Activation of p38- and CRM1-dependent nuclear export promotes E2F1 degradation during keratinocyte differentiation

Abstract

E2F factors modulate a plethora of cell functions, including proliferation, differentiation, DNA repair and apoptosis. We have shown that differentiation in primary epidermal keratinocytes leads to E2F1 downregulation via activation of protein kinase C and p38 mitogen-activated protein kinase. We now demonstrate that E2F1 downregulation in differentiating keratinocytes involves its ubiquitination, as well as proteasomal degradation subsequent to CRM1-dependent nuclear export. E2F1 nuclear export specifically in response to differentiation requires regions adjacent to the cyclin A-binding domain in the N-terminus of this protein. Significantly, inhibition of p38 interferes with nuclear export and degradation of E2F1 during differentiation, but has no effect on E2F1 in undifferentiated cells. Thus, induction of differentiation in epidermal keratinocytes activates a specific program for post-transcriptional downregulation of E2F1, which involves signaling through p38 and activation of nuclear export pathways.

CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes

“…The latter is necessary for degradation, as leptomycin B inhibition of CRM1 results in E2F1 stabilization and constitutive nuclear localization [12, 19]. Consistent with this notion, analyses of the subcellular distribution of the E2F1 mutants described above, and which exhibited differentiation-induced degradation, revealed nuclear export that was indistinguishable from that observed with the wild type protein (Figure 2 and Supplementary Figure S2).…”

“…E2F1 is downregulated to allow proper entry into quiescence and expression of differentiation markers in the epidermis [12, 13]. Our studies now describe a multifactorial mechanism of E2F1 degradation during keratinocyte differentiation that involves coordinate changes in subcellular localization and ubiquitylation patterns, and in which S403 and T433 play key modulatory roles.…”

“…E2F1 undergoes nucleocytoplasmic shuttling in undifferentiated keratinocytes and other cell types, although the balance of these movements favours a predominantly nuclear localization at steady-state [19]. Upon induction of differentiation, E2F1 is excluded from the nucleus and retained in the cytoplasm in keratinocytes and hippocampal neurons [12, 27]. Based on the ability of wild type and phosphomimetic E2F1 ST/D, but not E2F1 ST/A, to respond to Ca 2+ , we propose that such nucleocytoplasmic translocation is potentially dependent on phosphorylation of S403 and T433.…”

“…These events can be recapitulated in cultured primary keratinocytes by elevating the extracellular Ca 2+ concentration from ~0.05 to ≥ 0.1 mM. Under these conditions, E2F1 undergoes p38β-dependent phosphorylation at S403 and T433 and ubiquitylation, culminating with its degradation [7, 12, 13]. It is well established that E2F1 degradation is essential for proper keratinocyte differentiation [7, 12, 13].…”

Quantitative Proteomics Reveals Regulation of Karyopherin Subunit Alpha-2 (KPNA2) and Its Potential Novel Cargo Proteins in Nonsmall Cell Lung Cancer

“…E2F1 belongs to the E2F family of DNA-binding proteins (E2F1 to E2F6), which are central regulators of cell cycle progression, proliferation, differentiation, migration, and survival (81)(82)(83)(84)(85). Previous studies indicate that many E2F1 regulatory molecules are involved in lung cancer tumorigenesis, such as VEGF, HMGA2, UHRF1, TS, and SKP2 (52, 60, 86 -89).…”