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Cooperation of a Ubiquitin Domain Protein and an E3 Ubiquitin Ligase

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Cooperation of a Ubiquitin Domain Protein and an E3 Ubiquitin Ligase Research Paper 1569 Cooperation of a ubiquitin domain protein and an E3 ubiquitin ligase during chaperone/proteasome coupling Jens Demand*§, Simon Alberti‡, Cam Patterson† and Jo¨ rg Ho¨ hfeld‡ Background: Molecular chaperones recognize nonnative proteins and Addresses: *Abteilung fu¨r Molekulare Zellbiologie, orchestrate cellular folding processes in conjunction with regulatory Max-Planck-Institut fu¨r Biochemie, D-82152 † cofactors. However, not every attempt to fold a protein is successful, and Martinsried, Germany. Program in Molecular Cardiology and Lineberger Comprehensive misfolded proteins can be directed to the cellular degradation machinery Cancer Center, University of North Carolina, for destruction. Molecular mechanisms underlying the cooperation of Chapel Hill, North Carolina 27599, USA. ‡Institut molecular chaperones with the degradation machinery remain largely fu¨r Zellbiologie, Rheinische-Friedrich-Wilhelms- enigmatic so far. Universita¨t Bonn, Ulrich-Haberland-Str. 61a, D-53121 Bonn, Germany. Results: By characterizing the chaperone cofactors BAG-1 and CHIP, we Present address: §MediGene AG, D-82152 gained insight into the cooperation of the molecular chaperones Hsc70 Martinsried, Germany. and Hsp70 with the ubiquitin/proteasome system, a major system for protein degradation in eukaryotic cells. The cofactor CHIP acts as a ubiquitin Correspondence: Dr. Jo¨ rg Ho¨ hfeld ligase in the ubiquitination of chaperone substrates such as the raf-1 protein E-mail: [email protected] kinase and the glucocorticoid hormone receptor. During targeting of signaling molecules to the proteasome, CHIP may cooperate with BAG-1, Received: 26 July 2001 21 August 2001 a ubiquitin domain protein previously shown to act as a coupling factor between Revised: Accepted: 4 September 2001 Hsc/Hsp70 and the proteasome. BAG-1 directly interacts with CHIP; it accepts substrates from Hsc/Hsp70 and presents associated proteins Published: 16 October 2001 to the CHIP ubiquitin conjugation machinery. Consequently, BAG-1 promotes CHIP-induced degradation of the glucocorticoid hormone Current Biology 2001, 11:1569–1577 receptor in vivo. 0960-9822/01/$ – see front matter Conclusions: The ubiquitin domain protein BAG-1 and the CHIP ubiquitin 2001 Elsevier Science Ltd. All rights reserved. ligase can cooperate to shift the activity of the Hsc/Hsp70 chaperone system from protein folding to degradation. The chaperone cofactors thus act as key regulators to influence protein quality control. Background after ubiquitin activation mediated by the E1 enzyme. The concerted effects of molecular chaperones and energy- Multiubiquitinated proteins are recognized by the 26S pro- dependent proteases appear to be required to mediate pro- teasome,a proteolytic complex that catalyzes their degrada- tein quality control [1–4]. Molecular chaperones recognize tion [13]. aberrantly folded proteins via associations with exposed hy- drophobic stretches [5,6]. By stabilizing the aberrant confor- The CHIP protein was recently shown to stimulate the mation,the chaperoning pathway promotes folding to the degradation of chaperone substrates by the ubiquitin/protea- native state whenever possible. If the native state is not some system [3,4]. CHIP binds to the constitutively ex- attainable,the damaged protein is targeted for degradation pressed Hsc70,the heat-inducible Hsp70,and also to Hsp90 by energy-dependent proteases. In an analogous fashion,through multiple tetratricopeptide (TPR) repeats and inhib- signaling molecules,such as steroid hormone receptors and its the folding activity of the chaperones [3,14] (Figure 1). some protein kinases,associate with molecular chaperones CHIP possesse s a U box motif,which was originally identi- in their inactive state [7–10]. Interactions of these proteins fied in the yeast multiubiquitination factor Ufd2 [15]. Ufd2 with the chaperone machinery facilitate conformational cooperates with an E2 ubiquitin-conjugating enzyme and changes that allow the signaling molecules to attain a folding an E3 ubiquitin ligase in the formation of multiubiquitin state that is required for their activation. However,the chap- chains and was therefore termed E4. The presence of a U erone-associated signaling molecules can also be diverted box within CHIP suggested that the cochaperone might to a degradative pathway,suggesting an additional interplay participate in ubiquitination reactions, possibly as an E4 between molecular chaperones and energy-dependent pro- enzyme. In fact,CHIP promotes the ubiquitination of chap- teases [2,3]. A major pathway for protein degradation in erone substrates and stimulates their degradation by the the eukaryotic cell