also reflected by itscapacity to interact with apoptosis signal-regulating kinase 1, amitogen-activated protein kinase kinase kinase of the JNK andp38 MAPK (mitogen-activated protein kinase) signaling path-ways that is required for cytokine- and stress-induced apoptosis(56). In this case, Hsp70 binding prevents both oligomerizationof apoptosis signal-regulating kinase 1 (ASK1), an event

required for its activation, as well asASK1-dependent apoptosis (57).In mammalian cells Hsp70 inter-

acts with a multitude of co-chaper-ones that regulate its activity andsubstrate affinity in a positive ornegative manner (29). Such interac-tions have also been suggested tomodulate Hsp70 activity by mediat-ing its recruitment to specific pro-teins, protein complexes, and sub-cellular compartment (43). Hsp70co-chaperones, such asHsp40,Hop,and BAG-1, have been shown toserve as interaction partners forprotein involved in signal transduc-tion, growth control, and cell differ-entiation as well as apoptosis (58–60). Hsp70 also cooperates with agroup of co-chaperones that havedegradatory functions (61). Oneexample is CHIP, a protein thatinteracts with the C terminus ofHsp70 via a tetratricopeptide repeatdomain and negatively regulates itsATPase activity (33). CHIP also actsas a U-box type E3 ubiquitin ligasethat promotes the polyubiquitina-tion and subsequent degradation ofHsp70 substrates, such as the glu-cocorticoid hormone receptor andthe protein kinase Raf-1 (41). Thus,through its association with CHIP,Hsp70 is converted from a proteinfolding machine into a specific deg-radation factor (41). The resultspresented here provide strong evi-dence that DLK, when activated,serves as a new molecular target forthe degradative activity of theHsp70-CHIP complex. Initial sup-port for this conclusion came fromdata demonstrating that exog-enously expressed wild-type DLK,which exists as a constitutively acti-vated protein, but not kinase-defec-

tive DLK was able to associate with both endogenous Hsp70and endogenous CHIP in COS-7 cells. The DLK associationwith Hsp70 could be apparently modulated by posttransla-tional events such as chemical modifications of specific aminoacids, given that no direct interaction was observed in vitrobetween recombinant forms of these proteins. Alternatively, itis also possible that Hsp70 and DLKmay be physically linked inCOS-7 cells by one ormore intermediatemolecules which pos-sess the ability to interact with both proteins. By using transfec-tion-based experiments, we further show that overexpressionof Hsp70 in COS-7 cells dramatically down-modulates thesteady-state levels of exogenously expressed wild-type DLK,

FIGURE 8. Hsp70 down-regulation impairs the CHIP-dependent degradation of endogenous DLK. A,CHIP�/� and CHIP�/� cells transiently transfected with an empty vector (5 �g) or an antisense Hsp70mRNA-expression plasmid (5 �g) were heat-shocked at 43 °C for 2 h and allowed to recover at 37 °C for 24 h. Atthe end of the recovery period, cells were left untreated or were treated with 400 nM okadaic acid for 2 h. Cellswere subsequently lysed and subjected to immunoblot analysis (WB) with antibodies to DLK, Hsp70, and actin.B, CHIP�/� and CHIP�/� cells were heat-shocked at 43 °C for 2 h and allowed to recover at 37 °C for 24 h. At theend of the recovery period cells were left untreated or were treated with 10 �M lactacystin for 3 h beforeincubation with 400 nM okadaic acid for 2 h. Cells were subsequently lysed and subjected to immunoblotanalysis with antibodies to DLK, Hsp70, and actin. DLK activity was determined in the lysates from these cells byan immunocomplex kinase assay using myelin basic protein (MBP) as a substrate.



