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. Author manuscript; available in PMC: 2019 Feb 15.
Published in final edited form as: Methods Mol Biol. 2018;1709:209–219. doi: 10.1007/978-1-4939-7477-1_16

Detecting post-translational modifications of Hsp90

Rebecca A Sager 1,2,3, Mark R Woodford 1,2, Len Neckers 4, Mehdi Mollapour 1,2,3,†
PMCID: PMC6376113  NIHMSID: NIHMS1004304  PMID: 29177662

Summary

The molecular chaperone Heat Shock Protein 90 (Hsp90) is essential in eukaryotes. Hsp90 chaperones proteins that are important determinants of multistep carcinogenesis. The chaperone function of Hsp90 is linked to its ability to bind and hydrolyze ATP. Co-chaperones as well as post-translational modifications (phosphorylation, SUMOylation and ubiquitination) are important for its stability and regulation of the ATPase activity. Both mammalian and yeast cells can be used to express and purify Hsp90 and also detect its post-translational modifications by immunoblotting.

Keywords: Heat Shock Protein 90 (Hsp90), Molecular Chaperones, Post-translational modification, Phosphorylation, SUMOylation, Ubiquitination

1. Introduction

Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes 1,2. Its cellular functions have been most clearly identified in mammalian cells, Drosophilia, and baker’s yeast Saccharomyces cerevisiae 3. Hsp90 creates and maintains the functional conformation of a subset of proteins 4–6. These targets (or “clients”) are key components of signal transduction pathways and numerous transcription factors. Hsp90 and a discrete set of co-chaperone proteins “hold” these clients in a state from which they can respond to activating signals 7.

Hsp90 chaperone activity depends on ATP binding and hydrolysis 8–10. This is coupled to a conformational cycle involving the opening and closing of a dimeric “molecular clamp” via transient association of Hsp90’s N-terminal domain 11,12. The N-domain also binds the antitumor antibiotics geldanamycin and radicicol, both Hsp90 inhibitors 13–17.

Hsp90 ATPase activity is also regulated by co-chaperones. For example, HopSti1 18–20, p50Cdc37 21–23, and p23Sba1 24,25 have an inhibitory effect on the ATPase cycle of Hsp90, while Aha1 26–30, and Cpr6 31,32 have an activating effect.

Hsp90 is post-translationally modified (PTMs) by phosphorylation, acetylation, S-nitrosylation, ubiquitination, and SUMOylation 33–36. These PTMs, in concert with co-chaperones, fine-tune Hsp90 chaperone function, which ultimately leads to chaperoning of kinase and non-kinase client proteins of Hsp90 37,38. The most extensively studied Hsp90 PTM is phosphorylation 39–49. Early work has shown that cells treated with the serine/threonine phosphatase inhibitor, okadaic acid, demonstrate Hsp90 hyperphosphorylation and decreasesd association of the chaperone with pp60v-Src, suggesting a link between Hsp90 phosphorylation and chaperoning of its target proteins 50,51. Subsequent study has shown that c-Src directly phosphorylates Tyr300 of Hsp90 under basal conditions, reducing its ability to chaperone client proteins 48.

Recent work has shown that Hsp90 is also subject to SUMOylation, which is an addition of a small ubiquitin-like modifier to a lysine residue. This modification affects cellular localization or function of a protein rather than signal for its degradation like ubiquitination. SUMOylation of Lys191 in human Hsp90α (Lys178 in yeast) promotes its binding to the co-chaperone Aha1 and also increases cells’ sensitivity to Hsp90 inhibitors 33.

Lack of PTM-specific antibodies has made it difficult to study PTMs of Hsp90. There is currently only one phospho-Hsp90α antibody (Cell Signaling) available for detecting the phosphorylation of Hsp90α-Thr5/7 52. Also HSP90 gene knockouts are lethal in mammalian systems, therefore any PTM Hsp90 mutant must be investigated in a background of highly expressed native mammalian Hsp90 proteins.

Simple baker’s yeast, Saccharomyces cerevisiae, is a well-established and valuable tool for studying various aspects of conserved protein chaperone machinery. The yeast system has provided us with a powerful tool to study Hsp90 phosphorylation, since it readily allows plasmid exchange whereby any introduced Hsp90 gene – provided it is partially functional – can provide 100% of the Hsp90 of the cell (Figure 1). Such genetic modifications are simply not achievable in cultured mammalian cells. This plasmid exchange (Figure 1) was used to isolate temperature sensitive (ts) Hsp90 mutants.

Figure 1.

Figure 1.

