Activation of thyroxine by outer ring deiodination is the crucial first step of thyroid
hormone action. Substrate-induced ubiquitination of type 2 deiodinase (D2) is the
most rapid and sensitive mechanism known to regulate thyroid hormone activation. While
the molecular machinery responsible for D2 ubiquitination has been extensively studied,
the combination of molecular features sufficient and required to allow D2 ubiquitination
remained to be determined. To address this question we constructed chimeric deiodinases
by introducing different combinations of D2-specific elements into type 1 deiodinase
(D1), another member of the deiodinase enzyme family, which however does not undergo
ubiquitination in its native form. Studies on the chimeric proteins expressed transiently
in HEK-293T cells revealed that combined insertion of the D2-specific instability
loop and the K237/K244 D2 ubiquitin-carrier lysines into the corresponding positions
of D1 could-not ubiquitinate D1 unless the chimera was directed to the endoplasmic
reticulum (ER). Fluorescence resonance energy transfer measurements demonstrated that
the C-terminal globular domain of the ER-directed chimera was able to interact with
the E3 ligase subunit WSB1. However, this interaction did not occur between the chimera
and the TEB4 E3 ligase although a native D2 could readily interact with the N-terminus
of TEB4. In conclusion, insertion of the instability loop and ubiquitin-carrier lysines
in combination with direction to the ER are sufficient and required to govern WSB1-mediated
ubiquitination of an activating deiodinase enzyme.