at UN

IVE

RS

ITE

LAV

AL on O

ctober 16, 2006 w

ww

.jbc.orgD

ownloaded from

http://www.jbc.org

whereas its kinase-defective variant remains stable under thesame conditions. This finding is particularly significant becauseit suggests that Hsp70 antagonizes DLK expression only whenits kinase domain is functionally active. In addition, the ATPaseactivity of Hsp70, which is critical for its role in protein refold-ing, is not required for its effects onDLK since amutant form ofHsp70with theATPase domain deletedwas as effective aswild-type Hsp70 in promoting DLK down-regulation. Interestingly,the effects that Hsp70 has on wild-type DLK were blocked bytreatment of cells with the proteasome inhibitor lactacystin orexpression of a ubiquitin mutant incapable to form polyubiq-uitin chains, indicating the involvement of the ubiquitin-pro-teasome proteolytic pathway as the root cause of DLK down-regulation. Because Hsp70-induced DLK degradation was alsoreversed in COS-7 cells by a co-expressedCHIPmutant lackingubiquitin ligase activity, we then propose that CHIP may act asan important regulator of this process. In accordance with thispossibility, it has been found that Hsp70 fails to induce degra-dation of wild-type DLK when both proteins are co-expressedin CHIP�/� cells. However, adding back CHIP restores Hsp70ability to down-modulate wild-type DLK to levels comparablewith that seen in CHIP�/� cells. Thus, these results identifyCHIP as the ubiquitin ligase involved in the Hsp70-induceddegradation of exogenously expressed DLK.An important observation of this study is thatHsp70 andCHIP

also cooperate to promote the proteasomal degradation of endo-genously expressed DLK. This conclusion is supported by the fol-lowing arguments. First, endogenous DLK was ubiquitinated inCHIP�/� but not CHIP�/� cells. Second, okadaic acid, a serine/threonine phosphatase inhibitor that stimulates DLK activity inboth CHIP�/� and CHIP�/� cells, promoted down-regulation ofendogenous DLK only in CHIP�/� cells. Third, treatment ofCHIP�/� cells with the proteasome inhibitor lactacystin beforeokadaic acid stabilized endogenousDLKand favored the accumu-lation of ubiquitinated DLK conjugates. Finally, down-regulationof endogenous Hsp70 protein levels with an Hsp70 antisense wassufficient to prevent DLK degradation induced by okadaic acid inCHIP�/� cells. Therefore, our results indicate that endogenousDLK undergoes CHIP-dependent proteasomal degradation onceit is activated by okadaic acid and suggests that Hsp70 is requiredfor this process.DLK is the first example of a MLK family member whose

protein levels aremodulated byHsp70/CHIP-mediated protea-somal degradation. The significance of this observation is cur-rently unknown, but degradation of proteins via the protea-some is instrumental to various basic cellular processes, such asproliferation, cell cycling, differentiation, gene expression, andsignal transduction (62). Recent work has also established thatthe proteasome system plays a pivotal role in the regulation ofapoptosis by modulating the levels of proteins that have eitherpro-apoptotic or anti-apoptotic properties (63). Especiallyimportant among these are the tumor suppressor p53 (64), theBcl-2 family member Bax (65), and the inhibitor of apoptosis(IAP) proteins (66). It is clear from our data that activation ofboth exogenous and endogenous DLK serves as an essentialtrigger mechanism for its proteasomal-dependent proteolysis.Because DLK kinase activity modulates phosphorylation ofDLK in vivo, as reported previously (24), it will be very interest-

ing in future work to examine whether a specific phosphoryla-tion site(s) of DLK serves, at least in part, as a recognition signalfor the degradation machinery. Serine phosphorylation hasbeen found to be required for ubiquitination and degradation ofmany cell proteins that control proliferation and apoptosis,including the NF-�B inhibitor I�B�, which in its phosphoryla-ted form is targeted to the proteasome by the small heat shockprotein Hsp27 (67).In conclusion, the findings presented here point to an impor-

tant role for the Hsp70/CHIP degradation complex in the neg-ative regulation of DLK protein levels. It is possible that thecontrol of DLK levels by this complex might function as amechanism exploited by the cell to down-modulate DLK whenit becomes phosphorylated and activated in response to appro-priate stimulation. Given the role of DLK as a component of theJNK pathway, it is tempting to speculate that its degradationafter activation represents a novel regulatory mechanism bywhich Hsp70 modulates JNK-signaling events.

