SUMO-1 controls the protein stability and the biological function of phosducin.
Phosducin regulates Gbetagamma-stimulated signaling by binding to Gbetagamma 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 Gbetagamma. 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.
Klenk
Christoph
C
Institute of Pharmacology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany.
Humrich
Jan
J
Quitterer
Ursula
U
Lohse
Martin J
MJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins; D002417:Cattle; D018929:Cell Culture Techniques; D002460:Cell Line; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020555:GTP-Binding Protein Regulators; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009206:Myocardium / Q000737:chemistry; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012160:Retina / Q000737:chemistry; D012243:Rhodopsin / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D005136:Eye Proteins; C118981:G-protein Beta gamma; D020555:GTP-Binding Protein Regulators; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; C098789:PDCL protein, human; C498320:Pdcl protein, rat; D010750:Phosphoproteins; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D025842:SUMO-1 Protein; C065631:phosducin; D012243:Rhodopsin; D008239:Lysine