Caveolin-3 Undergoes SUMOylation by the SUMO E3 Ligase PIASy

Abstract

Caveolin (Cav) proteins in the plasma membrane have numerous binding partners, but the determinants of these interactions are poorly understood. We show here that Cav-3 has a small ubiquitin-like modifier (SUMO) consensus motif (ΨKX(D/E, where Ψ is a hydrophobic residue)) near the scaffolding domain and that Cav-3 is SUMOylated in a manner that is enhanced by the SUMO E3 ligase PIASy (protein inhibitor of activated STAT-y). Site-directed mutagenesis revealed that the consensus site lysine is the preferred SUMOylation site but that mutation of all lysines is required to abolish SUMOylation. Co-expression of a SUMOylation-deficient mutant of Cav-3 with β-adrenergic receptors (βARs) alters the expression level of β₂ARs but not β₁ARs following agonist stimulation, thus implicating Cav-3 SUMOylation in the mechanisms for β₂AR but not β₁AR desensitization. Expression of endothelial nitric-oxide synthase (NOS3) was not altered by the SUMOylation-deficient mutant. Thus, SUMOylation is a covalent modification of caveolins that influence the regulation of certain signaling partners.

Introduction

The caveolin (Cav)³ family has three members; Cav-1 and -2 are co-expressed in most cell types, whereas Cav-3 is muscle-specific (1). Cavs are the principal protein component of caveolae membrane microdomains and function as scaffolds that compartmentalize and regulate numerous receptors, channels, transporters, and signaling proteins that are enriched in caveolar membranes (e.g. G-proteins, G-protein-coupled receptors, RTKs, and eNOS) or undergo rapid translocation to or from caveolae in response to stimuli (2–5). Proteins interact with the caveolin scaffolding domain (CSD; Fig. 1, A and B) via a “caveolin-binding motif,” but how this interaction is dynamically regulated is poorly understood. Because of their rapid kinetics and reversibility, control by post-translational modifications is a likely mechanism; however, no regulatory post-translational modifications have been identified on Cav-3. We searched for motifs important for post-translational modifications in the amino acid sequence of Cav-3 and found a conserved, SUMO consensus motif in Cav-3 and -1 but not Cav-2 (Fig. 1A).

FIGURE 1.

Caveolin-3 has a SUMO consensus motif and is SUMOylated in vitro and in vivo. A, caveolin-3 and -1, but not caveolin-2, have a core SUMO consensus motif (ΨKX(D/E), where Ψ represents V/I/L) in their N termini. This motif also conforms to the extended “negatively charged amino acid-dependent SUMOylation motif.” The SUMO consensus motif (aa 37–40) lies near the CSD (aa 55–74) and transmembrane domain (TM; aa 75–104), which are highly conserved among the three caveolin family members. B, caveolin membrane topology. Caveolins form a hairpin loop in the plasma membrane such that both the N and C termini face the cytoplasm. Caveolin-3 (P51638 (CAV3_RAT)) has seven lysine residues that are potential SUMOylation sites. A putative SUMO consensus site at Lys³⁸ in the N terminus lies near the CSD. C, in vitro SUMOylation of caveolin-3; recombinant expression of Cav-3-V5 in insect cell extracts. Reactions (in 20 μl) included SUMOylation buffer (50 mm Tris-HCl, pH 7.5, 5 mm MgCl₂, 2 mm ATP), Mg²⁺/ATP, and recombinant SUMO E1 and E2 enzymes and were incubated at 30 °C for 1 h with either SUMO-1 or SUMO-3. Reactions omitting Cav-3-V5 (lane 2), SUMO (lanes 3), or Ubc9 (lane 4) were negative controls. Reactions were analyzed by immunoblotting with anti-V5-HRP antibodies. D, detection of endogenously SUMOylated caveolin-3. Rat myocyte lysates were immunoprecipitated with anti-Cav-3 antibodies, and immunoprecipitates (IP) and supernatants (SN) were analyzed along with whole cell lysates (WCL) by immunoblotting with anti-Cav-3 and anti-SUMO-2/3 antibodies. E, rat myocyte lysates were immunoprecipitated with anti-SUMO antibodies, and IP, SN, and WCL were analyzed by immunoblotting with anti-Cav-3 antibodies.

SUMOylation is the reversible modification of proteins by small ubiquitin-like modifier (SUMO) proteins and can regulate protein-protein interactions by masking or creating binding surfaces on target proteins (6, 7). SUMOylation, initially thought to occur only in nuclear or perinuclear compartments (8), also regulates cytoplasmic and plasma membrane proteins (9–15). TGFβ induces SUMOylation of the Type I TGFβ receptors (16), which localize to caveolae and interact with Cav (17–18). Other SUMOylated signaling proteins that interact with Cavs or localize to caveolae include Kv1.5, PTP1B, RGS-Rz, and PDE4D5 (19–24). Thus, modification by SUMO may contribute to the binding and regulation of proteins that interact with Cavs. Here we define properties of the SUMOylation of caveolin-3 and the contribution of this SUMOylation to the expression of β-adrenergic receptors (βARs).

EXPERIMENTAL PROCEDURES

Materials and Chemicals

Reagents and their sources were as follows: Protein-G-agarose (Roche Applied Science; catalog no. 11243233001), MG-132 (Calbiochem; catalog no. 474790), N-ethylmaleimide (NEM) and (S)-(−)-propranolol (Sigma; catalog nos. E3876 and P8688), Halt protease inhibitor mixture, EDTA-free (Pierce; catalog no. 87785), Ni²⁺-NTA-agarose resin (Qiagen; catalog no. 30210), and goat anti-V5-agarose (Bethyl Laboratories; catalog no. S190-119). Multi-Site and Lightning Mutagenesis kits were from Stratagene (catalog nos. 200515 and 210518), BL21(DE3) competent cells were from Novagen (catalog no. 70235-3), and isopropyl-β-d-1-thiogalactopyranoside was from Invitrogen (catalog no. 15529019). 1-O-Octyl-β-d-glucopyranoside was from A.G. Scientific (catalog no. 0-1036). For SDS-PAGE, NuPAGE BisTris 4–12% 12-well gels (Invitrogen) and PVDF membranes (Millipore) were used. SeeBlue Plus2 prestained standard (Invitrogen; catalog no. LC5925) was used for visualization of protein molecular weights.

