A Novel Mechanism of E2F1 Regulation Via Nucleocytoplasmic Shuttling: Determinants of Nuclear Import and Export

Abstract

E2F1 is a transcription factor central for cell survival, proliferation, and repair following genomic insult. Depending on the cell type and conditions, E2F1 can induce apoptosis in transformed cells, behaving as a tumour suppressor, or impart growth advantages favouring tumour formation. The pleiotropic functions of E2F1 are a likely consequence of its ability to transcriptionally control a wide variety of target genes, and require tight regulation of its activity at multiple levels. Although sequestration of proteins to particular cellular compartments is a well-established regulatory mechanism, virtually nothing is known about its contribution to modulation of E2F1 target gene expression. We have examined the subcellular trafficking of E2F1 and, contrary to the widely held notion that this factor is constitutively nuclear, we now demonstrate that it is subjected to continuous nucleocytoplasmic shuttling. We have also defined two nuclear localization domains and a nuclear export region, which mediates CRM1-dependent transit out of the nucleus. The predominant subcellular location of E2F1 is likely determined by the balance between the activity of nuclear import and export domains, and can be modulated by differentiation stimuli in epidermal cells. Thus, we have identified a hitherto unrecognized mechanism to control E2F1 function through modulation of its subcellular localization.

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).…”

“…This emphasizes the clear distinction between mechanisms activated in response to physiological and pathological signals. 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].…”

Epidermal growth factor induction of front–rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2

“…The cells were plated onto laminin 332 matrix–coated coverslips (Nakrieko et al , 2008) and were cultured for 3 h to allow attachment and spreading, prior to processing for fluorescence microscopy (Ivanova et al , 2007). For growth factor stimulation experiments, subconfluent cells cultured on laminin 332 matrix were transfected, and, 24 h later, the growth medium was replaced with FBS-, EGF-, and Ca 2+ -free EMEM supplemented with 2.5% BSA.…”

Pharmacological inhibition of ATM by KU55933 stimulates ATM transcription

“…The E2F family of transcription factors are known to be mostly nuclear, with some reports suggesting cell cycle dependent localization and continuous shuttling between the nucleus and cytoplasm. 37 We saw a distinct increase in nuclear and cytoplasmic E2F1 during cell cycle progression after serum release (Figure 2a). The nuclear fraction of E2F1 increased to a greater extent than the cytoplasmic fraction.…”