PubMed · 11731776
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J A Wolff, M G Sebestyén. 2001. Nuclear security breached.. https://doi.org/10.1038/nbt1201-1118
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LANA2 is a nuclear latent protein detected exclusively in Kaposi's sarcoma-associated herpesvirus-infected B cells. The protein inhibits p53-dependent transactivation and apoptosis, suggesting an important role in the transforming activity of the virus. To explore the molecular mechanisms of its nuclear localization, fusion proteins of green fluorescent protein (EGFP) and deletion constructs of LANA2 were expressed in HeLa cells. Only the fragment comprising amino acid residues 355-440 of LANA2 localized in the cell nucleus. This fragment contains two closely located basic domains and forms a putative bipartite nuclear localization signal (NLS). The putative LANA2 NLS was able to target EGFP to the nucleus consistently. Site-directed mutation analyses demonstrated that LANA2 contains a functional bipartite NLS between amino acid positions 367 and 384. In addition, analysis of cells transfected with a cytoplasmic LANA2 mutant revealed that an appropriate subcellular localization may be crucial to regulate p53 activity.
In the eukaryotic cell, exchange of biomolecules between nucleus and cytoplasm is a highly regulated process which responds sensitively to changes of the environment. One well-known cellular response to environmental challenges is cell death by apoptosis. In fact, apoptosis has been shown to affect the nucleocytoplasmic transport machinery, in particular the nuclear pore, by modulating its size exclusion limit for passive diffusion. The underlying molecular factors are still unknown, mainly because of the lack of a suitable system to detect and quantitate the apoptotic effects on the nuclear pore. Here we present an assay that was designed to measure alterations of the permeability of the nuclear envelope under apoptotic conditions. The assay is based on the well-established technique of selective permeabilization of the plasma membrane with digitonin and allows assessment of permeability changes in nonfixed samples. It comprises a computer program, called Nuclear Permeability Assay, for the quantitation of the nuclear fluorescence signal, which may be generally employed for the evaluation of in vitro transport systems using semipermeabilized cells, such as assays for nuclear import and export.
In the present study, we explored to determine whether insulin has any effect on the nuclear translocation of insulin receptor and IRS-1 in the nucleus of UMR-106 cells. Following insulin treatment, cells were subjected to subcellular fractionation. Each fraction containing equal amount of protein was subjected to Western blot analysis using antibodies against IR and IRS-1. Significant amounts of insulin receptors and IRS-1 were detected in the nucleus. Insulin receptor and IRS-1 appeared to be translocated to the nucleus in a time dependent manner by insulin whereas Akt levels remained unchanged by insulin treatment. The majority of insulin receptor and IRS-1 translocated to the nucleus appeared to associate with nuclear matrix. Tyrosine phosphorylation of a number of proteins with a molecular mass of 180, 95, 85, or 70 kDa in the nucleus was significantly stimulated by insulin, suggesting insulin signals to the nucleus and could regulate nuclear proteins. Confocal laser scanning microscope (CLSM) analysis also supports the nuclear translocation of insulin receptor and IRS-1. The nuclear insulin signaling may play an important role in the transcription control, differentiation, and growth of osteoblast cells.