Acknowledgments—We thank Drs. Luc Gaudreau and AlainLavigueur for critical reading of the manuscript.

REFERENCES1. Gallo, K. A., and Johnson, G. L. (2002)Nat. Rev.Mol. Cell Biol. 3, 663–6722. Ezoe, K., Lee, S. T., Strunk, K. M., and Spritz, R. A. (1994) Oncogene 9,

935–9383. Gallo, K. A.,Mark,M. R., Scadden, D. T.,Wang, Z., Gu, Q., andGodowski,

P. J. (1994) J. Biol. Chem. 269, 15092–151004. Hirai, S., Katoh,M., Terada,M., Kyriakis, J.M., Zon, L. I., Rana, A., Avruch,

J., and Ohno, S. (1997) J. Biol. Chem. 272, 15167–151735. Sakuma, H., Ikeda, A., Oka, S., Kozutsumi, Y., Zanetta, J. P., and Kawasaki,

T. (1997) J. Biol. Chem. 272, 28622–286296. Kiefer, F., Tibbles, L. A., Anafi, M., Janssen, A., Zanke, B. W., Lassam, N.,

Pawson, T., Woodgett, J. R., and Iscove, N. N. (1996) EMBO J. 15,7013–7025

7. Rasmussen, R. K., Rusak, J., Price, G., Robinson, P. J., Simpson, R. J., andDorow, D. S. (1998) Biochem. J. 335, 119–124

8. Nagata, K., Puls, A., Futter, C., Aspenstrom, P., Schaefer, E., Nakata, T.,Hirokawa, N., and Hall, A. (1998) EMBO J. 17, 149–158

9. Teramoto, H., Coso, O. A., Miyata, H., Igishi, T., Miki, T., and Gutkind,J. S. (1996) J. Biol. Chem. 271, 27225–27228

10. Cuenda, A., and Dorow, D. S. (1998) Biochem. J. 333, 11–1511. Hirai, S., Noda, K., Moriguchi, T., Nishida, E., Yamashita, A., Deyama, T.,

Fukuyama, K., and Ohno, S. (1998) J. Biol. Chem. 273, 7406–741212. Merritt, S. E., Mata, M., Nihalani, D., Zhu, C., Hu, X., and Holzman, L. B.

(1999) J. Biol. Chem. 274, 10195–1020213. Rana, A., Gallo, K., Godowski, P., Hirai, S.-i., Ohno, S., Zon, L., Kyriakis,

J. M., and Avruch, J. (1996) J. Biol. Chem. 271, 19025–1902814. Tibbles, L. A., Ing, Y. L., Kiefer, F., Chan, J., Iscove, N.,Woodgett, J. R., and

Lassam, N. J. (1996) EMBO J. 15, 7026–703515. Xu, Z., Maroney, A. C., Dobrzanski, P., Kukekov, N. V., and Greene, L. A.

(2001)Mol. Cell. Biol. 21, 4713–472416. Maroney, A. C., Finn, J. P., Connors, T. J., Durkin, J. T., Angeles, T., Gress-

ner, G., Xu, Z., Meyer, S. L., Savage, M. J., Greene, L. A., Scott, R. W., andVaught, J. L. (2001) J. Biol. Chem. 276, 25302–25308

17. Maroney, A. C., Glicksman,M.A., Basma, A.N.,Walton, K.M., Knight, E.,Jr., Murphy, C. A., Bartlett, B. A., Finn, J. P., Angeles, T., Matsuda, Y., Neff,N. T., and Dionne, C. A. (1998) J. Neurosci. 18, 104–111

18. Maroney, A. C., Finn, J. P., Bozyczko-Coyne, D., O’Kane, T.M., Neff, N. T.,Tolkovsky, A. M., Park, D. S., Yan, C. Y., Troy, C. M., and Greene, L. A.(1999) J. Neurochem. 73, 1901–1912