involves the ubiquitin/proteasome sys- ubiquitin/proteasome pathway [3,4]. However,until now, tem [11,12]. Substrate proteins are tagged with the degrada- the enzymatic function of CHIP has been uncertain [16]. tion marker ubiquitin through the action of multiple E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases,Besides CHIP,thechaperone cofactor BAG-1 has also been 1570 Current Biology Vol 11 No 20 Figure 1 Figure 2 A schematic presentation of the domain structure of CHIP and BAG-1. CHIP possesses an amino-terminal chaperone binding motif formed by three TPR repeats and an adjacent highly charged region. A U box required for ubiquitin ligase activity is present at the carboxyl terminus. The BAG-1 isoforms share a ubiquitin-like domain involved in proteasome binding and a BAG domain that mediates interaction CHIP acts as an E3 ubiquitin ligase to stimulate the ubiquitination of with Hsp70. In addition, multiple repeats of the “TRSEEX” peptide raf-1 kinase. In vitro ubiquitination of raf-1 was performed in the motif of unknown function are found at the amino terminus of the presence of CHIP or a deletion fragment of the cofactor lacking the BAG-1 isoforms. U box (CHIP⌬U), as indicated. For detection of raf-1 and ubiquitinated forms of the kinase (Ub(n)-raf-1), the immunoblot was probed with a specific anti-raf-1 antibody. implicated to provide a link between chaperones and en- ergy-dependent proteases. BAG-1 exists as multiple iso- forms in the mammalian cell and acts as a nucleotide ex- likely to cooperate with chaperone systems [24]. The capac- change factor for Hsc/Hsp70,stimulating the release of ity of these components to mediate ubiquitination of bacteri- protein substrates from these chaperones [17–21]. BAG-1 ally expressed raf-1 in the presence of purified ubiquitin binds to Hsc/Hsp70 via its carboxy-terminal BAG domain, and ATP was investigated. Raf-1 can be presented to the which associates with the ATPase domain of the chaperones ubiquitin/proteasome system by Hsp90 and may thus be [22] (Figure 1). In addition,BAG-1 possesses a ubiquitin- affected by the function of the chaperone cofactor CHIP [2]. like domain at its amino terminus that is utilized,at least In fact,CHIP,together with UbcH5b,formed a conjugation in part,to target the chaperone cofactor to the 26S protea- system that efficiently mediated the attachment of ubiquitin some [23]. This targeting also induces an association of Hsc/ moieties to the raf-1 protein kinase (Figure 2). This activity Hsp70 with the proteasome,revealing BAG-1 as a coupling was dependent on the U box of CHIP,revealing the essen- factor linking the chaperones and the proteolytic complex. tial role of this protein motif for ubiquitin conjugation. In contrast to UbcH5b,the ER-associated UbcH7 was unable Here,we provide evidence that BAG-1 and CHIP cooperate to cooperate with CHIP,indicating specificity with regard in targeting chaperone substrates for degradation by the to E2 coupling in this reaction. The CHIP protein is thus proteasome. In addition,we report that CHIP functions as a chaperone-associated E3 ubiquitin ligase that cooperates an E3 ubiquitin ligase for chaperone substrates. Despite its with a specific E2 enzyme in the ubiquitination of signaling structural relationship to the E4 multiubiquitination factor molecules. Ufd2,CHIP directly cooperates with E2 conjugation en- zymes in ubiquitination reactions. Our study sheds light onto BAG-1 interacts with the CHIP ubiquitin ligase molecular mechanisms regulating the interplay of molecular BAG-1 was recently shown to act as a coupling factor be- chaperones and energy-dependent proteases. tween Hsc/Hsp70 and the proteasome [23]. It was therefore conceivable that BAG-1 cooperates with CHIP during the Results targeting of chaperone substrates for degradation. As a first CHIP is a chaperone-associated ubiquitin ligase step to verify this notion,we investigated whether BAG- Although a role for CHIP in the ubiquitination of chaperone 1 associates with the CHIP protein. Purified BAG-1M was substrates has been previously demonstrated [3,4],the added to a HeLa cell extract,followed by immunoprecipi- mechanism by which CHIP promotes ubiquitination of tar- tation using an anti-BAG-1 antibody,to identify cofactor- get proteins has so far been elusive. We therefore established associated proteins. Significant amounts of CHIP were an in vitro ubiquitination reaction using purified compo- detected in the BAG-1 immunocomplexes (Figure 3a). nents,including rabbit E1,the human E2 proteins UbcH5b Notably,a major portion of the BAG-1-associated CHIP and UbcH7,and CHIP (or a deletion mutant of CHIP was released during ATP treatment of the immunocom- lacking the U box). UbcH5b was chosen because members plexes,which also stimulated a quantitative release of of the Ubc4/5 family of ubiquitin-conjugating enzymes me- BAG-1-bound
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