Antibodies

Antibodies and their sources were as follows: anti-FLAG M2 affinity gel and mouse monoclonal anti-FLAG M2 antibody (Sigma; catalog nos. A2220 and F1804); anti-FLAG-HRP and anti-Myc (Cell Signaling; catalog nos. 2044 and 2276); mouse monoclonal anti-V5 and anti-V5-HRP (Invitrogen; catalog nos. R960-25 and R961-25); rabbit polyclonal anti-PIASy, anti-Cav-3, anti-eNOS, anti-SUMO-1, and anti-rabbit secondary antibodies (Abcam; catalog nos. ab58416, ab2912, ab66127, ab11672, and ab6013); anti-SUMO-2/3, anti-Ubc9, and anti-ubiquitin (Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); catalog nos. sc-32873, sc-10759, and sc-47721); and rabbit polyclonal anti-SUMO-1 (Invitrogen; catalog no. 38-1900).

Plasmids

The mammalian expression plasmids for eNOS, SUMO-1, SUMO-3, Ubc9, PIAS3, and PIASy were obtained from Origene (catalog nos. SC108440, SC118050, SC115792, SC109867, SC11632, and SC114580), and the FLAG-β₁AR and FLAG-β₂AR mammalian expression plasmids were obtained from Addgene (catalog nos. 14697 and 14698). Myc-SUMO-1, Myc-SUMO-3, and PIAS1 were provided by Dr. Serena Ghisletti. The Escherichia coli expression plasmids pKRSUMO and pBADE12 were from Dr. Mario Mencia. The Cav-3-V5/His plasmid was provided by Dr. Brian Head and was constructed by cloning the rat caveolin-3 gene (AC_NM019155) into the pcDNA3.1/V5-His-TOPO vector (Invitrogen, catalog no. K4800). The E. coli expression plasmid pST39-Cav-3-V5/His (referred to as pST39-Cav-3-V5) was generated by subcloning the Cav-3-V5/His coding region from the pcDNA3.1-V5/His expression vector and inserting it into cassette 2 (EcoRI/HindIII) of the polycistronic vector pST39. The following primers were used to amplify Cav-3-V5/His and introduce EcoRI and HindIII restriction sites by PCR: Cav3Fw, 5′-GATCGAATTCATGATGACCGAAGAGCACAC-3′; Cav3Rv, 5′-GATCAAGCTTTCAATGGTGATGGTGATGATG-3′.

Site-directed Mutagenesis

The single and multiple Lys to Arg (KR) mutations were introduced into WT Cav-3-V5 using Multi-Site and Lightning QuikChange site-directed mutagenesis kits (Stratagene). The sequences for primers designed for creation of the KR mutants are available upon request. Cav-3-V5-K7R was used to create a series of Cav-3-V5 RK mutants. The sequences for primers designed for creation of the RK mutants are available upon request. All plasmids were sequenced to validate the presence of the desired mutations.

Cell Culture and Transfection

Human embryonic kidney cells (HEK AD-293) were obtained from Stratagene (catalog no. 240085) and cultured in a 37 °C, 5.0% CO₂ incubator. Cells were grown in “HEK medium” (DMEM with 4.5 g/liter glucose, l-glutamine, and sodium pyruvate, supplemented with 10% FBS) without antibiotics. HEK AD-293 cells are derived from HEK 293 cells. Confluent HEK AD-293 cells were transfected 2–3 days after plating in 6-well plates. 4.0 μg of plasmid DNA and 9 μl of Lipofectamine 2000 (Invitrogen) were each diluted separately in 250 μl of OptiMEM (Invitrogen) before being combined, incubated for 20 min at room temperature, and diluted with 500 μl of HEK medium. Transfection complexes (1-ml total volume) were pipetted onto cells in 6-well plates to which 2 ml of fresh HEK medium had been added. Cells were incubated with transfection complexes overnight, and medium was replaced with 3 ml of fresh HEK medium the following day. Except where stated, cells were analyzed 48 h post-transfection. For co-expression of the SUMOylation machinery with Cav-3-V5, the following ratio was used: Cav-3-V5/SUMO-3/Ubc9/PIASy = 1:1:0.5:0.15–0.25. For the experiments that assessed the dose dependence of PIASy (Fig. 3B, lanes 3–7), the range was 0–400 ng of plasmid. FLAG-β₂AR, FLAG-β₁AR, or eNOS was co-expressed in a 1:1 ratio with Cav-3-V5 using the same ratios of SUMOylation machinery components. The total DNA for all transfections was normalized to 4.0 μg with empty pcDNA3.1 vector.

FIGURE 3.

In vivo SUMOylation assays; SUMOylation of caveolin-3 is enhanced by interaction with the SUMO E3 ligase PIASy. A, PIASy stimulates SUMOylation of caveolin-3. HEK 293 cells were co-transfected with Cav-3-V5 (lanes 1–5), Myc-SUMO-3, and Ubc9 (lanes 1–4) and either PIAS1, PIAS3, or PIASy (lanes 2, 3, and 4, respectively). Cells transfected with empty vector DNA were included as a negative control (lane 6). Lysates were analyzed by immunoblotting with anti-Myc, anti-V5-HRP, and anti-PIASy antibodies. B, PIASy dose-dependently enhances caveolin-3 SUMOylation. HEK 293 cells were co-transfected with Cav-3-V5, SUMO-3, and Ubc9 with or without increasing amounts of PIASy (lanes 4–7; 0, 40, 100, or 200 ng of plasmid DNA). Cells transfected with just Cav-3-V5 and SUMO-3 or PIASy alone were negative controls (lanes 1 and 2, respectively). Lysates were analyzed by immunoblotting with anti-V5-HRP and anti-GAPDH antibodies. C and D, PIASy co-immunoprecipitates with caveolin-3. C, lysates from HEK 293 cells co-transfected with Cav-3-V5, Ubc9, SUMO-3, and PIASy were immunoprecipitated with anti-V5 and anti-Cav-3 antibodies and immunoblotted with anti-V5-HRP and anti-PIASy antibodies. D, the reciprocal immunoprecipitation was performed with anti-PIASy antibodies and immunoblotted with anti-V5-HRP and anti-PIASy antibodies. E, endogenous expression of PIASy in adult rat cardiac myocytes. Freshly isolated rat cardiac myocytes were lysed and analyzed by immunoblotting with anti-PIASy antibodies.

