Abstract
Proneural basic helix-loop-helix transcription factor, Atoh1, plays a key role in the development of sensory hair cells. We show here that the level of Atoh1 must be accurately controlled by degradation of the protein in addition to the regulation of Atoh1 gene expression to achieve normal cellular patterning during development of the cochlear sensory epithelium. The stability of Atoh1 was regulated by the ubiquitin proteasome system through the action of Huwe1, a HECT-domain, E3 ubiquitin ligase. An interaction between Huwe1 and Atoh1 could be visualized by a proximity ligation assay and was confirmed by co-immunoprecipitation and mass spectrometry. Transfer of a lysine 48-linked polyubiquitin chain to Atoh1 by Huwe1 could be demonstrated both in intact cells and in a cell-free system, and proteasome inhibition or Huwe1 silencing increased Atoh1 levels. The interaction with Huwe1 and polyubiquitylation were blocked by disruption of casein kinase 1 (CK1) activity, and mass spectrometry and mutational analysis identified serine 334 as an important phosphorylation site for Atoh1 ubiquitylation and subsequent degradation. Phosphorylation by CK1 thus targeted the protein for degradation. Development of an extra row of inner hair cells in the cochlea and an approximate doubling in the number of afferent synapses was observed after embryonic or early postnatal deletion of Huwe1 in cochlear-supporting cells, and hair cells died in the early postnatal period when Huwe1 was knocked out in the developing cochlea. These data indicate that the regulation of Atoh1 by the ubiquitin proteasome pathway is necessary for hair cell fate determination and survival.
Highlights
Loss of mammalian cochlear hair cells, caused by genetic mutations, autoimmune disease, ototoxic medications, exposure to noise, and aging, is usually permanent
E3 ubiquitin ligases are classified by the occurrence of HECT or RING domains, based on the identity of the domain involved in E2 enzyme interaction [13, 14]
We show that silencing of HECT-domain E3 ligase Huwe1 decreases the degradation of Atoh1 in the cochlea and in cell lines, which agrees with a previous study identifying Huwe1 as an E3 ligase for Atoh1 [15]
Summary
Lys-48-linked Polyubiquitin Targets Atoh for Proteasomal Degradation—We assessed the half-life of Atoh with and without proteasome inhibition. E-proteins, E12, and E47 (TCF3), which are known, interacting partners of Atoh, were high confidence candidates, indicating the validity of the immunoprecipitation/ mass spectrometry approach. Treatment of FLAG-HA-Atoh1 293T cells with Huwe shRNA extended the half-life of Atoh in an assay using a cycloheximide chase (Fig. 2, C and D). These results support the hypothesis that Huwe degrades Atoh through the ubiquitin-proteasome pathway. CK1 inhibition extended the half-life of Atoh to Ͼ2 h, compared with about 30 min for a control treated with DMSO in a cycloheximide-chase assay (Fig. 3, E and F), indicating a role in regulation of Atoh stability.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
