title: CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes.
abstract: The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma. In addition, E2F1 must be downregulated for proper keratinocyte differentiation, but the relevant mechanisms involved remain poorly understood. We show that differentiation signals induce a series of post-translational modifications in E2F1 that are jointly required for its downregulation. Analysis of the structural determinants that govern these processes revealed a central role for S403 and T433. In particular, substitution of these two amino acid residues with non-phosphorylatable alanine (E2F1 ST/A) interferes with E2F1 nuclear export, K11- and K48-linked polyubiquitylation and degradation in differentiated keratinocytes. In contrast, replacement of S403 and T433 with phosphomimetic aspartic acid to generate a pseudophosphorylated E2F1 mutant protein (E2F1 ST/D) generates a protein that is regulated in a manner indistinguishable from that of wild type E2F1. Cdh1 is an activating cofactor that interacts with the anaphase-promoting complex/cyclosome (APC/C) ubiquitin E3 ligase, promoting proteasomal degradation of various substrates. We found that Cdh1 associates with E2F1 in keratinocytes. Inhibition or RNAi-mediated silencing of Cdh1 prevents E2F1 degradation in response to differentiation signals. Our results reveal novel regulatory mechanisms that jointly modulate post-translational modifications and downregulation of E2F1, which are necessary for proper epidermal keratinocyte differentiation.
authors.affiliation: Department of Physiology and Pharmacology, Children's Health Research Institute and Lawson Health Research Institute, The University of Western Ontario, London, Ontario N6A 5C1, Canada.
authors.forename: Randeep K
authors.initials: RK
authors.lastname: Singh
authors.affiliation: Department of Physiology and Pharmacology, Children's Health Research Institute and Lawson Health Research Institute, The University of Western Ontario, London, Ontario N6A 5C1, Canada.
authors.forename: Lina
authors.initials: L
authors.lastname: Dagnino
mesh_terms: D021581:Active Transport, Cell Nucleus; D019943:Amino Acid Substitution; D000818:Animals; D064200:Cdh1 Proteins / Q000378:metabolism*; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D004249:DNA Damage; D015536:Down-Regulation; D050687:E2F1 Transcription Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D051379:Mice; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
chemical_list: D064200:Cdh1 Proteins; D050687:E2F1 Transcription Factor; C493754:E2f1 protein, mouse; C577585:Fzr1 protein, mouse; D013912:Threonine; D012694:Serine; D008239:Lysine
keywords: E2F1; epidermis; keratinocytes