Immunoprecipitation and Immunoblotting

Transfected HEK AD-293 cells were washed with ice-cold PBS and scraped into 500–800 μl of ice-cold “in vivo SUMOylation” lysis buffer (20 mm Tris-HCl, pH 7.8, 150 mm NaCl, 0.1% SDS, 0.5% deoxycholate, 0.5% Triton X-100, 1 mm EDTA) supplemented with 20 mm NEM (prepared fresh) and Halt protease inhibitor mixture (Pierce) and pipetted into prechilled 1.5-ml Eppendorf tubes. Cells were incubated on ice for 30 min and sonicated in an ice water bath (3 × 10 s). Lysates were clarified by centrifugation (5000 × g for 15 min at 4 °C), and protein concentrations were normalized by dilution with lysis buffer after performing BCA protein assays (Pierce). Clarified lysates were either directly resolved by SDS-PAGE and immunoblotted with appropriate antibodies or subjected to immunoprecipitation. Lysates were diluted with 4× LDS sample loading buffer (Invitrogen) supplemented with 100 mm DTT and heated (except where stated) to 80 °C for 5 min before being resolved by NuPAGE BisTris 4–12% 12-well gels. Proteins were then transferred for 1.5–2.5 h at 55 V onto PVDF membranes that were immediately blocked for 1 h at room temperature in 4% milk, 0.1% TBST. In general, primary antibodies were diluted at 1:1000 in 4% milk, 0.1% TBST except for V5-HRP (1:5000), Cav-3 (1:2000), and PIASy (1:2000). For anti-SUMO-1 and anti-SUMO-2/3 antibodies, 5% BSA, 0.1% TBST was used for blocking and dilution instead of milk to reduce background signal. PVDF membranes were incubated overnight at 4 °C with primary antibodies and washed at least three times for 10 min each with 0.1% TBST before incubation with secondary antibodies for 45–50 min at room temperature. Membranes were then washed (4 × 10 min) with 0.1% TBST and detected by Amersham Biosciences ECL (GE Healthcare; catalog no. RPN2135V2) or SuperSignal West Dura (Pierce; catalog no. 34075). Images were collected with a Sensicam QE High Performance CCD camera (Cooke Corp.) mounted on an Epi Chemi II Darkroom (UVP BioImaging Systems) and analyzed using LabWorks 4.0 image acquisition and analysis software (UVP BioImaging Systems).

For immunoprecipitations, 300–500 μl of clarified lysates were precleared with 40 μl of Protein G-agarose (Roche Applied Science), incubated overnight at 4 °C with primary antibodies (1–2 μg), followed by incubation with 40 μl of Protein G-agarose for 1 h. Complexes were centrifuged at 10,000 × g at 4 °C for 1 min; supernatants were transferred to prechilled 1.5-ml Eppendorf tubes. The beads were washed four times with lysis buffer and eluted by heating to 80–90 °C for 5 min in 50–100 μl of 2× LDS buffer with 100 mm DTT. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with appropriate antibodies. Immunoprecipitations with anti-FLAG M2 and anti-FLAG M2-agarose (Sigma) were performed per the manufacturer's instructions with FLAG immunoprecipitation lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100) supplemented with 20 mm NEM, 10% glycerol, and Halt protease inhibitor mixture. Isolation of adult rat cardiac myocytes for immunoprecipitation with anti-Cav-3, anti-SUMO-1, and anti-SUMO-2/3 antibodies was performed as described (25). Myocytes were washed with ice-cold PBS and incubated on ice with lysis buffer (0.2% SDS, 0.5% Nonidet P-40, 15 mm MgCl₂, 1 mm DTT) supplemented with Halt protease inhibitor mixture and 20 mm NEM. Lysates were sonicated, clarified by centrifugation, and incubated overnight with primary antibodies at 4 °C and processed by the Protein G-agarose method as described above.

In Vitro SUMOylation Assay

An in vitro protein synthesis kit (Qiagen EasyXpress Insect Kit II) was used to express recombinant Cav-3-V5 per the manufacturer's instructions. 2 μl of protein synthesis reactions containing Cav-3-V5 were used directly in 20-μl in vitro SUMOylation reactions (BioMol International) consisting of 2 μl of 10× SUMOylation buffer (50 mm Tris-HCl, pH 7.5, 5 mm MgCl₂, 2 mm ATP), 1 μl each of 20× SUMO E1 and SUMO E2 enzyme solutions, and recombinant SUMO-1 or SUMO-3 proteins. Reactions were incubated at 37 °C for 1 h and quenched with 2× LDS sample buffer with 100 mm DTT. 10 μl of the reaction product was analyzed by immunoblotting with anti-V5-HRP antibodies.

Expression and Purification of SUMOylated Caveolin-3 in E. coli

For production of SUMOylated Cav-3, BL21(DE3) competent cells were sequentially transformed with three E. coli expression plasmids: pKRSUMO, pBADE12, and pST39-Cav-3-V5. For negative controls, cells were transformed with pST39-Cav-3-V5 alone or with pKRSUMO and pBADE12. Transformed cells were stored as glycerol stocks at −80 °C. For protein expression, transformed cells were grown at 37 °C in LB supplemented with antibiotics (e.g. Amp, Cm, and Kan). The A₆₀₀ was monitored, and protein expression was induced at an A₆₀₀ of 0.5–0.6 for 3 h. Cultures were pelleted and resuspended in TNE buffer (25 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA). Purifications were performed with Ni²⁺-NTA-agarose (Qiagen) and anti-V5-agarose (Bethyl Laboratories) resins per the manufacturer's instructions.

RESULTS

Caveolin-3 Has a Conserved SUMO Consensus Motif and Is SUMOylated in Vitro and in Vivo

Cav-3 and -1 but not Cav-2 have conserved, core, and extended SUMO consensus motifs in their N termini near the CSD (Fig. 1A), suggesting that they are potential substrates for SUMOylation. The extended “negatively charged amino acid-dependent SUMOylation motif” (26, 27) specifies the presence of negatively charged amino acids distal to the core motif, and Cav-3 and -1 have motifs similar to other SUMO substrates (supplemental Fig. S1, A and B). In vitro, the SUMO E1, E2 (Ubc9), and Mg²⁺/ATP are sufficient to SUMOylate targets (28). To determine if Cav-3 undergoes SUMOylation, we performed in vitro SUMOylation assays using a dual, affinity-tagged construct (Cav-3-V5/His, which we refer to as Cav-3-V5; Fig. 4A) expressed in a transcription/translation system and analyzed by immunoblotting with anti-V5-HRP antibodies. Modification by SUMO-1 or -3 (but not if Ubc9, SUMO, or Cav-3-V5 were omitted) produced a ∼15–20 kDa mobility shift, from 25 to 40 kDa (Fig. 1C). To determine whether SUMOylation also occurs in vivo, lysates from rat cardiomyocytes were immunoprecipitated with anti-Cav-3 (*, Fig. 1D) or anti-SUMO antibodies (Fig. 1E). Immunoblotting for Cav-3 and SUMO-2/3 revealed bands at ∼40 kDa corresponding to SUMOylated Cav-3.

