No abstract

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3'-->5' DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

V-ATPases are proton-translocating enzymes, which are found not only in numerous intracellular organelles but also in the plasma membranes of many eukaryotic cells. Using differential display, we have identified one of the proton pump subunit genes, ATP6C, as a cisplatin-inducible gene. Northern blot analysis demonstrated that expression of other members of the subunit is inducible by cisplatin treatment. Proton pump gene expression is also upregulated in 3 independent cisplatin-resistant cell lines but not in vincristine-or etoposide-resistant cell lines. Cellular pH was significantly higher in cisplatin-resistant cells than in sensitive parental cells. In vitro DNA-binding activity of cisplatin was markedly increased in acidic conditions, suggesting that the cytotoxicity of cisplatin is modulated by cellular pH. Furthermore, the proton pump inhibitor bafilomycin can synergistically potentiate the cytotoxicity of cisplatin but not of etoposide or camptothecin. These results indicate that cellular pH is one of the critical parameters for effective cancer chemotherapy with cisplatin.

In flowering plants, light is one of the major environmental stimuli that determine the timing of the transition from the vegetative to reproductive phase. In Arabidopsis, phytochrome B (phyB); phyA; cryptochrome 2; and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 are major photoreceptors that regulate flowering. Unlike phyA; cryptochrome 2; and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1, phyB delays flowering mainly by destabilizing the CONSTANS (CO) protein, whose reduction leads to decreased expression of a florigen gene, FLOWERING LOCUS T. However, it remains unclear how the phyB-mediated CO destabilization is mechanistically regulated. Here, we identify a unique PHYTOCHROME-DEPENDENT LATE-FLOWERING (PHL) gene, which is mainly involved in the phyBdependent regulation of flowering. Plants with mutant phl exhibited a late-flowering phenotype, especially under long-day conditions. The late-flowering phenotype of the phl mutant was completely overridden by a phyB mutation, indicating that PHL normally accelerates flowering by countering the inhibitory effect of phyB on flowering. Accordingly, PHL physically interacted with phyB both in vitro and in vivo in a red light-dependent manner. Furthermore, in the presence of phyB under red light, PHL interacted with CO as well. Taken together, we propose that PHL regulates photoperiodic flowering by forming a phyB-PHL-CO tripartite complex.light signaling | FT

No abstract