Identification Of Nuclear Export Signal Research Articles

Identification of nuclear export signal in KLLN suggests potential role in proteasomal degradation in cancer cells.

Germline and somatic promoter hypermethylation of KLLN has been found in diverse heritable and sporadic cancers, respectively. KLLN has many identified tumor suppressor functions, and when first reported, was thought to be exclusively nuclear. Here, we report on KLLN localization in both the nucleus and cytoplasm and the identification of a putative nuclear export signal (NES) sequence. KLLN overexpression in colon and breast cancer cells showed both nuclear and cytoplasmic presence. Inhibition of the CRM1 export pathway increased nuclear sequestration of KLLN, confirming the prediction of an NES sequence. Point mutations introduced in the predicted NES sequence decreased the strength of the NES and increased the nuclear sequestration of KLLN. Contrary to expectations, the transcription regulation and cellular proliferation functions of KLLN were unaffected by increased KLLN nuclear sequestration. Instead, increased nuclear KLLN correlated with increased nuclear sequestration of TRIM25 and decreased inhibitory phosphorylation of MDM2. Computational analysis of The Cancer Genome Atlas (TCGA) dataset showed positive correlation among KLLN, TRIM25 and MDM2 expression; pathway analysis of the common genes downstream of these three genes revealed protein degradation as one of the top canonical pathways. Together, our observations suggest that CRM1 pathway-based nuclear export of KLLN may impact proteasomal degradation.

Identification of Nuclear Export Signals in Antizyme-1

Antizyme-1 (AZ1) is a protein that negatively regulates polyamine synthesis by inhibiting the key synthetic enzyme ornithine decarboxylase and targeting it for degradation by the 26 S proteasome. Recent work shows that antizyme protein translocates to the nucleus during mouse development (Gritli-Linde, A., Nilssom, J., Bohlooly, Y. M., Heby, O., and Linde, A. (2001) Dev. Dyn. 220, 259-275). However, the significance and mechanism of this phenomenon remain unclear. In this study, we expressed AZ1 fused with enhanced green fluorescent protein (EGFP) to study its localization in a living cell. We found that EGFP-AZ1 was predominantly localized in the cytoplasm and that treatment with leptomycin B, a specific inhibitor of chromosomal region maintenance 1 (CRM1) induced nuclear accumulation of EGFP-AZ1 in Chinese hamster ovary and NIH3T3 cells. Two independent nuclear export signal (NES) sequences, each containing essential hydrophobic residues, were identified in the 50 N-terminal amino acid residues and in the central part of AZ1. The activity of the second NES was inhibited by an N-terminal adjacent region and was only revealed in N-terminal truncated constructs. Both NESs were active when fused to an artificial nuclear protein SV40-NLS-EGFP-EGFP. The ability of AZ1 to shuttle between the nucleus and the cytoplasm suggests that it has a novel function in the nucleus.

Identification of Nuclear Export Signal in UL37 Protein of Herpes Simplex Virus Type 2

The UL37 gene of herpes simplex virus (HSV) encodes a 120-kDa phosphoprotein associated with the virion. In this study, we have generated a rabbit polyclonal antiserum against HSV-2 UL37 protein, and examined its intracellular localization by immunofluorescence study. In infected cells, specific fluorescence was detectable in the perinuclear region. In transfected cells, UL37 protein was observed mainly in the cytoplasm. Transfection assays of deletion mutants of UL37 protein suggested that the leucine rich region (LRR) containing amino acids 263–273 may be important for cytoplasmic localization. Deletion of the LRR or substitution of the leucine residues resulted in nuclear remaining of UL37 protein. Moreover, the LRR could export green fluorescent protein (GFP) to the cytoplasm as a fusion protein and this export was blocked by leptomycin B treatment, indicating that the LRR acted as a nuclear export signal. These results suggest that UL37 protein fulfills a role as a shuttle between the nucleus and the cytoplasm through the LRR.