Cell-Based Assays for Smoothened Ubiquitination and Sumoylation

Abstract

The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis by regulating the abundance, localization, and activity of the GPCR family protein Smoothened (Smo). Smo trafficking and subcellular accumulation are controlled by multiple posttranslational modifications (PTMs) including phosphorylation, ubiquitination, and sumoylation, which appears to be conserved from Drosophila to mammals. Smo ubiquitination is dynamically regulated by E3 ubiquitin ligases and deubiquitinases (dubs) and is opposed by Hh signaling. By contrast, Smo sumoylation is stimulated by Hh, which counteracts Smo ubiquitination by recruiting the dub USP8. We describe cell-base assays for Smo ubiquitination and its regulation by Hh and the E3 ligases in Drosophila. We also describe assays for Smo sumoylation in both Drosophila and mammalian cultured cells.

1. Introduction

Hedgehog (Hh) family of secreted proteins control many key processes in both embryonic development and adult tissue homeostasis; not surprisingly, deregulation of Hh signaling contributes to a wide range of human disorders including congenital diseases and cancer [1, 2]. Hh signaling regulates cell growth and patterning through a conserved signal transduction cascade that initiates from the GPCR family protein Smoothened and culminates in the activation of latent transcription factors Cubitus interruptus (Ci) and Gli proteins [2, 3].

Smo is regulated through changes of its conformation and subcellular localization [4]. In Drosophila, the Hh receptor Patched (Ptc) inhibits Smo by keeping it in a closed and inactive conformation and preventing its accumulation on the cell surface [5, 6]. Upon Hh stimulation, Smo is phosphorylated by multiple kinases including protein kinase A (PKA), casein kinase 1 (CK1), CK2, and Gprk2, which promotes Smo cell surface accumulation and triggers a conformational change of Smo C-terminal intracellular tail (Smo-CT) into an open conformation, leading to Smo-CT clustering [6–11].

Ubiquitination and subsequent degradation of Smo via both proteasome- and lysosome-dependent mechanisms are responsible for preventing Smo cell surface accumulation in quiescent cells [12–14]. Upon Hh stimulation, Smo ubiquitination is inhibited by PKA/CK1-mediated phosphorylation of Smo-CT as well as by the deubiquitinating enzyme USP8/UBPY [12, 13]. Smo ubiquitination is promoted by multiple E3 ubiquitin ligases, including the Smurf family of HECT-domain containing E3s as well as the modular ubiquitin ligase complex CRL4^Gβ, which contains Cul4, DDB1, and G protein β subunits (Gβ) (Fig. 1) [15, 16]. Hh inhibits Smo ubiquitination largely by blocking the recruitment of multiple E3 ubiquitin ligases. PKA/CK1-phosphorylation of Smo-CT inhibits the recruitment of Smurf family members while PKA-mediated phosphorylation of DDB1 inhibits the recruitment of CRL4^Gβ (Fig. 1) [15, 16]. A recent study has revealed that ubiquitination of mammalian Smo promotes its ciliary removal and that Shh signaling decreases Smo ubiquitination and ciliary removal although the E3 ligases responsible for the ubiquitination of mammalian Smo remain unidentified [17].

Fig. 1.

Hh inhibits Smo ubiquitination while promoting its sumoylation. (a) In the absence of Hh, Smo is ubiquitinated by Smurf family of E3s and CRL4^Gβ, leading to its internalization and degradation by both proteasome and lysosome-dependent mechanisms. Gprk2 phosphorylates Smurf to promote its binding to Smo. Ulp1 binds to Smo C-tail to prevent Smo sumoylation. (b) In the presence of Hh, PKA/CK1-mediated phosphorylation of Smo C-tail inhibit Smurf binding while PKA-mediated phosphorylation of DDB1 causes the disassembly of CRL4^Gβ, thereby inhibiting Smo ubiquitination. In addition, Hh inhibits Ulp1 binding to Smo, leading to Smo sumoylation. Sumoylated Smo recruits USP8 antagonize Smo ubiquitination. As a consequence, Smo is accumulated on the cell surface

While Hh inhibits Smo ubiquitination, it stimulates sumoylation of Smo, which is also required for Smo cell surface accumulation [18, 19]. Sumoylation of Smo occurs on Lys851 that conforms the sumoylation consensus sites: Ψ-K-x-E, where Ψ is an aliphatic branched amino acid, K is the SUMO receptor, and x is any amino acid [20]. The sumoylation-deficient Smo variant, SmoK851R, exhibited reduced cell surface accumulation and Hh pathway activity, both of which could be rescued by fusing a SUMO moiety to the C-terminus of SmoK851R [18]. Hh stimulates sumoylation of Smo in a manner independent of its phosphorylation by PKA and CK1. Hh does not regulate the recruitment of sumoylation enzymes but rather dissociate the desumoylation enzyme Ulp1 from Smo [18]. Interestingly, Sonic Hedgehog (Shh) also stimulates sumoylation of mammalian Smo (mSmo) to facilitate its ciliary accumulation; however, the underlying mechanism has remained unexplored [18]. Because ubiquitination of mSmo inhibits its ciliary accumulation [17], it would be interesting to determine whether sumoylation promotes mSmo ciliary accumulation by antagonizing its ubiquitination.