FIGURE 4.

Modification of Cav-3-V5 by SUMO-3 is reduced by mutation of the consensus site lysine (K38R) and abolished by mutating all lysines (K7R). A, schematic of the Cav-3-V5 construct. Each lysine residue is shown (K15, K20, K30, K38, K69, K108, and K144) in context with the N terminus, scaffolding domain, transmembrane domain, C terminus, and V5/His affinity tags. B, mutation of consensus site lysine 38 (K38). Wild-type Cav-3-V5 (WT; lanes 1 and 2) or the consensus site Lys to Arg mutant (K38R, lane 3) was co-transfected into HEK 293 cells with Ubc9, PIASy (lanes 1–3), and either SUMO-1 (lane 2) or SUMO-3 (lanes 1 and 3). Lysates were analyzed by immunoblotting with anti-V5-HRP antibodies. C, single KR mutants. HEK 293 cells were co-transfected with Cav-3-V5 KR mutants, K20R, K30R, K38R, K69R, and K144R (lanes 2–6) and the SUMOylation machinery, SUMO-3, Ubc9, and PIASy (lanes 1–7). Lysates were analyzed by immunoblotting with anti-V5-HRP antibodies (long exposure (top) and a short exposure (bottom)). The WT (lane 1) and Cav-3-V5-K7R mutant (lane 7) are included as positive and negative controls for Cav-3-V5-SUMO-3 conjugates. D, multiple KR mutants. WT or the K7R mutant were transfected with and without the SUMOylation machinery (lanes 1–5). MG-132 was preincubated with cells 3 h prior to lysis (lane 5). Multiple KR mutants were also co-transfected with the SUMOylation machinery (lanes 6–11). Labels indicate residues not mutated (i.e. K144 has a single Lys at position 144).

Production of SUMOylated Caveolin-3 in E. coli

To identify and purify SUMOylated caveolin proteins, we used an E. coli system (29–31), which has the advantages of scalability and the absence of SUMO-specific proteases (SENPs). Cav-3-V5/His, cloned into a bacterial expression vector (pST39-Cav-3-V5) (32), was co-transformed into BL21(DE3) cells along with plasmids expressing SUMO-1 (pKRSUMO) and the SUMO E1 and E2 enzymes (pBADE12) (Fig. 2A). We tested the ability of BL21(DE3) cells sequentially transformed with all three plasmids, pST39-Cav-3-V5 alone, or pKRSUMO and pBADE12 to produce SUMOylated Cav-3. We analyzed the supernatant (Fig. 2B) and insoluble pellet (data not shown) and found that the majority of SUMOylated Cav-3-V5 was soluble. BL21(DE3) cells transformed with all three plasmids, but not with Cav-3-V5 alone, showed electrophoretic mobility shifts at 40 and 50 kDa (*, Fig. 2B); the 40 kDa band is consistent with in vitro and in vivo data (Fig. 1, C–E).

FIGURE 2.

In vitro SUMOylation of caveolin-3; expression and purification of Cav-3-V5 in E. coli. A, strategy for expression of SUMOylated Cav-3-V5 in E. coli. BL21(DE3) cells were sequentially transformed with three plasmids with compatible origins of replication and selection markers, pKRSUMO, pBADE12, and pST39-Cav-3-V5, which allow inducible expression by isopropyl-β-d-1-thiogalactopyranoside of SUMO-1, the SUMO E1 and E2 enzymes, and Cav-3-V5, respectively (plasmid maps adapted from Ref. 29). B, small scale expression to determine solubility of Cav-3-V5 and validate expression of each plasmid. BL21(DE3) cells transformed with all three plasmids (lanes 5 and 6) were lysed before and after isopropyl-β-d-1-thiogalactopyranoside induction of protein expression. Cells transformed with pST39-Cav-3-V5 alone (lanes 1 and 2) or just pKRSUMO and pBADE12 (lanes 3 and 4) were negative controls. The soluble supernatant and insoluble pellet (data not shown) from cell lysates were analyzed by immunoblotting with anti-V5-HRP antibodies. C, Ni²⁺-NTA purification of SUMOylated Cav-3-V5. Cells expressing all three plasmids were scaled up for Ni²⁺-NTA purification. Cav-3-V5 was eluted with 250 mm imidazole (lanes 8–12). Cells transformed with pST39-Cav-3-V5 alone or with pKRSUMO and pBADE12 were processed in parallel as negative controls (data not shown) (IN, input; SN, supernatant; W, wash; E, elution). D, incubation of E. coli lysates with the catalytic domain of SENP1. Purified lysates from BL21(DE3) cells transformed with all three plasmids (C) were incubated for 1 h at 37 °C with and without the catalytic domain of SENP1 (SENP1_CD) to validate that electrophoretic mobility shifts in anti-V5-HRP immunoblots were caused by covalent modification of Cav-3-V5 by SUMO-1.

We purified SUMOylated Cav-3-V5 using Ni²⁺-NTA resin (Fig. 2C). Elution fractions (E3–E5) contained unmodified Cav-3-V5 (25 kDa) and multiple slower migrating bands corresponding to SUMOylated Cav-3-V5 (*, Fig. 2C) that probably represent poly-SUMOylated Cav-3-V5. To confirm that these bands were a result of SUMOylation, the elution fractions were incubated with the catalytic domain of SENP1 (SENP1_CD; Fig. 2D). The bands at 40, 50, and 60 kDa disappeared with SENP1 treatment; however, it is unclear whether the multiple bands are indicative of multiple sites of SUMOylation or modification by poly-SUMO chains.

