title: Destabilization of Atoh1 by E3 Ubiquitin Ligase Huwe1 and Casein Kinase 1 Is Essential for Normal Sensory Hair Cell Development.
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.
authors.affiliation: From the Program in Speech and Hearing Bioscience and Technology, Harvard University/Massachusetts Institute of Technology, Cambridge, Massachusetts 02138, the Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, and the Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Yen-Fu
authors.initials: YF
authors.lastname: Cheng
authors.affiliation: the Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, and the Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Mingjie
authors.initials: M
authors.lastname: Tong
authors.affiliation: From the Program in Speech and Hearing Bioscience and Technology, Harvard University/Massachusetts Institute of Technology, Cambridge, Massachusetts 02138, the Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, and the Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts 02115 albert_edge@meei.harvard.edu.
authors.forename: Albert S B
authors.initials: AS
authors.lastname: Edge
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D047389:Casein Kinase I / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival / Q000502:physiology; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D057809:HEK293 Cells; D006199:Hair Cells, Auditory, Inner / Q000166:cytology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D025821:Polyubiquitin / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D059748:Proteolysis; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D047389:Casein Kinase I; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; C500942:ATOH1 protein, human; C502830:HUWE1 protein, human
keywords: Atoh1; E3 ubiquitin ligase; casein kinase; hair cell; inner ear; protein degradation; protein kinase; protein stability