In this chapter, we describe cell-base assays for Smo ubiquitination and its regulation by Hh and the E3 ligase Smurf in cultured Drosophila cells. We also describe assays for Smo sumoylation in both Drosophila and mammalian cultured cells.

2. Materials

2.1. Cell Culture and Transfection

1. Drosophila Schneider 2 cells (S2 cells), store in a 25 °C incubator.

2. S2 culture medium: Schneider’s Drosophila Medium, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin.

3. Hh-conditioned medium: collected from S2 stable cell line expressing HhN (S2-Hh) with the induction of 0.7 mM copper sulfate after 24 h.

4. S2-Hh stable cell medium: Schneider’s Drosophila Medium with 10% fetal bovine serum and 500 μg/mL hygromycin.

5. Nucleobond Xtra Midi Plus Kit.

6. S2 cells were transfected with regular calcium phosphate method.

7. NIH/3T3 cells.

8. NIH/3T3 cell culture medium: DMEM Medium: 10% bovine calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin.

9. Cell culture incubator at 37 °C.

10. Polyjet in vitro DNA transfection reagent.

11. Transfection-grade plasmids: desired protein coding sequences were subcloned into pGE and pcDNA3.1(+) vectors for mammalian cell transfection and into pUAST vector for S2 cell transfection.

12. 10 cm Cell culture dishes.

13. 10% SDS stock solution.

14. SDS PAGE gel.

2.2. Smo Ubiquitination and Its Regulation by Hh and Smurf

1. Lysis Buffer: 20 mM Tris HCl pH8.0, 150 mM NaCl, 5 mM EDTA, 1% IGPAL CA-630,10% glycerol, 1X Complete protease inhibitors cocktail, 1X phosSTOP phosphatase inhibitors cocktail.

2. SDS loading buffer (4x): 0.25 M Tris–HCl (pH 6.8), 8% SDS, 20% 2-mercaptoethanol, 40% glycerol, and 0.008% bromophenol blue.

3. Running buffer: 25 mM Tris base, 190 mM glycine, 0.1% SDS.

4. Transfer buffer: 25 mM Tris base, 190 mM glycine, 20% methanol.

5. Nitrocellulose Membrane.

6. TBST solution: 20 mM Tris–HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween-20.

7. Blocking buffer: 5% nonfat dried milk or bovine serum albumin (BSA) in TBST.

8. Recombinant Protein G Sepharose 4B.

9. Antibodies:

   1. Mouse anti-Myc (9E10).
   2. Mouse anti-Ubiquitin (P4D1).
   3. Mouse anti-HA (F7) (see Note 1).

10. Coding sequence of Myc Smo, Flag Smurf, and HA Ubiquitin (and its mutants) were subcloned into pUAST vector (see Note 2).

11. Proteasome inhibitor MG132 (see Note 3).

2.3. Detect Smo SUMOylation in Drosophila S2 Cells

1. Lysis Buffer: 20 mM Tris HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% IGPAL CA-630, 10% glycerol, 20 mM NEM, 1X Complete protease inhibitors cocktail, 1X phosSTOP phosphatase inhibitors cocktail.

2. SDS loading buffer (4x): 0.25 M Tris–HCl (pH 6.8), 8% SDS, 20% 2-mercaptoethanol, 40% glycerol, and 0.008% bromophenol blue.

3. Running buffer: 25 mM Tris base, 190 mM glycine, 0.1% SDS.

4. Transfer buffer: 25 mM Tris base, 190 mM glycine, 20% methanol.

5. Nitrocellulose Membrane.

6. TBST solution: 20 mM Tris–HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween-20.

7. Blocking buffer: 5% nonfat dried milk or bovine serum albumin (BSA) in TBST.

8. Coding sequence of Myc Smo and HA SUMO were subcloned into pUAST vector.

9. Recombinant Protein G Sepharose 4B.

10. Antibodies:

    1. Mouse anti-Myc (9E10).
    2. Mouse anti-HA (F7).

11. N-Ethylmaleimide.

2.4. Detect Smo SUMOylation in Mammalian NIH/3T3 Cells

1. Lysis Buffer: 20 mM Tris HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% IGPAL CA-630, 10% glycerol, 20 mM NEM, 1X Complete protease inhibitors cocktail, 1X phosSTOP phosphatase inhibitors cocktail (see Note 4).

2. SDS loading buffer (4x): 0.25 M Tris–HCl (pH 6.8), 8% SDS, 20% 2-mercaptoethanol, 40% glycerol, and 0.008% bromophenol blue.

