p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV-40 T antigen proteins.

Abstract

Abstract. The nuclear import of transcription regulatory proteins appears to be used by the cell to trigger transitions in cell cycle, morphogenesis, and transformation . We have previously observed that the rate at which SV-40 T antigen fusion proteins containing a functional nuclear localization sequence (NLS; residues 126-132) are imported into the nucleus is enhanced in the presence of the casein kinase R (CK-II) site Si"ai2 . In this study purified p34cdc2 kinase was used to phosphorylate T antigen proteins specifically at T'24 and kinetic measurements at the single-cell level performed to assess its effect on nuclear protein import . Tí24 phosphorylation, which could be functionally simulated by a T-to-13 124 substitution, was found to reduce the maximal extent of nuclear accumulation whilst negligibly affecting the import C LOSE correlations between the nuclear cytoplasmic distribution ofcertain transcription factors (Lenardo and

Mutual exclusivity of DNA binding and nuclear localization signal recognition by the yeast transcription factor GAL4: implications for nonviral DNA delivery

“…40,41 Mammalian expression vectors A 44 bp oligonucleotide (5Ј-GATCGGAAGACTCTCCTC CGAGCGCTCGGAAGACTCTCCTCCGC-3Ј), containing tandem repeats of the 17mer shown to be specifically recognised by the GAL4 DNA binding domain, 25,42 was annealed with the complementary oligonucleotide (5Ј-GATCGCGGAGGAGAGTCTTCCGAGCGCTCGGAGG AGAGTCTTCC-3Ј) to yield a double stranded oligomer (17m) which was then ligated into the unique BamHI sites of the reporter plasmids pSV2neo 43 and pCH110. 44 These confer SV40 promoter directed expression of the neomycin resistance gene that confers resistance to the antibiotic G418, and the E. coli enzyme ␤-galactosidase, respectively.…”

BK Virus as a Cofactor in the Etiology of Prostate Cancer in Its Early Stages

“…Wildtype p53 has been shown to be retained in the cytoplasm as a result of interactions with proteins such as Hdm-2 and heat shock proteins (81). Phosphorylation of serine residues adjacent to the NLS of SV40 TAg by casein kinase II facilitates the nuclear translocation of TAg, and this process of nuclear import is inhibited by the p34 cdc2 -mediated phosphorylation of a nearby threonine residue (38,70,74). Complexes of p34 cdc2 and p53 have been observed in TAg-transformed cells (50), and TAg has been shown to stimulate the expression of the cdc2 gene (12,55).…”

Supramolecular structure and nuclear targeting efficiency determine the enhancement of transfection by modified polylysines

“…12 To determine nuclear import kinetics, the labelled pLy derivatives, subsequent to precomplexation with or without DNA, were added to mechanically perforated HTC cells together with cytosolic extracts (untreated reticulocyte lysate) and an ATP-regenerating system. 12,29,30 CLSM imaging, analysis and curve fitting was carried out as previously. 12,28,30 EM and AFM Plasmid DNA and its complexes with pLy and pLyP101 at various Ly/Nu ratios were prepared for EM and rotary shadowing as previously.…”