PIASy Interacts with Caveolin-3 and Stimulates SUMO-3 Modification

To validate the in vitro results and further investigate the determinants of Cav-3 SUMOylation, we performed SUMOylation assays in eukaryotic cells. Although SUMO-1 and SUMO-3 covalently modified Cav-3 in vitro (Fig. 1C), only SUMO-3 did so when co-expressed in HEK cells (Fig. 4B). The SUMO E1 and E2 enzymes (SAE1/2 and Ubc9) SUMOylate Cav-3 in vitro; however, in vivo, a specific SUMO E3 ligase is often required. To identify a SUMO E3 ligase specific for Cav-3, we co-transfected PIAS1, PIAS3, or PIASy with Cav-3-V5, Ubc9, and Myc-SUMO-3 (Fig. 3A). When PIASy was co-expressed, bands at 40 and 50 kDa were enhanced in V5-HRP and Myc immunoblots. The 45 kDa band in anti-V5-HRP immunoblots between these Cav-3-V5-SUMO conjugates is probably dimerized Cav-3-V5 because it does not vary with co-expressed SUMO, Ubc9, or PIASy (Fig. 3, A–D).

Transfection of HEK cells with increasing amounts of PIASy yielded increased modification of Cav-3-V5 by SUMO-3 (Fig. 3B, lanes 4–7). A ladder of bands corresponding to Cav-3 modified at multiple lysine residues or by poly-SUMO-3 chains was seen (*, Fig. 3B) at 65 kDa and higher in addition to the 40 and 50 kDa bands observed in vitro (Fig. 2, B–D). Transfecting >400 ng of PIASy had no further effect on Cav-3-V5 SUMOylation and produced cell toxicity (data not shown).

HEK cells transfected with Cav-3-V5, SUMO-3, Ubc9, and PIASy were immunoprecipitated with anti-V5 and anti-Cav-3 antibodies. Immunoblotting revealed that Cav-3-V5 co-immunoprecipitates with PIASy (Fig. 3C). Reciprocal immunoprecipitation, performed with anti-PIASy antibodies, showed that PIASy interacts with unmodified Cav-3-V5 (25 kDa) and SUMOylated Cav-3-V5 (*, Fig. 3D). Thus, PIASy interacts with Cav-3 and stimulates its modification at multiple lysine residues or by poly-SUMO-3 chains. PIASy expression in cardiomyocytes was confirmed by immunoblotting of adult rat cardiomyocytes with anti-PIASy antibodies (Fig. 3E).

Mutation of the Consensus Site Lysine Reduced but Did Not Abolish SUMOylation of Caveolin-3

We next sought to identify the lysine(s) modified and to create a SUMOylation-deficient mutant that would facilitate identification of biological functions influenced by SUMOylation of Cav-3. We initially mutated the consensus site lysine in Cav-3-V5 (Fig. 4A) to arginine (K38R) and assessed SUMOylation (Fig. 4B). K38R reduced but did not abolish SUMOylation of Cav-3-V5, indicating that either 1) multiple lysine residues are modified or 2) if the consensus site is mutated, a different lysine can be modified.

Identification of a SUMOylation-deficient Mutant

We mutated the seven lysines of Cav-3-V5 to arginine one at a time (KR mutants) and assessed SUMOylation in transfected HEK cells (Fig. 4C). K38R reduced SUMOylation relative to WT Cav-3-V5, but SUMOylation of other KR mutants was equal to or greater than that of WT. It is unclear whether this is due to increased SUMOylation or differences in stability or expression of the mutants (e.g. K30R and K69R). Lys³⁸ appeared to be the major site of SUMOylation; however, other sites can be modified, suggesting that two or more lysines might need to be mutated. Two rounds of multisite mutagenesis were performed to generate multiple KR mutants. None of these mutants completely prevented SUMOylation of Cav-3 (Fig. 4D, lanes 6–10). SUMOylation was abolished when all seven lysines were mutated (Cav-3-V5-K7R; Fig. 4, C (lane 7) and D (lanes 1–4)), implying that unavailability of the preferred SUMOylation site(s) allows other lysines to be SUMOylated.

Lys³⁸ Is the Preferred SUMOylation Site

To define precisely which lysines are potential versus preferred SUMOylation sites and determine their relative ability to be SUMOylated, we used Cav-3-V5-K7R (Fig. 5A) to create a series of RK mutants. HEK cells were transfected with WT and the Cav-3-V5-K7R mutant in parallel with the RK mutants (Fig. 5B). Immunoblotting with anti-V5-HRP antibodies detected SUMOylation at each lysine, but reintroduction of the consensus site lysine in the R38K mutant most effectively restored the SUMOylation of Cav-3-V5 (Fig. 5B, lane 6). Moreover, R38K was the only mutant that, akin to WT Cav-3-V5, was poly-SUMOylated (*, Fig. 5B, lane 1 versus lane 6), suggesting that PIASy stimulates SUMOylation by poly-SUMO-3 chains at this site.

FIGURE 5.

Reversal of each arginine mutation in K7R back to lysine one at a time reveals that Lys³⁸ is the preferred SUMOylation site when PIASy is co-expressed. A, schematic of the Cav-3-V5-K7R construct. Each arginine mutation present in K7R (R15, R20, R30, R38, R69, R108, and R144) is shown in context with the N terminus, scaffolding domain, transmembrane domain, C terminus, and V5/His affinity tags. B, identification of the preferred SUMOylation site RK mutants. HEK 293 cells were co-transfected with each of the seven RK mutants (lanes 3–9) along with the SUMOylation machinery (SUMO-3, Ubc9, and PIASy). WT and K7R (lanes 1 and 2, respectively) served as positive and negative controls for Cav-3-V5-SUMO-3 conjugates. Lysates were analyzed by immunoblotting with anti-V5-HRP antibodies. C, PIASy-dependent SUMOylation of Lys³⁸. The four N-terminal RK mutants (R15K, R20K, R30K, and R38K) (lanes 5–12) were transfected with and without the SUMOylation machinery. WT and Cav-3-V5-K7R (lanes 1–4) were included as positive and negative controls for Cav-3-V5-SUMO-3 conjugates. Lysates were analyzed by immunoblotting with anti-V5-HRP, anti-PIASy, and anti-GAPDH antibodies.

To confirm that PIASy was required for SUMOylation at Lys³⁸, we transfected the four N-terminal RK mutants with and without co-expression of the SUMOylation machinery (SUMO-3, Ubc9, and PIASy). Immunoblotting with anti-V5-HRP, anti-PIASy, and anti-GAPDH revealed that co-expression of the SUMOylation machinery had no effect on the low level of modification of R15K, R20K, or R30K but strongly enhanced modification of R38K (Fig. 5C, lane 6), which closely resembled WT (*, Fig. 5C, lane 2 versus lane 6). Thus, PIASy is necessary for SUMOylation, including poly-SUMOylation, of Lys³⁸.

