Fibroblast growth factor receptor 3 (FGFR3) is a strong heat shock protein 90 (Hsp90) client: implications for therapeutic manipulation - PubMed (original) (raw)
Fibroblast growth factor receptor 3 (FGFR3) is a strong heat shock protein 90 (Hsp90) client: implications for therapeutic manipulation
Melanie B Laederich et al. J Biol Chem. 2011.
Abstract
Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of growth and differentiation, whose aberrant activation causes a number of genetic diseases including achondroplasia and cancer. Hsp90 is a specialized molecular chaperone involved in stabilizing a select set of proteins termed clients. Here, we delineate the relationship of Hsp90 and co-chaperone Cdc37 with FGFR3 and the FGFR family. FGFR3 strongly associates with these chaperone complexes and depends on them for stability and function. Inhibition of Hsp90 function using the geldanamycin analog 17-AAG induces the ubiquitination and degradation of FGFR3 and reduces the signaling capacity of FGFR3. Other FGFRs weakly interact with these chaperones and are differentially influenced by Hsp90 inhibition. The Hsp90-related ubiquitin ligase CHIP is able to interact and destabilize FGFR3. Our results establish FGFR3 as a strong Hsp90 client and suggest that modulating Hsp90 chaperone complexes may beneficially influence the stability and function of FGFR3 in disease.
Figures
FIGURE 1.
FGFR3 associates with Hsp90 and Cdc37. A, Coomassie stain of immunoprecipitated CA-FGFR3-GFP or GFP from 293 cells. Arrow 1, FGFR3; arrow 2, Hsp90A/B; arrow 3, Hsp70; arrow 4, IgG heavy chain; arrow 5, GFP. B, IP of transfected V5-tagged WT-, CA-, or KD-FGFR3 and immunoblotting (IB) for Hsp90, Cdc37, V5 (FR3) or phosphotyrosine (PY20) in 293 cells. C, IP of endogenous Hsp90 and blot for transfected FGFR3 (V5) in 293 cells. D, IP of endogenous Cdc37 and blot for transfected FGFR3 (V5) in 293 cells. E, IP of endogenous FGFR3 (FR3) from RT112 cells and blot for Hsp90, Hsp70, and Cdc37. IgG, rabbit IgG used as control. F, IP of Hsp90 from RT112 cells and blot for FGFR3 and EGFR. IgG, mouse IgG used as control. G, 293 cells inducibly expressing WT-FGFR3-GFP were treated for 1 h with increasing concentrations of Hsp90 inhibitors (17, 17-AAG; Rad, radicicol; Cel, celastrol; Que, quercetin), immunoprecipitated for GFP, and blotted as indicated.
FIGURE 2.
FGFRs differentially associate with Hsp90 complexes. A, sequence alignment of part of the EGFR, ErbB2, and FGFR family kinase domains. Gray shading indicates identical/related residues. Boxed residues and the asterisk mark the conserved glycine (FGFR3 Gly-533 in humans). Double asterisks indicate a negatively charged residue (Gly-548) absent in FGFR3 relative to the other FGFRs. Secondary structures are identified below by black boxes based on the analysis by Chen et al. (18). B, transfection of empty vector (V) or FGFR1–4 V5 in 293 cells followed by V5 IP and blotting. An arrow denotes the Cdc37 band running just below the IgG heavy chain. C, transfection in 293 cells of empty vector (V), WT-FGFR3 (WT), G533D FGFR3 (533), or G548D FGFR3 (548) followed by IP and blotting. −Avg indicates the averaged ratio of the amount of Hsp90 pulldown by mutated receptor relative to the wild-type receptor. D, 293 transfection of empty vector (V) or FGFR1–4 with a swapping mutation of the respective residue found in FGFR3 (548) as indicated by an asterisk (R1/2, D to G; R4, E to G; R3, G to D). E, 293 transfection followed by IP and blotting of FGFR3 (R3), FGFR2 (R2), or chimeras FGFR3/kinase-FGFR2 (K), FGFR3/N-terminal lobe-hinge-FGFR2 (N), and FGFR3/C-terminal lobe-kinase insert-FGFR2 (C).
FIGURE 3.