3. Running buffer: 25 mM Tris base, 190 mM glycine, 0.1% SDS.

4. Transfer buffer: 25 mM Tris base, 190 mM glycine, 20% methanol.

5. Nitrocellulose Membrane.

6. TBST solution: 20 mM Tris–HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween-20.

7. Blocking buffer: 5% nonfat dried milk or bovine serum albumin (BSA) in TBST.

8. Coding sequence of Myc mSmo was subcloned into pGE vector and HA SUMO2 was subcloned into pcDNA3.1(+) (see Note 5).

9. Cell starving medium: DMEM, 0.5% BCS.

10. Recombinant Protein G Sepharose 4B.

11. Antibodies:

    1. Mouse anti-Myc (9E10).
    2. Mouse anti-HA (F7).

12. N-Ethylmaleimide.

13. Recombinant Human Shh-N (see Note 6).

3. Methods

3.1. Detect Smo Ubiquitination in Drosophila S2 Cells and Its Regulation by Hh and Smurf

1. Seed one to two million S2 cells in a 10 cm culture dish.

2. Transfect S2 cells with Myc-Smo, HA-Ub, and ub-Gal4 constructs with or without Flag-Smurf (5 μg for each plasmid) on the second day. Add pUAST empty vector to bring the total amount of DNA to 20 μg for each transfection.

3. Treat the cells with Hh-conditioned medium or control medium on the third day (see Note 7).

4. The next day, treat the cells with 50 μM MG132 for 4 h. Then collect the cells with centrifugation and resuspend the cells with lysis buffer and incubate at 4 °C with shaking for 5 min.

5. Remove the cell debris with centrifugation.

6. Add 25 μL 10% SDS to 225 μL cell lysate, mix well, and boil the mixture for 3–5 min.

7. Cool down and mix with 1 mL cold lysis buffer.

8. Add Myc antibody and incubate at 4 °C for 1–2 h with agitation.

9. Add 15–20 μL (bed volume) Protein G Sepharose beads and incubate for another hour at 4 °C with agitation.

10. Wash the beads with cold lysis buffer 3 times at 4 °C for 5 min each.

11. Elute the protein with 2X SDS loading buffer at 95 °C for 5 min.

12. Load the eluted protein onto an SDS PAGE gel and perform the standard immunoblot with either anti-Ub or anti-HA (in the case of co-expressed with HA-Ub) antibodies as primary antibodies.

3.2. Detect Smo SUMOylation in Drosophila S2 Cells

1. Seed one to two million S2 cells in a 10 cm culture dish.

2. Transfect S2 cells with Myc-Smo, HA-SUMO, and ub-Gal4 constructs the next day.

3. 24 h after transfection, treat the cells with Hh-conditioned medium or control medium for another day.

4. 24 h after Hh treatment, add MG132 (50 μM) for 4 h and then collect the cells with centrifugation.

5. Resuspend the cells with lysis buffer containing 20 mM NEM and incubate at 4 °C with shaking for 5 min.

6. Remove the cell debris with centrifugation.

7. Add 25 μL 10% SDS to 225 μL cell lysate, mix well, and boil the mixture for 3–5 min.

8. Cool down and mix with 1 mL cold lysis buffer.

9. Add Myc antibody and incubate at 4 °C for 1–2 h with agitation.

10. Add 15–20 μL (bed volume) Protein G Sepharose beads and incubate for another hour at 4 °C with agitation.

11. Wash the beads with cold lysis buffer 3 times at 4 °C for 5 min each.

12. Elute the protein with 2X SDS loading buffer at 95 °C for 5 min.

13. Load the eluted protein onto an SDS PAGE gel and perform the standard immunoblot with mouse anti-HA antibody as primary antibodies.

3.3. Detect Smo SUMOylation in NIH/3T3 Cells

1. Seed appropriate number of NIH/3T3 cells.

2. The next day, transfect Myc-Smo and HA-SUMO2 plasmids into NIH/3T3 cells with polyJet in vitro DNA transfection reagent.

3. On the third day, serum starve the cells with cell starving medium for 24 h.

4. Add 0.1 μg/mL of Shh-N and incubate for 12–16 h.

5. Treat the cells with 50 μM MG132 for 4 h.

6. Resuspend the cell pellet with Lysis buffer with 20 mM NEM at 4 °C for 10 min with shaking.

7. Remove the cell debris with centrifugation.

8. Add 25 μL 10% SDS to 225 μL cell lysate, mix well, and boil the mixture for 3–5 min.

9. Cool down and mix with 1 mL cold lysis buffer.

10. Add Myc antibody and incubate at 4 °C for 1–2 h with agitation.

11. Add 15–20 μL (bed volume) Protein G Sepharose beads and incubate for another hour at 4 °C with agitation.

12. Wash the beads with cold lysis buffer 3 times at 4°C for 5 min each.

13. Elute the protein with 2X SDS loading buffer at 95 °C for 5 min.

14. Load the eluted protein onto an SDS PAGE gel and perform the standard immunoblot with mouse anti-HA antibody as primary antibody.