Etoposide-induced protein Ei24 is a novel negative regulator of nuclear import.

Abstract

Nucleocytoplasmic trafficking mediated by the importin (IMP)/karyopherin superfamily of transporters is central to the transfer of signals between the nucleus and cytoplasm in response to diverse stimuli. Although much research has focused on mechanisms of regulation specific to cargoes that play a role in the nucleus, little is known regarding more general mechanisms that may control IMP localisation/activity. Although initially identified as a p53-induced pro-apoptotic gene, the function of the etoposide-induced protein Ei24 has not been determined. The work described in this thesis reports for the first time that Ei24 is a negative regulator of nuclear import (NRNI) that modulates IMP localisation/activity, and hence can influence p53 localisation and function. Close similarity was noted for the first time between the region of IMPα2 that binds IMPβ1 (the IMPβ-binding - IBB - domain), and Ei24 residues 44-87 (the IBB-like - IBBL - domain). Subcellular fractionation/colocalisation studies showed that ectopically expressed Ei24 is localised predominantly at the ER with IMPβ1 and IMPα2, whilst in vitro binding studies indicated high affinity direct binding of Ei24 to IMPs. The N-terminal 225 residues of Ei24, including the IBBL, was shown to be sufficient for IMP binding, with activity similar to that of the IBB of IMPα2. Further, binding of RanGTP to IMPβ1, but not RanGDP, disrupted binding to Ei24, suggesting that the IBBL has similar properties to other IBB-containing proteins. Ei24 was shown to sequester IMPs at the ER and thereby inhibit IMPα2/β1-facilitated nuclear accumulation of p53 upon etoposide-induced upregulation of Ei24 in primary murine embryonic fibroblasts. Analogously, ectopically expressed Ei24 inhibited nuclear import of IMPα2/β1- and IMPβ1-recognised cargoes such as SV40 large tumour antigen and the telomeric-repeat binding factor TRF1, respectively, as determined by quantitative CLSM analysis in cotransfected cells. In contrast, nuclear accumulation of a non IMP-recognised control molecule aF10 (ALL1 fused gene from chromosome 10) was not affected. The N-terminus of Ei24 was also sufficient to inhibit IMPα2/β1- and IMPβ1-mediated nuclear import, with site-directed mutagenesis of basic sequences within the IBBL domain indicating that a highly conserved polyarginine motif RRRR62 is required for Ei24’s NRNI activity. Regulation of Ei24 activity by a conserved glycogen synthase kinase (GSK) 3β phosphorylation motif within Ei24’s C-terminus (344SPHPS348) was examined using wortmannin to activate GSK3β or by analysing a phosphomimetic derivative (S344/348E). In both cases, IMPβ1 binding by Ei24 was enhanced concomitant with inhibition of IMPα2/β1- and IMPβ1-mediated nuclear import. Conversely, LiCl-induced inhibition of GSK3β activity or expression of a non-phosphorylatable derivative of Ei24 (S344/348A) resulted in reduced IMPβ1 binding and lack of an effect on nuclear transport. Intriguingly, Ei24 was found to bind to inactive GSK3β, which may contribute to steric hindrance of the IBBL, especially in its non-phosphorylated form. GSK3β may thus play a key role in regulating Ei24 NRNI activity. This study implicates Ei24 in the negative regulation of nuclear transport through transiently sequestering IMPβ1 and IMPα2 at the ER. The findings are important in that many breast cancers retain Ei24 mutations, implying that impaired Ei24 NRNI activity may contribute to oncogenesis.