FGFR3 requires Hsp90 function for stability. A, time course of 1 μ
m
17-AAG or vehicle (DMSO, D) in whole cell lysates from RT112 cells. B, immunofluorescence of stably expressed WT-FGFR3-GFP in COS7 cells in the presence of DMSO or 1 μ
m
17-AAG for 3 h. C, 35S-labeled half-life of transfected WT-FGFR3 and or CA-FGFR3 in 293 cells in the presence or absence of 1 μ
m
17-AAG. The graph represents background subtracted densitometry relative to the percentage remaining at time 0. D, cycloheximide half-life analysis of immunoprecipitated and blotted FGFR3 in the presence or absence of 0.5 μ
m
17-AAG in RT112. The graph represents background subtracted band densitometry expressed as the percent remaining relative to 0.5 h. E, representative 35S-labeled pulse-chase half-life analysis scan of transfected FGFR1–4 V5 in 293 cells and chased in the presence or absence of 1 μ
m
17-AAG. The graph represents the average percent change in half-life in response to 1 μ
m
17-AAG relative to DMSO control. Error bars are mean ± S.D. (n = 3).
FIGURE 4.
Hsp90 inhibition results in FGFR3 ubiquitination. A, time course of total ubiquitin blotting of immunoprecipitated FGFR3 from RT112 in the presence of 0.5 μ
m
17-AAG or DMSO (D) for the indicated times. B, transiently expressed V5 tagged FGFR1–4 (R1–R4) in 293 cells for 48 h. Cells were treated for 1 h with 1 μ
m
17-AAG (17) or vehicle (D) followed by IP and blotting for polyubiquitin (Poly Ub). C, cotransfection (24 h) of WT- or CA-FGFR3-V5 with empty vector (V), CHIP-FLAG (C), or H260Q-FLAG (Q) in 293 cells, followed by V5 IP, and probed as indicated. D, equal protein loaded from whole cell lysates of cotransfected (48 h) WT-FGFR3-V5 with CHIP-FLAG (C), H260Q-FLAG (Q), or empty vector (V) E, 293 cells were cotransfected with WT-FGFR3-V5 and empty vector (V), CHIP-FLAG (C), or H260Q-FLAG (Q). After 24 h, cells were treated for 1 h with 1 μ
m
17-AAG or DMSO followed by IP and polyubiquitin blotting (Poly Ub). F, transfection and IP of WT-FGFR3-V5 in the presence of 5 μ
m
lactacystin (L) or vehicle (D, DMSO) for 5 h, with the last hour in the presence of 1 μ
m
17-AAG (17) or vehicle (D, DMSO).
FIGURE 5.
Loss of Hsp90 function reduces FGFR3 bioactivity. A, biotinylated RT112 cell surface proteins were chased in 1 μ
m
17-AAG (17) or DMSO (D) for indicated times, affinity purified, and blotted as indicated. Actin exposure serves as a control cell surface specific biotinylation. B, 35S-labeled pulse-chase of transfected WT-FGFR3-V5 in 293 cells in the presence of BFA (−B, no BFA control) and DMSO or 1 μ
m
17-AAG (17). −R3 is empty vector control. C, serum-starved RT112 incubated with 1 μ
m
17-AAG (17) or DMSO for 1.5 h and then treated with FGF1 for 10 min. D, MTT assay in RT112 cells after 48 h of growth in the presence of increasing concentrations of inhibitors. E, rat chondrosarcoma cells were pretreated for 4 h with DMSO, 17-AAG, or PD173074 (PD) and then stimulated with 50 ng/ml FGF9 in the continued presence of the drugs for 48 h. Data are graphed relative to the average vehicle absorbance (no FGF, DMSO). An asterisk indicates statistical significance by a two-tailed paired t test (p < 0.01). NS, not statistically different. Error bars are mean ± S.D.
References
- Eswarakumar V. P., Lax I., Schlessinger J. (2005) Cytokine Growth Factor Rev. 16, 139–149 - PubMed
- Knights V., Cook S. J. (2010) Pharmacol. Ther 125, 105–117 - PubMed
- Cappellen D., De Oliveira C., Ricol D., de Medina S., Bourdin J., Sastre-Garau X., Chopin D., Thiery J. P., Radvanyi F. (1999) Nat. Genet. 23, 18–20 - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources