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.

Dual Role of the Anaphase Promoting Complex/Cyclosome in Regulating Stemness and Differentiation in Human Primary Keratinocytes

“…Interestingly, DNp63a gradually decreased with the differentiation status of the clones to reach dramatically low levels in paraclones. The same phenomenon was observed for E2F1-a positive regulator of the G1/S transition, recently shown to be an APC/C Cdh1 substrate in keratinocytes (Singh and Dagnino, 2017).…”

“…Our data provide more clues as to relevant APC/C Cdh1 substrates. E2F1 is an APC/C Cdh1 substrate when overexpressed, and its translocation to the cytoplasm followed by degradation correlates with keratinocyte differentiation (Ivanova and Dagnino, 2007;Singh and Dagnino, 2017). Here, the E2F1 protein expression was lower in paraclones than holoclones, and appeared regulated at the transcriptional level.…”

The role of FZR1 in tumorigenesis: Focus on cell-cycle control

“…Abnormal expression of E2F1 can promote non-melanoma skin cancer, and FZR1 binds to E2F1 in keratinocytes. Inhibition of FZR1 prevents its binding to E2F1, co-regulating post-translational modification, and down-regulating E2F1, resulting in a response to differentiation signals (Singh and Dagnino, 2017).…”