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With plasmid shuffling, a mutant hsp90 gene can be made to provide all the Hsp90 of the yeast cell (yHsp90=Hsp82 and yHsc90=Hsc82). This involves introducing the mutation into yHsp90 on Leu2 plasmid and then introducing it into haploid yeast cells (yHsp90Δ, yHsp90Δ). Growth of these cells on 5-fluoroorotic acid (5-FOA) will “cure” the yeast cells of the wild type yHsc90 therefore creating hsp90 mutant.

This chapter describes the isolation and identification of yeast Hsp90 phosphorylation using immunoblotting procedures. Using the yeast system it is possible to show that Hsp90 is constitutively phosphorylated on serine and threonine residues. However, Hsp90 threonine phosphorylation is lost upon either heat shock stress or treatment with the Hsp90 inhibitor GA (Figure 2).

Figure 2.

Figure 2.

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Yeast Hsp90 phosphorylation on serine (phos-S) and threonine (p-T) residues. yHsp90-His6 was purified from yeast cell that were heat shocked at 39˚C for 40 min or treated with 100 μM GA for 1hr. Wild type cells containing the empty plasmid were used as negative control.

This chapter also describes the isolation and analysis of the human (h)Hsp90-N-domain from mammalian cells. This is achieved by introducing a PreScission protease cleavage site between the N-domain and adjacent charged linker region of hHsp90α, allowing isolation of the N-domain. Separation of the N-domain containing either wild-type or non-SUMOylated hHsp90α-K191R mutant from the full-length Hsp90 protein allows for better detection of SUMOylated Hsp90 by immunoblotting (Figure 3).

Figure 3.

Figure 3.

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Schematic representation of Hsp90-FLAG showing the amino (N-), charged linker (CL), middle (M-) and carboxy (C-) domains. Mammalian lysate with the Hsp90-FLAG will be attached to anti-FLAG agarose and Hsp90 N-domain can be isolated by PreScission protease digestion.

2. Materials

  1. YPD (2% (wt/vol) Bacto peptone, 1% (wt/vol) yeast extract, 2% (wt/vol) glucose, 20 mg/liter adenine).

  2. Yeast protein extraction buffer (yEB): 50mM Tris-HCI, pH6.8, 100mM NaCl, 50mM MgCl2. One tablet of complete EDTA-free protease inhibitor cocktail (Roche) and one tablet of PhosphoSTOP (Roche) are added to 50mL mEB.

  3. 425–600 μm glass beads (acid washed) (Sigma).

  4. Dulbecco’s Modified Eagle’s Medium – high glucose (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS).

  5. Mammalian protein extraction buffer (mEB): 0.1% NP-40, 20mM Tris-HCl, pH7.4, 100mM NaCl, 1mM MgCl2, 20mM Na2MoO4. One tablet of complete EDTA-free protease inhibitor cocktail (Roche) and one tablet of PhosphoSTOP (Roche) are added to 50mL mEB. (For detection of SUMO, mEB should also contain 20mM N-ethylmaleimide (NEM), see Note 8).

  6. TransIT-2020 transfection reagent (Mirus).

  7. Bio-Rad Protein Assay Dye solution (Bio-Rad).

  8. Ni-NTA agarose (Qiagen).

  9. Imidazole (Sigma).

  10. Anti-FLAG M2 Affinity Gel agarose (Sigma).

  11. PreScission protease (GE Healthcare).

  12. PreScission protease cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT (pH 7.0)

  13. SDS-PAGE sample buffer (2X): 125 mM Tris-HCI pH6.8, 20% glycerol, 2% SDS, 10% 2-mercaptoethanol, 0.01% bromophenol blue, stable at −20˚C. Aliquot and avoid freeze-thaw cycles.

  14. Protran BA85, 0.45 μm Nitrocellulose membrane (Whatman).

  15. Ponceau S solution (Sigma).

  16. Tris-buffered saline (TBS): 150 mM NaCl, 25 mM Tris-Base. Adjust pH to 7.4 using HCI. Sterile filter and incubate at 4˚C.

  17. Albumin, bovine serum (minimum purity 98%).

  18. Dried skimmed milk.

  19. Phospho-serine antibody (Sigma).

  20. Phospho-threonine antibody (Sigma).

  21. Phospho-tyrosine antibody (4G10; Millipore).

  22. Acetylated lysine antibody (Cell Signaling).

  23. Ubiquitin antibody (Santa Cruz).

  24. SUMO-1 or SUMO-2/3 antibody (Cell Signaling).

  25. 6X-His antibody (Invitrogen).

  26. FLAG epitope antibody (ThermoScientific).

  27. Anti-secondary mouse and/or rabbit antibody; ECL™ anti-mouse or anti-rabbit IgG, Horseradish Peroxidase-linked whole antibody (GE Healthcare).