19. Saporito, M. S., Hudkins, R. L., and Maroney, A. C. (2002) Prog. Med.Chem. 40, 23–62



at UN

IVE

RS

ITE

LAV

AL on O

ctober 16, 2006 w

ww

.jbc.orgD

ownloaded from

http://www.jbc.org

20. Nihalani, D., Merritt, S., and Holzman, L. B. (2000) J. Biol. Chem. 275,7273–7279

21. Fukuyama, K., Yoshida, M., Yamashita, A., Deyama, T., Baba, M., Suzuki,A., Mohri, H., Ikezawa, Z., Nakajima, H., Hirai, S., and Ohno, S. (2000)J. Biol. Chem. 275, 21247–21254

22. Nihalani, D., Meyer, D., Pajni, S., and Holzman, L. B. (2001) EMBO J. 20,3447–3458

23. Nihalani, D., Wong, H. N., and Holzman, L. B. (2003) J. Biol. Chem. 278,28694–28702

24. Mata, M., Merritt, S. E., Fan, G., Yu, G. G., and Holzman, L. B. (1996)J. Biol. Chem. 271, 16888–16896

25. Nakata, K., Abrams, B., Grill, B., Goncharov, A., Huang, X., Chisholm,A. D., and Jin, Y. (2005) Cell 120, 407–420

26. Beere, H. M. (2004) J. Cell Sci. 117, 2641–265127. Garrido, C., Schmitt, E., Cande, C., Vahsen, N., Parcellier, A., and Kro-

emer, G. (2003) Cell Cycle 2, 579–58428. Kiang, J. G., and Tsokos, G. C. (1998) Pharmacol. Ther. 80, 183–20129. Takayama, S., Reed, J. C., and Homma, S. (2003)Oncogene 22, 9041–904730. Douziech, M., Laberge, G., Grondin, G., Daigle, N., and Blouin, R. (1999)

J. Histochem. Cytochem. 47, 1287–129631. Tanguay, R. M., Wu, Y., and Khandjian, E. W. (1993) Dev. Genet. 14,

112–11832. Laplante, A. F., Moulin, V., Auger, F. A., Landry, J., Morrow, G., Tanguay,

R. M., and Germain, L. (1998) J. Histochem. Cytochem. 48, 1291–130133. Ballinger, C. A., Connell, P., Wu, Y., Hu, Z., Thompson, L. T., Yin, L.-Y.,

and Patterson, C. (1999)Mol. Cell. Biol. 19, 4535–454534. Hunt, C., and Morimoto, R. I. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,

6455–645935. Ward, C. L., Omura, S., and Kopito, R. R. (1995) Cell 83, 121–12736. Connell, P., Ballinger, C. A., Jiang, J., Wu, Y., Thompson, L., Hohfeld, J.,

and Patterson, C. (2001) Nat. Cell Biol. 18, 93–9637. Jaattela,M.,Wissing, D., Kokholm, K., Kallunki, T., and Egeblad,M. (1998)

EMBO J. 17, 6124–613438. Milarski, K. L., and Morimoto, R. I. (1989) J. Cell Biol. 109, 1947–196239. Wegele, H., Muller, L., and Buchner, J. (2004) Rev. Physiol. Biochem. Phar-

macol. 151, 1–4440. Stebbins, C. E., Russo, A. A., Schneider, C., Rosen, N., Hartl, F. U., and

Pavletich, N. P. (1997) Cell 89, 239–25041. Demand, J., Alberti, S., Patterson, C., and Hohfeld, J. (2001)Curr. Biol. 11,

1569–157742. Hayes, S. A., and Dice, J. F. (1996) J. Cell Biol. 132, 255–25843. Hohfeld, J., Cyr, D. M., and Patterson, C. (2001) EMBO Rep. 21, 885–89044. Imai, J., Yashiroda, H., Maruya, M., Yahara, I., and Tanaka, K. (2003) Cell

Cycle 2, 585–590

45. Glickman, M. H., and Ciechanover, A. (2002) Physiol. Rev. 82, 373–42846. McDonough, H., and Patterson, C. (2003) Cell Stress Chaperones 8,

303–30847. Meacham, G. C., Patterson, C., Zhang, W., Younger, M., and Cyr, D. M.

