Nuclear translocation of green fluorescent protein‐nuclear factor κB with a distinct lag time in living cellsTenjinbaru, Kazuyoshi; Furuno, Tadahide; Hirashima, Naohide; Nakanishi, Mamoru
doi: 10.1016/S0014-5793(99)00002-2pmid: 10037137
A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the human nuclear factor κB (NF‐κB) p50 and p105 (p50/IκBγ), a precursor protein of NF‐κB p50. GFP‐p50 and GFP‐p105 were expressed in monkey COS‐7 cells and human HeLa cells. Translocation of these chimeric proteins was observed by confocal laser scanning microscopy. GFP‐p50 (without IκBγ) in the transfected cells resided in the nucleus. On the other hand, GFP‐p105 (GFP‐p50 with IκBγ) localized only in the cytoplasm before stimulation and translocated to the nucleus with stimulant specificity similar to that of native NF‐κB/IκB. In addition, the translocation of NF‐κB to the nucleus had a distinct lag time (a quiescent time) in the target cells. The lag time lasted 10–20 min after stimulation with hydrogen peroxide or tumor necrosis factor α. It was suggested that this might be due to the existence of a limiting step where NF‐κB is released from NF‐κB/IκB by the proteasome.
Characterization of the calcyclin (S100A6) binding site of annexin XI‐A by site‐directed mutagenesisSudo, Toshiki; Hidaka, Hiroyoshi
doi: 10.1016/S0014-5793(99)00014-9pmid: 10037139
Residues in annexin XI‐A essential for binding of calcyclin (S100A6) were examined by site‐directed mutagenesis. GST fusion proteins with the calcyclin binding site of annexin XI‐A, GST‐AXI 34–62 and GST‐AXI 49–77 bound to calcyclin‐Sepharose Ca2+‐dependently. The mutants GST‐AXI L52E, M55E, A56E and M59E lost the binding ability, whereas GST‐AXI A57E retained the ability. These results demonstrate that the hydrophobic residues L52, M55, A56 and M59 on one side surface of the α‐helix are critical for the binding. Assays with GST fusion proteins and synthesized peptides corresponding to the calcyclin binding site indicated that other regions around the calcyclin binding site are important to stabilize the conformation.
Isocitrate lyase of Ashbya gossypii – transcriptional regulation and peroxisomal localizationMaeting, Ines; Schmidt, Georg; Sahm, Hermann; Revuelta, José Luis; Stierhof, York-Dieter; Stahmann, K.-Peter
doi: 10.1016/S0014-5793(99)00017-4pmid: 10037140
The isocitrate lyase‐encoding gene AgICL1 from the filamentous hemiascomycete Ashbya gossypii was isolated by heterologous complementation of a Saccharomyces cerevisiae icl1d mutant. The open reading frame of 1680 bp encoded a protein of 560 amino acids with a calculated molecular weight of 62 584. Disruption of the AgICL1 gene led to complete loss of AgIcl1p activity and inability to grow on oleic acid as sole carbon source. Compartmentation of AgIcl1p in peroxisomes was demonstrated both by Percoll density gradient centrifugation and by immunogold labeling of ultrathin sections using specific antibodies. This fitted with the peroxisomal targeting signal AKL predicted from the C‐terminal DNA sequence. Northern blot analysis with mycelium grown on different carbon sources as well as AgICL1 promoter replacement with the constitutive AgTEF promoter revealed a regulation at the transcriptional level. AgICL1 was subject to glucose repression, derepressed by glycerol, partially induced by the C2 compounds ethanol and acetate, and fully induced by soybean oil.
Calcitonin gene‐related peptide decreases expression of acetylcholinesterase in mammalian myotubesBoudreau-Larivière, Céline; Jasmin, Bernard J
doi: 10.1016/S0014-5793(99)00015-0pmid: 10037141
Nerve‐derived trophic factors are known to modulate expression of acetylcholinesterase (AChE) in skeletal muscle fibers, yet the precise identity of these factors remains elusive. In the present study, we treated mouse C2 myotubes with calcitonin gene‐related peptide (CGRP). Compared to non‐treated myotubes, cell‐associated AChE activity levels were decreased by ∼60% after 48 h of treatment. A parallel reduction in AChE total protein levels was also observed as determined by Western blot analysis. The reduction in AChE activity was due to a decrease in the levels of the G1 molecular form and to an elimination of G4. By contrast, levels of secreted AChE remained unchanged following CGRP treatment. Finally, the overall decrease in AChE activity was accompanied by a reduction in AChE transcripts which could not be attributed to changes in the transcriptional rate of the ACHE gene.
