Hsp90 provides a platform for kinase dephosphorylation by PP5

Abstract

The Hsp90 molecular chaperone collaborates with the phosphorylated Cdc37 cochaperone for the folding and activation of its many client kinases. As with many kinases, the Hsp90 client kinase CRaf is activated by phosphorylation at specific regulatory sites. The cochaperone phosphatase PP5 dephosphorylates CRaf and Cdc37 in an Hsp90-dependent manner. Although dephosphorylating Cdc37 has been proposed as a mechanism for releasing Hsp90-bound kinases, here we show that Hsp90 bound kinases sterically inhibit Cdc37 dephosphorylation indicating kinase release must occur before Cdc37 dephosphorylation. Our cryo-EM structure of PP5 in complex with Hsp90:Cdc37:CRaf reveals how Hsp90 both activates PP5 and scaffolds its association with the bound CRaf to dephosphorylate phosphorylation sites neighboring the kinase domain. Thus, we directly show how Hsp90's role in maintaining protein homeostasis goes beyond folding and activation to include post translationally modifying its client kinases.

Fig. 1. Kinase sterically blocks Cdc37 dephosphorylation.

a Mammalian purified Hsp90:Cdc37:CRaf complex (1.5 mM) was incubated with PP5 (75 nM) at 25 °C. The dephosphorylation of CRaf^pS338, CRaf^pS621 and Cdc37^pS13 was assayed by phospho-specific blotting (n = 3, rate ± SEM). CRaf^pS338 was preferentially dephosphorylated (yellow, 0.147 ± 0.012 min^–1), CRaf^pS621 was more slowly dephosphorylated (red, 0.063 ± 0.006 min^–1), while Cdc37^pS13 dephosphorylation was not apparent (blue, 0.001 ± 0.002 min^–1). Both native and tagged Cdc37 are visible in the α-Cdc37^pS13 blot. b Cdc37^pS13 (3 mM) was incubated with equimolar complex components (Hsp90^{open dimer}, Hsp90^{closed dimer}, BRaf) and PP5 (750 nM) at 25 °C (n = 3, rate ± SEM). Cdc37^pS13 dephosphorylation was assayed by α-Cdc37^pS13 blotting. There is no dephosphorylation when BRaf kinase is added to the Hsp90^open:Cdc37^pS13 complex (magenta, 0 min⁻¹). PP5 dephosphorylates Cdc37^pS13 bound to Hsp90^open (green, 0.024 ± 0.009 min⁻¹) faster than when bound to Hsp90^closed (gray, 0.018 ± 0.01 min⁻¹). PP5 does not dephosphorylate Cdc37^pS13 when Hsp90 is absent (blue, 0.004 ± 0.37 min⁻¹). All western blot data is available in Supplementary Information, and quantified data available in the provided Source Data file.

Fig. 2. Hsp90 activates PP5, acting as a phosphatase scaffold.

a Cryo-EM composite map and Hsp90:Cdc37:CRaf:PP5 complex model (b) show unfolded CRaf kinase (magenta) threaded through the closed Hsp90 dimer lumen (green), and embraced by Cdc37 (blue). PP5 is in an active conformation as its TPR domain (lavender) interacts with the Hsp90 CTDs, while the PP5 catalytic domain (purple) is near the Hsp90^MD and the CRaf C-lobe. c In the PP5 crystal structure (PDB: [1WAO]), the PP5 active site is occluded when not bound to Hsp90. d Overlaying the autoinhibited PP5 structure with the activated structure reveals PP5-Hsp90 steric clashes (e, in red) that would require rearrangement of the PP5 domain interface, possibly driving PP5 activation.

Fig. 3. Distinct TPR α7 helix – Hsp90 C-terminal domain interaction mode.

a The PP5 TPR domain interacts with the amphipathic Hsp90 dimer C-terminal groove. b Hydrophobic residues PP5^F148 and PP5^I152 bind the Hsp90 hydrophobic CTD groove, while c, d PP5^R113 and PP5^R150 electrostatically interact with the Hsp90 acidic tail. e Mutations to key TPR residues significantly decrease PP5 dephosphorylation of both Cdc37^pS13 and CRaf^pS338 substrates (mean rates ± SEM (min⁻¹) reported, CRaf: PP5^WT = 0.19 ± 0.016, PP5^R113D = 0.046 ± 0.013, PP5^F148K = 0.05 ± 0.02, PP5^R150D = 0.04 ± 0.03, Cdc37: PP5^WT = 0.043 ± 0.006, PP5^R113D = 0.006 ± 0.001, PP5^F148K = 0.009 ± 0.001, PP5^R150D = 0.012 ± 0.001). The differences between PP5 mutants and PP5^WT were significant in an ordinary one-way ANOVA (CRaf: n = 3, p < 0.0001; Cdc37: n^mutants = 3, n^WT = 4, p < 0.0001) with a Dunnett test for multiple hypothesis testing (PP5^WT vs PP5^muts p < 0.0001). f Mutations to key TPR residues decrease Hsp90^closed:PP5 complex formation measured by FCS hydrodynamic radius. Hsp90^closed dimer concentrations were used for K_D evaluation. Hsp90:PP5 K_D’s were solved by nonlinear regression (K_D ± SEM (mM) reported, n^mutants = 4, n^WT = 3: PP5^WT = 0.23 ± 0.04; PP5^R113D = 4.3 ± 0.8; PP5^F148K = 2.0 ± 0.4; PP5^R150D = 3.3 ± 0.6). R_hydmax and R_hydPP5 were fit globally, an ordinary one-way ANOVA showed significant differences (F = 9.8, p < 0.0001) with Dunnett’s multiple hypothesis test for comparison of individual interactions (PP5^WT vs PP5^F148K p = 0.09, vs PP5^R113D p = <0.0001, vs PP5^R150D p = 0.0006). Source data are provided as a Source Data file.