WT Caveolin-3 Increases, but the K7R Mutant Decreases, β₂AR Expression Levels

We used the K7R mutant to probe the effect of SUMO modification on protein-protein interactions of caveolin with a known G-protein-coupled receptor binding partner, the β2-adrenergic receptor (β₂AR). β₂ARs predominantly reside in caveolar microdomains in cardiac myocytes and interact with Cav-3 (33–35). To determine whether SUMOylation of Cav-3 modulates its interaction with β₂ARs, we transfected FLAG-β₂AR into HEK 293 cells either alone or with WT or Cav-3-V5-K7R. Cells were grown in DMEM plus 10% FBS, harvested 48 h post-transfection, and immunoprecipitated with anti-FLAG-agarose beads. Immunoprecipitates were analyzed by immunoblotting with anti-FLAG-HRP, ubiquitin, V5-HRP, and PIASy antibodies (Fig. 6A). We anticipated that SUMOylation might change the ability of FLAG-β₂AR to bind Cav-3-V5, perhaps by blocking access to the CSD (Figs. 1A and 8A). We found, however, that we were unable to determine the relative ability of FLAG-β₂AR to interact with WT Cav-3-V5 or the K7R mutant, because the level of FLAG-β₂AR expression was increased or decreased depending on co-expression of WT or Cav-3-V5-K7R, respectively (Fig. 6A, lanes 1, 3, and 5). In addition, co-expression of the SUMOylation machinery (SUMO-3, Ubc9, and PIASy) increased the level of FLAG-β₂AR expression (Fig. 6A, lanes 2, 4, and 6) independent of Cav-3-V5 expression.

FIGURE 6.

Wild-type Cav-3-V5 stabilizes, whereas the SUMOylation-deficient K7R mutant destabilizes, FLAG-β₂AR. A and B, co-expression of Cav-3-V5-K7R reduces the expression of FLAG-β₂AR protein, and WT has an opposite effect when co-transfected with the SUMOylation machinery. HEK 293 cells were co-transfected with FLAG-β₂AR (lanes 1–11) and either WT Cav-3-V5 (lanes 3 and 4 and lanes 8 and 9) or the K7R mutant (lanes 5 and 6 and lanes 10 and 11), both with and without the SUMOylation machinery, SUMO-3, Ubc9, and PIASy (even versus odd lanes, respectively). The proteasomal inhibitor MG-132 was preincubated with cells 3 h prior to lysis (lanes 7–11). 48 h post-transfection, lysates were immunoprecipitated (IP) with anti-FLAG antibodies and analyzed by immunoblotting with anti-FLAG-HRP, anti-ubiquitin, anti-V5-HRP, and anti-PIASy antibodies. B, HEK 293 cells were transfected identically to those in lanes 1–6 of A; however, cells were harvested 24 h, rather than 48 h, post-transfection. Lysates were immunoprecipitated with anti-FLAG antibodies and analyzed by immunoblotting with anti-FLAG-HRP and anti-V5-HRP antibodies. C, eNOS expression is not affected by co-transfection WT or Cav-3-V5. HEK 293 cells were co-transfected with eNOS (lanes 1–6) and either WT Cav-3-V5 (lanes 3 and 4) or the K7R mutant (lanes 5 and 6) with and without the SUMOylation machinery, SUMO-3, Ubc9, and PIASy. Cells were harvested 24 h post-transfection, and anti-eNOS immunoprecipitates were analyzed by immunoblotting with anti-eNOS and anti-V5-HRP antibodies.

FIGURE 8.

A “SUMO Switch”; regulation by SUMOylation of protein-protein interaction by the CSD. Proposed interaction of basic residues in the α-helical CSD with an “acidic patch” surrounding the SUMOylation site of caveolin-3. A, membrane topology and α-helical secondary structure of the CSD of caveolin-3. The CSD (aa 55–72) forms an α-helix, whereas the remainder of the N terminus lacks secondary structure; it has been proposed that the N-terminal tail region (aa 1–54) wraps back around the CSD (47). This interaction might occur through electrostatic interactions of an acidic patch (³⁴EDIVKVDFED⁴³) (underline denotes acidic residues) with basic residues in the CSD (α-helix image adapted from Ref. 47). B, helical wheel projection of the Cav-3 CSD. The amino acid sequence of the CSD (⁵⁵VWKVSYTTFTVSKYWCYR⁷²) is color-coded by each residue's side chain properties and represented linearly and as a helical wheel projection to illustrate the clustering of basic (blue), polar (purple), and non-polar (yellow) residues along different sides of the helix (see the HeliQuest Web site). The single-letter amino acid codes in the helical wheel are proportional to amino acid volume.

Endogenous β₂ARs have a half-life of 24 h if unstimulated (36). Thus, we repeated the experiment with cells harvested 24 h post-transfection. WT Cav-3-V5 increased expression levels of FLAG-β₂AR when the SUMOylation machinery was co-expressed (Fig. 6B, lanes 1 and 2 versus lanes 3 and 4), whereas the K7R mutant decreased FLAG-β₂AR (lanes 1 and 2 versus lanes 5 and 6). Moreover, there was no effect of co-expression of the SUMOylation machinery independent of Cav-3-V5. Thus, co-expression of the K7R mutant decreases expression levels of FLAG-β₂AR.

The mechanisms of agonist-induced desensitization of the β₂AR have been extensively studied in HEK 293 cells (37, 38). β-Adrenergic agonists (norepinephrine and epinephrine) are present in fetal bovine serum (FBS) (39). Therefore, during these experiments, the FLAG-β₂AR-transfected HEK cells were exposed to β-agonists by use of FBS in the culture media. The data shown thus far suggest that modification of Cav-3 by SUMO-3 may alter FLAG-β₂AR expression levels by inhibiting, whereas the SUMOylation-deficient Cav-3 mutant may promote agonist-induced desensitization of β₂ARs. To determine whether the effect of the K7R mutant of Cav-3 is specific for β₂AR, we assessed the impact of K7R on expression of eNOS (NOS3), a well studied, caveolin-regulated protein (40, 41). HEK 293 cells were transfected with eNOS alone or with WT or Cav-3-V5-K7R and with or without the SUMOylation machinery for 24 h (Fig. 6C). The expression level of eNOS was unaffected by expression of either WT Cav-3 or Cav-3-V5-K7R, suggesting that the effect on FLAG-β₂AR is not a nonspecific inhibition of protein expression caused by the Cav-3-V5-K7R mutant.

