Journal of Molecular Biology
Volume 395, Issue 3, 22 January 2010, Pages 587-594
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Identification of Residues on Hsp70 and Hsp90 Ubiquitinated by the Cochaperone CHIP

https://doi.org/10.1016/j.jmb.2009.11.017Get rights and content

Abstract

Molecular chaperones Hsp70 and Hsp90 are in part responsible for maintaining the viability of cells by facilitating the folding and maturation process of many essential client proteins. The ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) has been shown in vitro and in vivo to associate with Hsp70 and Hsp90 and ubiquitinate them, thus targeting them to the proteasome for degradation. Here, we study one facet of this CHIP-mediated turnover by determining the lysine residues on human Hsp70 and Hsp90 ubiquitinated by CHIP. We performed in vitro ubiquitination reactions of the chaperones using purified components and analyzed the samples by tandem mass spectrometry to identify modified lysine residues. Six such ubiquitination sites were identified on Hsp70 (K325, K451, K524, K526, K559, and K561) and 13 ubiquitinated lysine residues were found on Hsp90 (K107, K204, K219, K275, K284, K347, K399, K477, K481, K538, K550, K607, and K623). We mapped the ubiquitination sites on homology models of almost full-length human Hsp70 and Hsp90, which were found to cluster in certain regions of the structures. Furthermore, we determined that CHIP forms polyubiquitin chains on Hsp70 and Hsp90 linked via K6, K11, K48, and K63. These findings clarify the mode of ubiquitination of Hsp70 and Hsp90 by CHIP, which ultimately leads to their degradation.

Introduction

Protein turnover is an essential cellular process needed for health and survival. It is a way to ensure proper function of proteins, regulate cellular mechanisms, and prevent the accumulation of the aberrant proteins. Here, we focus on the turnover of the mammalian molecular chaperones Hsp70 and Hsp90. Many proteins are dependent upon these chaperones for proper folding and maturation. Numerous kinases and transcription factors, for example, are ‘clients’ of Hsp70 and Hsp90.1

Hsp70 is a monomer with two domains: an N-terminal ATPase domain and a C-terminal client protein binding domain.2 Hsp90 functions as a dimer and contains three domains: an N-terminal ATPase domain, a middle domain, and a C-terminal dimerization domain.3 Through their C-terminal sequences, Hsp70 and Hsp90 bind the tetratricopeptide repeat 1 (TPR1) and TPR2A domains of Hsp70/Hsp90 organizing protein, respectively.4 An unfolded client protein first associates with Hsp70, where it is partially folded, and is then passed to Hsp90, where its maturation is completed.5 During the folding process, both chaperones undergo conformational changes that are coupled to their ATPase activities.

Hsp70 and Hsp90 turnover is dependent upon the ubiquitin–proteasome system. In this pathway, ubiquitin ligases attach the C-terminus of a ubiquitin molecule to typically a lysine side chain on a target protein via an isopeptide bond.6 In many cases, a polyubiquitin chain, in which the C-terminus of one ubiquitin is linked to a lysine on another ubiquitin, is attached to a target protein. It is known that a target protein modified by a chain of four or more ubiquitin molecules linked via K48 is recognized by the proteasome and subsequently degraded.7 It has recently been demonstrated that polyubiquitin chains linked via other lysine residues may also target the modified protein for degradation.8

CHIP (C-terminus of Hsc70 interacting protein) is the ubiquitin ligase that ubiquitinates Hsp70 and Hsp90. This enzyme contains an N-terminal TPR domain, a central α-helical domain, and a C-terminal U-box ubiquitin ligase domain.9, 10 By associating, via its TPR domain, with the C-terminal sequences of Hsp70 and Hsp90, CHIP has been proposed to ubiquitinate the chaperones' client proteins and target them to the proteasome for degradation.11 CHIP is thus the quality control regulator of the folding pathway. We and others have observed that Hsp70 and Hsp90 are also ubiquitinated by CHIP in vitro (L.K. and L.R., unpublished results).12, 13 In addition, CHIP-mediated turnover of Hsp70 and Hsp90 has been studied extensively in vivo.14 Over-expression of CHIP leads to a decrease in Hsp70 levels; conversely, an increase in Hsp70 was noted in the absence of CHIP. Furthermore, addition of exogenous CHIP to CHIP knock-out cells greatly decreased the half-life of both Hsp70 and Hsp90. Our data suggest that the cellular concentration of CHIP is fine-tuned relative to the concentrations of the chaperones, so that there is a constant, low level of CHIP-dependent Hsp70 and Hsp90 ubiquitination and consequent proteasome-mediated degradation (L.K. and L.R., unpublished results).

Here, we investigate the mechanism of Hsp70 and Hsp90 degradation by identifying the lysine residues on the chaperones ubiquitinated by CHIP and mapping these residues on homology models of Hsp70 and Hsp90 to find any structural regions where the ubiquitination sites cluster. We also determine that CHIP forms polyubiquitin chains on Hsp70 and Hsp90 and identify the types of ubiquitin–ubiquitin linkages in these chains.

Section snippets

Results

We performed CHIP-mediated in vitro ubiquitination of Hsp70 and Hsp90 using purified components. UbcH5c was used as the ubiquitin-conjugating enzyme because it has previously been shown to function with CHIP in vitro.13 Ubiquitination of Hsp70 and Hsp90 can be detected by the formation of higher-molecular-weight species that are evident following electrophoresis on SDS polyacrylamide gels (Fig. 1a). To ensure that these bands predominantly correspond to ubiquitinated Hsp70 or Hsp90 and not free

Discussion

In summary, we have identified not only the specific lysine residues on Hsp70 and Hsp90 ubiquitinated by CHIP but also the structural regions of the chaperones where these ubiquitination sites cluster. It is possible that CHIP also ubiquitinates other Hsp70 and Hsp90 residues not identified by these experiments as Hsp70 peptides, which include 27 out of the total 50 lysine residues, and Hsp90 peptides, which include 32 out of the total 75 lysine residues were not sequenced. For example, there

DNA constructs

Full-length human CHIP was cloned as a BamHI-NcoI restriction fragment from pcDNA3-CHIP (a gift from Cam Patterson)11 into pET11a (Stratagene), which had been modified to include a TEV (tobacco etch virus) protease cleavable His6 tag between the promoter and cloning cassette to create pET11a-His6-TEV-hCHIP. Full-length human His6-Hsp90β in pET-14b28 and human His6-Hsp70 in pET28 were gifts from Sophie Jackson. Full-length human UbcH5c in pET28a (without a tag) was a gift from Rachel Klevit.29

Recombinant protein purification

Acknowledgements

We thank TuKiet Lam at the Yale WM Keck Foundation Biotechnology Resource Laboratory for performing the mass spectrometry experiments and database searches as well as for helping analyze the data. We also thank Genaro Pimienta-Rosales for helpful discussions about experimental set up/data analysis and Tijana Grove, Robielyn Ilagan, Meredith Jackrel, and Aitziber López Cortajarena for their help in manuscript preparation.

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