Fig. 4. Heterogeneous PP5 catalytic domain is in position for CRaf^pS621 dephosphorylation.

a The PP5 catalytic domain sits by Hsp90^MD allowing for CRaf kinase dephosphorylation. b Multiple PP5 conformations can be teased apart through focused classification. Anchored by its TPR domain, PP5 is flexibly held in proximity to the CRaf^pS621. c In some classes, the PP5 linker can be seen leaving the Hsp90 CTD groove and leading up to the PP5 active site. d A frame from a 3D variability analysis movie (Supplementary Movie 3) shows density spanning between the C-terminus of CRaf and the PP5 active site. e Focused classification of the interface between the CRaf C-lobe and the catalytic domain yields a similar class. A model was fit based on the substrate-bound PP5 crystal structure (PDB: [5HPE]) to show how CRaf^pS621 substrate may be positioned right at the PP5 active site. f Low-resolution volumes from the longer Hsp90:Cdc37:CRaf^{304–648,S338E}:PP5^{αJ del} complex used in the biochemical experiments show density for the PP5 catalytic domain interacting with both the CRaf N-lobe and the CRaf C-lobe multiple PP5 orientations. The heterogeneity seen in the upper panel (gray) can be resolved into complexes having either the N-terminal or C-terminal interactions (bottom panel, purple and blue).

Fig. 5. Deletion of PP5’s αJ helix increases rate of dephosphorylation.

a In its inhibited conformation, the PP5 αJ helix (orange) lies at the hydrophobic interface between the PP5 catalytic and TPR domains. b While initial focused classification around PP5 showed no αJ helix density, c further classification showed potential αJ helix density near the PP5 catalytic domain. d PP5^{αJ del} does not dephosphorylate Cdc37 without Hsp90 present (Rates ± S.E.M. reported (min⁻¹), 0.0 ± 0.0), but PP5^{αJ del} dephosphorylates Cdc37 while bound to Hsp90 at a slightly higher rate than PP5^WT (0.055 ± 0.007 vs 0.043 ± 0.006). An ordinary one-way ANOVA found differences significant (F = 29.40, p < 0.0001), while Dunnett’s multiple comparisons test showed significance between Hsp90-free complexes and Hsp90:PP5^WT complexes (p < 0.0001), but not between PP5^{αJ del} and PP5^WT (0.055 ± 0.007 and 0.043 ± 0.006, p = 0.17). e PP5^{αJ del} led to a slight increase in CRaf^pS338 dephosphorylation as compared to PP5^WT (0.28 ± 0.03 vs 0.187 ± 0.016 min⁻¹, two-tailed unpaired t-test: t = 2.9, d.f. = 20, p = 0.009). f The binding affinity of fluorescently labeled PP5^WT or PP5^{αJ del} to Hsp90^open or Hsp90^closed was measured through FCS (n = 3, K_D ± S.E.M. (μM) reported). Significant difference between the K_D’s of PP5^WT and PP5^{αJ del} to Hsp90^open and Hsp90^closed was found in an ordinary one-way ANOVA (F = 5.3, p = 0.002), with a Šidák’s multiple comparisons test comparing individual interactions (Hsp90^open:PP5^WT vs PP5^{αJ del} = 0.62 ± 0.17 vs 0.12 ± 0.018, p = 0.0022 and Hsp90^closed:PP5^WT vs PP5^{αJ del} = 0.43 ± 0.09 vs 0.13 ± 0.03, p = 0.11). Source data has been provided as a Source Data file.

Fig. 6. Model of PP5’s role in kinase and Cdc37 dephosphorylation.

Steric blocking of Cdc37^pS13 dephosphorylation by kinase clients suggests that Cdc37 can only be dephosphorylated once the kinase has exited the Hsp90 complex. Our model goes as follows: (1) Cdc37^pS13 first recruits the kinase to Hsp90 for folding or modification. (2) PP5 can then dephosphorylate CRaf, binding to Hsp90 in either direction to allow for the removal of phosphate groups on either side of the Hsp90-bound kinase. (3) After dephosphorylation of CRaf and potential conformational modifications by Hsp90, the phosphorylation-free kinase can be released. (4) As the kinase is released from Hsp90, Cdc37^pS13 may remain bound to Hsp90 until it’s dephosphorylated by PP5. This would allow for a unidirectional Hsp90-kinase cycle.