Abstract
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.
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