β₁ARs and β₂ARs are highly homologous both structurally and functionally (35), and a portion of β₁ARs also localize to caveolae and interact with Cav-3 but do not undergo agonist-induced ubiquitination, internalization, and desensitization (38, 42). FLAG-tagged β₁AR or β₂AR were co-transfected into HEK 293 cells either alone or with WT or Cav-3-V5-K7R. Unlike FLAG-β₂ARs, expression of FLAG-β₁ARs was not altered by co-transfection of the Cav-3-V5-K7R mutant (Fig. 7A, lanes 1–3 versus lanes 4–6). Thus, SUMOylation of Cav-3 appears to influence the expression level of β₂ARs but not β₁ARs, and this effect may depend on agonist-induced desensitization of the receptor.

FIGURE 7.

The SUMOylation-deficient K7R mutant does not affect the stability of β₁AR and destabilization of β₂AR by K7R is blocked by β-adrenergic antagonist. A, FLAG-β₁AR is not destabilized by the K7R mutant. HEK 293 cells were co-transfected with FLAG-β₁AR (lanes 1–3) or FLAG-β₂AR (lanes 4–6), the SUMOylation machinery (lanes 1–6), and either WT Cav-3-V5 (lanes 2 and 5) or the K7R mutant (lanes 3 and 6). Cell lysates were immunoprecipitated (IP) with anti-FLAG antibodies and analyzed by immunoblotting with anti-FLAG-HRP and anti-V5-HRP antibodies. B–D, the destabilization of FLAG-β₂AR by Cav-3-V5-K7R is blocked by (−)-propranolol and reduced serum. B, HEK 293 cells were co-transfected with FLAG-β₂AR and the SUMOylation machinery (lanes 1–6) with either WT Cav-3-V5 (lanes 2 and 5) or the K7R mutant (lanes 3 and 6). Cells were grown in media plus 2 μm (−)-propranolol for 48 h (lanes 1–3). Cells were also grown with reduced serum for 24 h prior to harvest (lanes 4–6). Anti-FLAG immunoprecipitates were analyzed by immunoblotting with anti-FLAG-HRP and anti-V5-HRP antibodies. C, HEK 293 cells were transfected as in A and B and were treated or not for 48 h with 2 μm (−)-propranolol. Lysates were immunoprecipitated with anti-FLAG antibodies, but proteins were not heated prior to SDS-PAGE to reduce formation of FLAG-β₂AR aggregates and aid in quantification. Immunoprecipitates and cell lysates were analyzed by immunoblotting with anti-FLAG-HRP and anti-GAPDH antibodies, respectively. D, FLAG-HRP immunoblots (C) were quantified by densitometry, normalized to GAPDH, and expressed as -fold change relative to vehicle-treated controls (lane 1).

Propranolol Attenuates the Effect of Cav-3-V5-K7R on FLAG-β₂AR Expression Levels

We used the βAR antagonist propranolol to determine whether the decrease in FLAG-β₂AR expression is a result of β-agonist stimulation of FLAG-β₂AR by catecholamines present in the culture media. HEK 293 cells transfected with the SUMOylation machinery (SUMO-3, Ubc9, and PIASy) were co-transfected with either FLAG-β₂AR alone, WT Cav-3-V5, or the K7R mutant in the absence or presence of 2 μm propranolol. Treatment with propranolol blunted the effect of the Cav-3-V5-K7R mutant on FLAG-β₂AR expression (Fig. 7B, lanes 1–3). Incubation of cells in media with reduced serum (1%) had a similar effect as did propranolol (Fig. 7B, lanes 4–6). In each case, WT Cav-3-V5 increased FLAG-β₂AR expression levels (Fig. 7B, lanes 2 and 5), presumably by inhibiting the agonist-induced desensitization of the receptor.

To quantify this effect, HEK 293 cells were co-transfected with FLAG-β₂AR alone, WT Cav-3-V5, or the K7R mutant and treated with or without 2 μm propranolol; FLAG-HRP immunoblots of anti-FLAG immunoprecipitates were then quantified by densitometry (Fig. 7, C and D). Immunoprecipitates and protein samples are typically heated prior to analysis by SDS-PAGE; however, this can cause self-aggregation of membrane proteins, such as G-protein-coupled receptors (43). To achieve accurate quantification, we did not heat the anti-FLAG immunoprecipitates prior to SDS-PAGE to reduce the formation of >100-kDa FLAG-β₂AR aggregates (Fig. 7, A and B). We found that K7R reduced FLAG-β₂AR expression levels by about 50% (p = 0.005) in vehicle-treated cells and that treatment with propranolol returned the expression to control levels (Fig. 7, C and D). WT Cav-3-V5 had the opposite effect, increasing FLAG-β₂AR expression by ∼2-fold; expression was further increased by propranolol. Cav-3 and its ability to undergo SUMOylation thus appear to modulate the agonist-induced desensitization of β₂ARs.

DISCUSSION

These studies demonstrate for the first time that a caveolin, Cav-3, is post-translationally modified by SUMO proteins in vitro and in vivo. SUMOylation of Cav-3 appears to be a novel regulatory mechanism for agonist-induced desensitization of β₂ARs. In vitro SUMOylation assays and co-expression of recombinant Cav-3 in E. coli with the SUMOylation machinery showed that conjugation with SUMO-1 or SUMO-3 is possible. However, in mammalian cells, conjugation is specific for SUMO-3. In E. coli transformed with the SUMOylation machinery, Cav-3 was modified at multiple sites or by poly-SUMO-1 (Fig. 2, B–D). There is debate regarding the ability of SUMO-1 to form chains in vivo (44); this possibility cannot be ruled out because SENPs (which reverse SUMO conjugation and edit poly-SUMO chains) are absent in E. coli. The tandem SUMO-interacting motifs (SIMs) of Cav-3 (supplemental Fig. S1, C and D) may assist in the modification by poly-SUMO chains by recruiting Ubc9 thioesters containing polymerized SUMO. The specificity for modification by SUMO-3 in HEK 293 cells may result from its greater cytosolic localization or preferential interaction of Cav-3 SIMs with SUMO-3 rather than SUMO-1 (45–47).

