Insights into the structural dynamics of the Hsp110–Hsp70 interaction reveal the mechanism for nucleotide exchange activity

Andréasson, Claes; Fiaux, Jocelyne; Rampelt, Heike; Druffel-Augustin, Silke; Bukau, Bernd

doi:10.1073/pnas.0804187105

Cited by 78 publications

(65 citation statements)

References 24 publications

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“…These data establish HSP105 as a potential target for the treatment of colon cancer patients, the majority of whom have APC mutations and active ␤-catenin signaling (3). Furthermore, like many other heat shock proteins, HSP105 has an ATPase domain at its N terminus (49,50). It will be interesting to see if the weak ATPase activity of HSP105 is required for its regulation of Wnt signaling and therefore has potential as a novel target for anticancer therapy.…”

Section: Discussionmentioning

confidence: 99%

HSP105 Recruits Protein Phosphatase 2A To Dephosphorylate β-Catenin

Yu¹,

Kakunda²,

Pham³

et al. 2015

Molecular and Cellular Biology

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The Wnt/␤-catenin pathway causes accumulation of ␤-catenin in the cytoplasm and its subsequent translocation into the nucleus to initiate the transcription of the target genes. Without Wnt stimulation, ␤-catenin forms a complex with axin (axis inhibitor), adenomatous polyposis coli (APC), casein kinase 1␣ (CK1␣), and glycogen synthase kinase 3␤ (GSK3␤) and undergoes phosphorylation-dependent ubiquitination. Phosphatases, such as protein phosphatase 2A (PP2A), interestingly, also are components of this degradation complex; therefore, a balance must be reached between phosphorylation and dephosphorylation. How this balance is regulated is largely unknown. Here we show that a heat shock protein, HSP105, is a previously unidentified component of the ␤-catenin degradation complex. HSP105 is required for Wnt signaling, since depletion of HSP105 compromises ␤-catenin accumulation and target gene transcription upon Wnt stimulation. Mechanistically, HSP105 depletion disrupts the integration of PP2A into the ␤-catenin degradation complex, favoring the hyperphosphorylation and degradation of ␤-catenin. HSP105 is overexpressed in many types of tumors, correlating with increased nuclear ␤-catenin protein levels and Wnt target gene upregulation. Furthermore, overexpression of HSP105 is a prognostic biomarker that correlates with poor overall survival in breast cancer patients as well as melanoma patients participating in the BRIM2 clinical study.

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Section: Discussionmentioning

confidence: 99%

HSP105 Recruits Protein Phosphatase 2A To Dephosphorylate β-Catenin

Yu¹,

Kakunda²,

Pham³

et al. 2015

Molecular and Cellular Biology

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“…Hydrogen-Deuterium Exchange Experiments and Mass Spectrometry-HX experiments were performed in a similar manner as described previously (9,28,29). Briefly, 100 -350 pmol of purified monomeric Ssa1 NBD, or Ssa1 NBD in complex with Lhs1 or Sse1 were preincubated for 3 min at 30°C and diluted 20-fold into D 2 O-based buffer (25 mM Hepes-KOH, pH 7.6, 50 mM KCl, 5 mM MgCl 2 ) to initiate amide proton-deuterium exchange.…”

Section: Methodsmentioning

confidence: 99%

“…Recent structural and biochemical analysis of the NEFs GrpE, Bag1, Bag2, HspBP1, and Hsp110 have revealed that each of these NEFs uses a unique Hsp70 NBD interaction interface (3)(4)(5)(6)(7)(8)(9). However, despite unique modes of binding, the actual mechanisms used to trigger nucleotide release fall into two principle classes.…”

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confidence: 99%

“…GrpE, Bag1, Bag2, and Hsp110 all promote nucleotide release by stabilizing similar Hsp70 NBD conformations that involve tilting NBD lobe II outwards. This tilting opens the NBD structure and facilitates nucleotide exchange (3)(4)(5)(7)(8)(9). In contrast, HspBP1/Fes1 associates with lobe II subdomain IIB and elicits a near global loss of tertiary structure of the NBD and thereby promotes nucleotide exchange (6,9).…”