  28. ELC plus Western Blotting Detection System (GE Healthcare).

  29. X-ray film, X-ray cassette, and X-ray film developing machine.

3. Methods

3.1. Extraction of Total Yeast Protein

  • 1.

    Grow PP30 cells 8 expressing His6 linked at the N domain of Hsp82 (yHsp90) on 150ml YPD overnight a 28°C.

  • 2.

    Harvest and wash cells 2–3 times in ice-cold deionized water (dH2O).

  • 3.

    Transfer the cell pellet into a screw cap 2ml tube.

  • 4.

    Pellet the cells and remove the supernatant (see Note 1).

  • 5.

    Add equal volume of cell pellets, ice-cold glass beads.

  • 6.

    Add half the volume of pellet/glass beads, yEB.

  • 7.

    Bead beat the cells using the mini-beadbeater (BioSpec Products, Inc.) for 30 sec.

  • 8.

    Incubate the cells on ice for 30 sec.

  • 9.

    Repeat (7 and 8), 10–12 times.

  • 10.

    Centrifuge the tubes at (10,000g) for 10 min at 4°C (see Note 2).

  • 11.

    Transfer the supernatants into fresh 1.5ml micro-centrifuge tubes,

  • 12.

    Centrifuge the tubes at (10,000g) for 10 min at 4°C (see Note 2).

  • 13.

    Transfer the supernatants (soluble protein) into fresh 1.5ml micro-centrifuge tubes.

  • 14.

    Determine protein concentrations using Bio-Rad Protein Assay solution (Bio-Rad).

  • 15.

    Transfer 40 μl of Ni-NTA Agarose slurry into a 1.5ml micro-centrifuge tube. (see Note 3).

  • 16.

    Add 1.0 ml of yEB to the Ni-NTA Agarose and spin at 10,000g for 1min at 4˚C.

  • 17.

    Remove supernatant and add 1.0 ml of yEB to the Ni-NTA Agarose (see Note 4).

  • 18.

    Repeat step (16–17) four times.

  • 19.

    Re-suspend the Ni-NTA Agarose in 30 μM imidazole in yEB and incubate at 4°C for 30 min (see Note 5).

  • 20.

    Repeat step 16–17 twice and remove supernatant.

  • 21.

    Add 1mg of total protein to the Ni-NTA Agarose in a total volume of 600 μl.

  • 22.

    Incubate the total proteins/Ni-NTA Agarose at 4°C for 2 hr (see Note 6).

  • 23.

    Centrifuge the tubes at (1,000g) for 1 min at 4°C.

  • 24.

    Gently remove the supernatant (see Note 7).

  • 25.

    Add 1 ml of yEB to the Ni-NTA Agarose.

  • 26.

    Repeat (23–25) five times.

  • 27.

    Wash the Ni-NTA agarose with 30 μM imidazole in yEB.

  • 28.

    Wash the Ni-NTA agarose with yEB once.

  • 29.

    Centrifuge the micro-centrifuge tube at 15,000g for 1 min at 4˚C.

  • 30.

    Remove as much supernatant as possible.

  • 31.

    Add 40μl of the protein sample buffer.

  • 32.

    Boil the samples for 3–5 min.

  • 33,

    Proceed to section 3.3 for Western blotting and PTM detection.

3.2. Extraction of total protein from HEK293 cells and immunoprecipitation (IP) of hHsp90:

  1. Transfect HEK293 cells (~40% confluent in a 10cm dish; growing in DMEM +10% FBS) with 2μg hHSP90-FLAG using TransIT-2020 reagent (Mirus) and incubate overnight at 37°C, 5%CO2.