We find that PIASy preferentially enhances SUMOylation of Cav-3 at a consensus site, but mutation of this site does not abolish SUMOylation. This may result from a lack of secondary structure of the N terminus of Cav. Amino acids 79–96 in Cav-1, which comprise the CSD (aa 55–72 in Cav-3), form an α-helix, whereas the remainder of the N terminus lacks secondary structure (48). Perhaps non-consensus site lysines become accessible when Lys³⁸ is mutated. Fernandez et al. (48) proposed that the N-terminal tail region (aa 1–54 in Cav-3) wraps back around the α-helical region (Fig. 8). Mutation of Cav-1 aa 66–70 (IDFED) to alanine dramatically affects oligomerization (48); Fernandez et al. (48) speculated that this acidic patch binds basic residues in the α-helix, thereby causing the tail to wrap around the CSD. The corresponding region in Cav-3 (aa 39–43) is next to the SUMOylated residue in Cav-3, Lys³⁸ (Fig. 8A, EDIVK(SUMO)VDFED). A helical wheel projection illustrates clustering of basic residues along one side of the α-helical CSD (Fig. 8B). Thus, modification by SUMO at this site could block the interaction of the N-tail region with the CSD, acting as a switch to regulate binding of proteins to the CSD or oligomerization of caveolins.

The tandem SIMs in the N terminus of Cav-3 and the ability of caveolin to oligomerize may also contribute to the SUMOylation of other lysines, albeit at a much lower level than WT or R38K (Figs. 4 and 5). Proteins can be SUMOylated on non-consensus sites that are in close proximity to a SIM (e.g. USP25 (47)). This suggests that stabilization of the binding between Ubc9 ∼ SUMO thioesters and the target by a SUMO-SIM interaction or by substrate-E3 ligase interactions may facilitate the interaction of Ubc9 with non-consensus site lysines.

PIASy and other PIAS proteins have been considered nuclear proteins (49, 50), whereas Cav-3 is primarily found in plasma membrane microdomains, with some cytosolic and perinuclear localization (51). A study in PIASy^+/+ MEFs showed that although PIASy was predominantly found in the nucleus, it could also be detected in the cytoplasm (52). PIASy accumulates in the cytoplasm and co-localizes with the E3 ubiquitin ligase TRIM32 when MG-132 is used to inhibit proteasomal degradation (50), which may explain why it is normally detected at low levels in the cytoplasm. Consistent with this idea, treatment of HEK 293 cells with MG-132 for 3 h enhanced SUMOylation of Cav-3 relative to untreated cells (Fig. 4D, lane 5), presumably by increasing the cytosolic expression of PIASy. This finding is also consistent with data obtained with HEK 293 cells in which we sought to manipulate the expression of PIASy. TGFβ stimulates a 6–8-fold increase in PIASy mRNA expression in Hep2B cells that peaks after 12 h (53). We incubated HEK 293 cells with TGFβ in the presence or absence of MG-132 (treatment for 12 h) and found that cells treated with MG-132 have a 3-fold increase in PIASy protein expression and an increase in 40-kDa Cav-3-V5 (data not shown).

PIASy appears to be highly expressed in cardiac myocytes (Fig. 3E), and transfection of HEK 293 cells (which express low levels of PIASy (Fig. 3A)), with as little as 40 ng of PIASy greatly enhances SUMOylation of Cav-3 (Fig. 3B). PIASy co-immunoprecipitates with Cav-3 in transfected HEK 293 cells (Fig. 3, C and D), and immunofluorescence studies of HEK 293 cells transfected with Cav-3-V5 alone or with the SUMOylation machinery show that the affinity-tagged Cav-3 construct localizes in the cytosol and plasma membrane puncta, similar to endogenous Cav-3, consistent with the idea that PIASy co-localizes with Cav-3 in these compartments (data not shown).

PIASy interacts with the type I TGF-β receptor (TβRI) in MCF7 human breast cancer cells (54), and TβRIs localize to caveolae and interact with Cav-1 and eNOS in endothelial cells (18). Whether Cav-3 and PIASy co-localize in vivo has not been investigated. The E3 ubiquitin ligase TRIM32 can control cytosolic levels of PIASy and, akin to what has been observed for Cav-3, TRIM32 is mutated in Limb-Girdle muscular dystrophy. The latter mutation prevents TRIM32 from binding PIASy (50), suggesting that the regulation of cytosolic SUMOylation of Cav-3 and other targets by PIASy has functional consequences in terms of the pathophysiology of muscular dystrophy.

The findings presented here are consistent with recent data that show PIASy-dependent SUMOylation of a phosphodiesterase subtype, PDE4D5, which is recruited to activated β₂ARs (24, 55, 56). Caveolin and this PDE subtype share a high degree of similarity in their SUMOylation motifs (supplemental Fig. S1B). Perhaps Cav-3, β₂AR, PDE4D5, and PIASy are part of a signaling complex localized in caveolae that regulates cellular responses to agonist stimulation.

The mechanism by which SUMOylation of caveolin affects β₂AR expression levels is not clear. Cav-3 might affect β₂ARs through its ability to couple to the machinery that regulates the agonist-induced internalization and desensitization of the receptors. For example, β-arrestin-2 and Mdm2 are recruited to activated receptors. Cav-1 interacts with Mdm2 following H₂O₂ treatment (57), and oxidative stress caused by H₂O₂ can stimulate altered patterns and increases in SUMO-2/3 conjugation (58). Moreover, Mdm2 and β-arrestin-2 are targets of SUMOylation (37, 59–60), and PIASy cooperates with Mdm2 to regulate SUMOylation of p53 (61). β-Arrestin-2 preferentially interacts with the SUMOylated form of Mdm2 (37) (p90Mdm2). A search for SIMs similar to those in Cav-1 and -3 reveals the presence of tandem SIMs in β-arrestin-1 and β-arrestin-2 (supplemental Fig. S1C, ARRB1 and ARRB2), suggesting that these motifs may be responsible for the preferential binding of p90Mdm2. It will thus be of interest to test if the decreased expression of β₂AR caused by the Cav-3 K7R mutant results from SUMOylation-dependent alterations of Mdm2-β-arrestin-2 interactions.

In summary, our findings are the first to show that Cavs are SUMOylated and that SUMOylation influences the expression level of a binding partner of Cav. These results thus define a previously unappreciated mechanism that contributes to the interaction of Cavs, presumably by the CSD, with such binding partners. It will be of interest to define the full range of binding partners whose interaction is influenced by SUMOylation and whether SUMOylation of Cavs is altered during physiological and pathophysiological settings.