SUMO-1 Controls the Protein Stability and the Biological Function of Phosducin

• Published in: The Journal of biological chemistry Vol. 281; no. 13; pp. 8357 - 8364
• Main Authors: Klenk, Christoph, Humrich, Jan, Quitterer, Ursula, Lohse, Martin J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 31-03-2006; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Animals; Binding Sites; Blotting, Western; Carrier Proteins; Cattle; Cell Culture Techniques; Cell Line; Chlorocebus aethiops; Consensus Sequence; COS Cells; Eye Proteins - chemistry; Eye Proteins - genetics; Eye Proteins - isolation & purification; Eye Proteins - metabolism; GTP-Binding Protein beta Subunits - metabolism; GTP-Binding Protein gamma Subunits - metabolism; GTP-Binding Protein Regulators; Humans; Lysine - chemistry; Molecular Chaperones; Molecular Sequence Data; Molecular Weight; Mutation; Myocardium - chemistry; Nerve Tissue Proteins; Phosphoproteins - chemistry; Phosphoproteins - genetics; Phosphoproteins - isolation & purification; Phosphoproteins - metabolism; Phosphorylation; Precipitin Tests; Protein Binding; Rats; Receptors, G-Protein-Coupled - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Retina - chemistry; Rhodopsin - metabolism; Sequence Homology, Amino Acid; SUMO-1 Protein - genetics; SUMO-1 Protein - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M513703200

Phosducin regulates Gβγ-stimulated signaling by binding to Gβγ subunits of heterotrimeric G-proteins. Control of phosducin activity by phosphorylation is well established. However, little is known about other mechanisms that may control phosducin activity. Here we report that phosducin is regulated at the posttranslational level by modification with the small ubiquitin-related modifier, SUMO. We demonstrate modification with SUMO for phosducin in vitro expressed in cells and for native phosducin purified from retina and the heart. A consensus motif for SUMOylation was identified in phosducin at amino acid positions 32–35. Mutation of the conserved lysine 33 to arginine in this motif abolished SUMOylation of phosducin, indicating that SUMO is attached to lysine 33 of phosducin. In transfected cells the steady-state levels of the K33R mutant protein were much lower compared with wild-type phosducin. The investigation of the stability of wild-type phosducin and of phosducinK33R showed a decreased protein stability of the SUMOylation-deficient mutant. The decreased protein stability correlated with increased ubiquitinylation of the SUMOylation-deficient mutant. These findings indicate that SUMOylation protects phosducin from proteasomal degradation. SUMOylation of phosducin decreased its ability to bind Gβγ. PhlP, a closely related member of the phosducin family, was not a target for SUMOylation, but its SUMOylation can be achieved by a single amino acid insertion in the conserved N terminus of PhlP. Together, these findings show that phosducin is a previously unrecognized target of SUMO modification and that SUMOylation controls phosducin stability in cells as well as its functional properties.