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confidence: 99%

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The Endoplasmic Reticulum Grp170 Acts as a Nucleotide Exchange Factor of Hsp70 via a Mechanism Similar to That of the Cytosolic Hsp110

Andréasson

Rampelt²,

Fiaux³

et al. 2010

Journal of Biological Chemistry

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Grp170 and Hsp110 proteins constitute two evolutionary distinct branches of the Hsp70 family that share the ability to function as nucleotide exchange factors (NEFs) for canonical Hsp70s. Although the NEF mechanism of the cytoplasmic Hsp110s is well understood, little is known regarding the mechanism used by Grp170s in the endoplasmic reticulum. In this study, we compare the yeast Grp170 Lhs1 with the yeast Hsp110 Sse1. We find that residues important for Sse1 NEF activity are conserved in Lhs1 and that mutations in these residues in Lhs1 compromise NEF activity. As previously reported for Sse1, Lhs1 requires ATP to trigger nucleotide exchange in its cognate Hsp70 partner Kar2. Using site-specific cross-linking, we show that the nucleotide-binding domain (NBD) of Lhs1 interacts with the NBD of Kar2 face to face, and that Lhs1 contacts the side of the Kar2 NBD via its protruding C-terminal ␣-helical domain. To directly address the mechanism of nucleotide exchange, we have compared the hydrogen-exchange characteristics of a yeast Hsp70 NBD (Ssa1) in complex with either Sse1 or Lhs1. We find that Lhs1 and Sse1 induce very similar changes in the conformational dynamics in the Hsp70. Thus, our findings demonstrate that despite some differences between Hsp110 and Grp170 proteins, they use a similar mechanism to trigger nucleotide exchange.The Hsp70 chaperones are essential components of the cellular machinery that controls the conformational states of proteins. They are involved in diverse functions, including folding of proteins, transport of proteins across membranes, and regulation of signal transduction components (1, 2). The common activity of Hsp70 chaperones that underlies these diverse functions is the transient binding to peptide segments of protein substrates. The association of Hsp70 with substrates is controlled by its ATPase cycle. When ATP is bound to the N-terminal nucleotide-binding domain (NBD), 4 interdomain communication ensures that the C-terminal substrate-binding domain (SBD) exhibits low affinity for substrates, which consequently bind and release with high rates. Hydrolysis of ATP converts Hsp70 to the ADP-bound conformation, which traps the substrate with higher affinity. Exchange of ADP for ATP and concomitant substrate release completes this chaperone cycle.The chaperone cycle of Hsp70s is regulated by cofactors that facilitate either ATP hydrolysis or exchange of ADP for ATP. ATP hydrolysis is stimulated by J-domain-harboring co-chaperones of the Hsp40 class that provide substrate specificity to the Hsp70 chaperone by delivering clients. Release of substrate from Hsp70 is facilitated by nucleotide-exchange factors (NEFs) that accelerate nucleotide dissociation, thereby allowing ATP to rebind. NEFs function by associating with and stabilizing specific conformations of the Hsp70 NBD that exhibit low affinity for nucleotide. Rebinding of ATP to the Hsp70 results in dissociation of the NEF.Several structurally unrelated proteins function as NEFs for Hsp70s. Recent structural and biochemical ana...

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“…This unusually fast exchange kinetics is indicative of a highly dynamic and loosely folded protein conformation. Because nucleotide binding affects the conformational dynamics of other Hsp70 family members (15,19,20), we added 0.6 mM ATP to the reaction. The presence of ATP resulted in strongly decreased deuteron incorporation to 45% after a 2-min HX reaction (Fig.…”

Section: Rac Formation Decreases the Conformational Dynamics Ofmentioning

confidence: 99%