  2. Place plates on ice and aspirate media. Wash 2x with cold PBS. Remove all remaining PBS from plate.

  3. Add 200μL cold mEB to plate. Scrape cells and transfer to 1.5mL micro-centrifuge tube on ice (see Note 8).

  4. Sonicate the lysate for 15 seconds. Incubate on ice for 15 seconds. Repeat ten times.

  5. Centrifuge the tubes at (10,000xg) for 8min at 4°C (see Note 2).

  6. Transfer supernatants (soluble protein) into fresh 1.5mL micro-centrifuge tubes.

  7. Determine protein concentrations using Bio-Rad Protein Assay solution (Bio-Rad).

  8. Transfer 50μL anti-FLAG M2 Affinity Gel agarose (Sigma) into a 1.5mL micro-centrifuge tube.

  9. Add 500μL mEB to the anti-FLAG agarose and spin at 10,000xg for 1 min. Remove supernatant (see Note 4).

  10. Repeat step 9 four times.

  11. Add 1mg of total protein to the anti-FLAG agarose in a total volume of 500μL.

  12. Incubate the total protein/anti-FLAG agarose at 4°C for 2hr on a rotator (see Note 6).

  13. Centrifuge the tubes at 1,000xg for 1 min.

  14. Gently remove the supernatant (see Note 7).

  15. Add 500μL mEB to the anti-FLAG agarose.

  16. Repeat (steps 13–15) five times.

  17. Add 500μL mEB to the anti-FLAG agarose.

  18. Centrifuge at 15,000xg for 1min.

  19. Remove as much supernatant as possible. (see Note 9 for optional PreScission protease cleavage, Figure 3).

  20. Add 40μL of the protein sample buffer.

  21. Boil the samples for 3–5min.

  22. Proceed to section 3.3 for Western blotting and PTM detection.

3.3. Western blotting and detection of Hsp90 PTMs:

  1. Centrifuge the samples at 1,000g and load the supernatant on to a 7.5% SDS-PAGE Tris-HCI gel (see Note 10).

  2. Transfer the proteins from SDS-PAGE gel on to ProtranBA85, 0.45 μm nitrocellulose membrane (Whatman) (see Note 11).

  3. Examine the quality and efficiency of the transfer by staining the membrane with Ponceau S solution (Sigma) for 2 min (see Note 12).

  4. Wash the membrane with dH2O.

  5. Incubate the membrane in 5% milk in TBS-T for 15–20 min at room temperature.

  6. Wash the membrane with 1XTBS-T for 5 min at room temperature.

  7. Repeat (38) three times

  8. Incubate the membrane with primary antibody (see Table 1).

  9. Wash the membrane three times with 1XTBS-T for 5 min at room temperature.

  10. Incubate the membrane with 1:2000 dilution of secondary anti-mouse or anti-rabbit antibody in 5% milk-TBS-T for 1 h at room temperature.

  11. Wash the membrane three times with 1XTBS-T for 5 min at room temperature.

  12. Remove 1XTBS-T and then apply ECL plus (GE Healthcare) to nitrocellulose membrane for 2–3min.

  13. Drain nitrocellulose membrane of excess developing solution (do not let dry).

  14. Wrap the blot in saran wrap.

  15. Place the blot in the X-ray film cassette (see Note 13).

  16. Expose the blots to X-ray films by placing X-ray film directly against the western blot at different length of time.

Table 1.

Post-translational modifications antibodies for immunoblotting. o/n (over-night).

PTM Antibody Manufacturer Dilution Diluent Time and
Temperature		 Species
Phosphorylation Phospho-serine (PSR-45) Sigma (cat no. P5747) 1:500–1:1000 1% BSA in TBS-T o/n 4°C Mouse
Phospho-threonine (PTR-8) Sigma (cat no. P6623) 1:500–1:1000 1% BSA in TBS-T o/n 4°C Mouse
Phospho-tyrosine (4G10) Millipore (cat no. 05–321) 1:2000 5% milk in TBS-T o/n 4°C Mouse
Acetylation Acetylated lysine Cell Signaling (#9441) Rabbit
Ubiquitination Ubiquitin (P4D1) Santa Cruz (sc-8017) 1:500–1:2000 5% milk in TBS-T o/n 4°C Mouse
SUMOylation SUMO-1 (2A12) Cell Signaling (#5718) Mouse
SUMO-2/3 (18H8) Cell Signaling (#4971) Rabbit
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Notes

  1. The cell pellet must be kept on ice.

  2. At this stage, Bio-Rad Protein Assay solution (Bio-Rad) should be prepared.

  3. Ni-NTA Agarose is precharged with Ni2+ ions and appears blue in color. It is provided as a 50% slurry in 30% ethanol.

  4. Do not disturb the Ni-NTA or anti-FLAG Agarose pellet.

  5. Imidazole at low concentrations is commonly used in the binding and wash buffer to minimize binding of unwanted host cell proteins.

  6. Use Eppendorf Thermomixer R to gently mix total proteins/Ni-NTA Agarose solution.

  7. Avoid disturbing the Ni-NTA or anti-FLAG Agarose.

  8. For detection of SUMO, mEB should always contain 20mM N-ethylmaleimide (NEM).

  9. PreScission protease cleavage: incubate hHsp90α-FLAG bound to anti-FLAG agarose with 2 units of PreScission Protease in 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT (pH 7.0) at 10°C for 16 h.

  10. Criteron precast gels from Bio-Rad are suitable for this purpose.

  11. The high MW setting on the Bio-Rad Trans-Blot Turbo transfer system is suitable for this purpose.

  12. Prepare 5% dry milk (LabScientific Inc.) in 1x TBS-T (O.1% Tween-20, Sigma) buffer before examining the membrane.

  13. This procedure must be performed in the dark.

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