Caveolin-3 Undergoes SUMOylation by the SUMO E3 Ligase PIASy: SUMOYLATION AFFECTS G-PROTEIN-COUPLED RECEPTOR DESENSITIZATION

Paul A Insel,Stephen R Fuhs

doi:10.1074/jbc.m110.214270

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 β(2)ARs but not β(1)ARs following agonist stimulation, thus implicating Cav-3 SUMOylation in the mechanisms for β(2)AR but not β(1)AR 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.

Highlights

The caveolin (Cav)3 family has three members; Cav-1 and -2 are co-expressed in most cell types, whereas Cav-3 is musclespecific [1]
We show here that Cav-3 has a small ubiquitin-like modifier (SUMO) consensus motif (⌿KX(D/E, where ⌿ 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
To determine whether SUMOylation occurs in vivo, lysates from rat cardiomyocytes were immunoprecipitated with anti-Cav-3 (*, Fig. 1D) or anti-SUMO antibodies (Fig. 1E)

Summary

Introduction

The caveolin (Cav)3 family has three members; Cav-1 and -2 are co-expressed in most cell types, whereas Cav-3 is musclespecific [1]. 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 (SENP1CD) to validate that electrophoretic mobility shifts in anti-V5-HRP immunoblots were caused by covalent modification of Cav-3-V5 by SUMO-1.

Results

Conclusion