Cell-dependent efficiency of reiterated nuclear signals in a mutant simian virus 40 oncoprotein targeted to the nucleus.

Abstract

We investigated the requisites for, and functional consequences of, the relocation to the nucleus of a transforming nonkaryophilic mutant of the simian virus 40 large T antigen (a natural deletion mutant lacking an internal large-T-antigen domain that includes the signal for nuclear transport). Synthetic oligonucleotides were used to obtain gene variants with one or more copies of the signal-specifying sequence inserted near the gene 3' end, in a region dispensable for the main large-T-antigen functions. The analysis of stable transfectant populations showed that mouse NIH 3T3 ceUls, rat embryo fibroblasts, and simian CS cells (a subclone of CV1 ceUls) differed considerably in their ability to localize some variant molecules into the nucleus. CS cells were always the most efficient, and NIH 3T3 ceUs were the least efficient. The nuclear localization improved either with reiteration of the signal or with a left-flank modification of the signal amino acid context. Three signals appeared to be necessary and sufficient, even in NIH 3T3 ceUs, to obtain a nuclear accumulation comparable to that of wild-type simian virus 40 large T antigen; other signal-cell combinations caused a large variability in subcellular localization among ceUls of the same population, as if the nuclear uptake of some molecules depended on individual cel states. The effect of the modified location on the competence of the protein to alter cell growth was examined by comparing the activity of variants containing either the normal signal or a signal with a mutation (corresponding to large-T-antigen amino acid 128) that prevented nuclear transport. It was found that the nuclear variant was slightly more active than the cytoplasmic variants in rat embryo fibroblasts and NIH 3T3 cells and was notably less active in CS cells.The nuclear localization of a number of eucaryotic proteins is known to depend on the presence in their polypeptide chains of determinants that vary in sequence and number; these are generally referred to as nuclear signals (3,

Firefly luciferase mutants as sensors of proteome stress

“…The NLS sequence was derived from the SV40 large T antigen and contains two repeats of a positively charged stretch rich in lysine residues (Fischer-Fantuzzi and Vesco, 1988) Figure 36: Design of Fluc-based sensors to study proteostasis in the cytosol and the nucleus.…”

Intracellular Localization and Activity State of Tissue Transglutaminase Differentially Impacts Cell Death

“…To make the NLS-tagged constructs, the cDNA for human tTG or [C277S]tTG was amplified from the pcDNA3.1 vectors by Pfu DNA polymerase-catalyzed PCR using a pair of DNA primers, the forward (5Ј-CCC GAC CAT GGC CGA GGA GCT GGT CTT AG-3Ј) containing an NcoI site and reverse (5Ј-CCG CTC GAG GGC GGG GCC AAT GAT GAC ATT C-3Ј) containing an XhoI site, digested with NcoI and XhoI and subsequently ligated into the NcoI/XhoI cloning sites of pShooter vector pCMV/myc/nuc (Invitrogen). The resulting DNA constructs, pCMV-tTG-NLS, pCMV-C277S-tTG-NLS contained cDNA for tTG or [C277S]tTG fused in-frame Cterminally with the 3ϫ NLS from SV40 large T antigen (28).…”

α-Catenin Inhibits β-Catenin Signaling by Preventing Formation of a β-Catenin·T-cell Factor·DNA Complex

“…GFP-␣N-catenin cDNA (23) was provided by Ravinder Sehgal and Louis Reichardt (University of California, San Francisco). To make GFP-␣-NLS, a double-stranded oligonucleotide (MWG Biotech, Germany) encoding the nuclear localization signal from SV40 large T antigen (24) and appropriate restriction sites was ligated into GFP-␣N-catenin cut with AflII and HindIII. This resulted in the insertion of the sequence AARDPKKKRKV after residue 902 of avian ␣N-catenin, followed by a stop codon.…”