(2001) Nat. Cell Biol. 3, 100–10548. Fan, G., Merritt, S. E., Kortenjann, M., Shaw, P. E., and Holzman, L. B.

(1996) J. Biol. Chem. 271, 24788–2479349. Mayer, M. P., and Bukau, B. (2005) Cell. Mol. Life Sci. 62, 670–68450. Bukau, B., and Horwich, A. L. (1998) Cell 92, 351–36651. Beere, H. M., Wolf, B. B., Cain, K., Mosser, D. D., Mahboubi, A., Kuwana,

T., Tailor, P., Morimoto, R. I., Cohen, G. M., and Green, D. R. (2000)Nat.Cell Biol. 2, 469–475

52. Mosser, D. D., Caron, A. W., Bourget, L., Meriin, A. B., Sherman, M. Y.,Morimoto, R. I., and Massie, B. (2000)Mol. Cell. Biol. 20, 7146–7159

53. Saleh, A., Srinivasula, S. M., Balkir, L., Robbins, P. D., and Alnemri, E. S.(2000) Nat. Cell Biol. 2, 476–483

54. Ravagnan, L., Gurbuxani, S., Susin, S. A., Maisse, C., Daugas, E., Zamzami,N., Mak, T., Jaattela, M., Penninger, J. M., Garrido, C., and Kroemer, G.(2001) Nat. Cell Biol. 3, 839–843

55. Park, H. S., Lee, J. S., Huh, S. H., Seo, J. S., and Choi, E.-J. (2001) EMBO J.20, 446–456

56. Takeda, K., Matsuzawa, A., Nishitoh, H., and Ichijo, H. (2003) Cell Struct.Funct. 28, 23–29

57. Park, H. S., Cho, S. G., Kim, C. K., Hwang, H. S., Noh, K. T., Kim, M. S.,Huh, S. H., Kim, M. J., Ryoo, K., Kim, E. K., Kang, W. J., Lee, J. S., Seo, J. S.,Ko, Y. G., Kim, S., and Choi, E. J. (2002)Mol. Cell. Biol. 22, 7721–7730

58. Alberti, S., Esser, C., and Hohfeld, J. (2003) Cell Stress Chaperones 8,225–231

59. Fan, C. Y., Lee, S., andCyr, D.M. (2003)Cell Stress Chaperones 8, 309–31660. Odunuga, O. O., Longshaw, V. M., and Blatch, G. L. (2004) BioEssays 26,

1058–106861. Cyr, D. M., Hohfeld, J., and Patterson, C. (2002) Trends Biochem. Sci. 27,

368–37562. Naujokat, C., and Hoffman, S. (2002) Lab. Investig. 82, 965–98063. Jesenberger, V., and Jentsch, S. (2002)Nat. Rev. Mol. Cell Biol. 3, 112–12164. Maki, C. G., Huibregtse, J. M., and Howley, P. M. (1996) Cancer Res. 56,

2649–265465. Li, B., and Dou, P. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3850–385566. Yang, Y., Fang, S., Jensen, J. P., Weissman, A., and Ashwell, J. D. (2000)

Science 288, 874–87767. Parcellier, A., Schmitt, E., Guruxani, S., Seigneurin-Berny, D., Pance, A.,

Chantome, A., Plenchette, S., Khochbin, S., Solary, E., and Garrido, C.(2003)Mol. Cell. Biol. 23, 5790–5802



at UN

IVE

RS

ITE

LAV

AL on O

ctober 16, 2006 w

ww

.jbc.orgD

ownloaded from

http://www.jbc.org

Down-regulation of the Mixed-lineage Dual Leucine Zipper-bearing Kinase by Heat Shock Protein 70 and Its Co-chaperone CHIP

Documents