Transcription factor AP‐2 activity is modulated by protein kinase A‐mediated phosphorylationGarcı́a, Miguel Angel; Campillos, Mónica; Marina, Anabel; Valdivieso, Fernando; Vázquez, Jesús
doi: 10.1016/S0014-5793(99)00021-6pmid: 10037142
We recently reported that APOE promoter activity is stimulated by cAMP, this effect being mediated by factor AP‐2 [Garcı́a et al. (1996) J. Neurosci. 16, 7550–7556]. Here, we study whether cAMP‐induced phosphorylation modulates the activity of AP‐2. Recombinant AP‐2 was phosphorylated in vitro by protein kinase A (PKA) at Ser239. Mutation of Ser239 to Ala abolished in vitro phosphorylation of AP‐2 by PKA, but not the DNA binding activity of AP‐2. Cotransfection studies showed that PKA stimulated the effect of AP‐2 on the APOE promoter, but not that of the S239A mutant. Therefore, cAMP may modulate AP‐2 activity by PKA‐induced phosphorylation of this factor.
Involvement of p21 in the PKC‐induced regulation of the G2/M cell cycle transitionBarboule, Nadia; Lafon, Corinne; Chadebech, Philippe; Vidal, Simone; Valette, Annie
doi: 10.1016/S0014-5793(99)00022-8pmid: 10037143
Activation of protein kinase C (PKC) inhibits cell cycle progression at the G1/S and G2/M transitions. We found that phorbol 12‐myristate 13‐acetate (PMA) induced upregulation of p21, not only in MCF‐7 cells arrested in the G1 phase as previously shown, but also in cells delayed in the G2 phase. This increase in p21 in cells accumulated in the G1 and G2/M phases of the cell cycle after PMA treatment was inhibited by the PKC inhibitor GF109203X. This indicates that PKC activity is required for PMA‐induced p21 upregulation and cell cycle arrest in the G1 and G2/M phases of the cell cycle. To further assess the role of p21 in the PKC‐induced G2/M cell cycle arrest independently of its G1 arrest, we used aphidicolin‐synchronised MCF‐7 cells. Our results show that, in parallel with the inhibition of cdc2 activity, PMA addition enhanced the associations between p21 and either cyclin B or cdc2. Furthermore, we found that after PMA treatment p21 was able to associate with the active Tyr‐15 dephosphorylated form of cdc2, but this complex was devoid of kinase activity indicating that p21 may play a role in inhibition of cdc2 induced by PMA. Taken together, these observations provide evidence that p21 is involved in integrating the PKC signaling pathway to the cell cycle machinery at the G2/M cell cycle checkpoint.
Expression of Mash1 in basal cells of rat circumvallate taste buds is dependent upon gustatory innervationSeta, Yuji; Toyono, Takashi; Takeda, Shinobu; Toyoshima, Kuniaki
doi: 10.1016/S0014-5793(99)00023-Xpmid: 10037145
Mash1, a mammalian homologue of the Drosophila achaete‐scute proneural gene complex, plays an essential role in differentiation of subsets of peripheral neurons. In this study, using RT‐PCR and in situ RT‐PCR, we investigated if Mash1 gene expression occurs in rat taste buds. Further, we examined dynamics of Mash1 expression in the process of degeneration and regeneration in denervated rat taste buds. In rat tongue epithelium, Mash1 gene expression is confined to circumvallate, foliate, and fungiform papilla epithelia that include taste buds. In taste buds, Mash1‐expressing cells are round cells in the basal compartment. In contrast, the mature taste bud cells do not express the Mash1 gene. Denervation and regeneration experiments show that the expression of Mash1 requires gustatory innervation. We conclude that Mash1 is expressed in cells of the taste bud lineage, and that the expression of Mash1 in rat taste buds is dependent upon gustatory innervation.