Molecular cell biology research communications : MCBRC最新文献

Signal transducers and activators of transcription (STATs) are a family of transcription factors that were originally identified as mediators of cytokine-induced gene expression. We and others have recently shown that STAT5 also plays a major role in cellular transformation by the Bcr-Abl oncogene. Here we show that the antiapoptotic bcl-xL gene product and the cell cycle regulator cyclin D1 are targets of STAT5 in Bcr-Abl-transformed cells. In the CML cell line K562 and in BaF3 cells ectopically expressing Bcr-Abl, both the cyclin D1 and bcl-x promoters are highly active. The activity of these promoters can be strongly repressed by cotransfection of a dominant negative (DN) mutant of STAT5. Moreover, the cyclin D1 and bcl-x promoters contain STAT binding sites to which STAT5 constitutively binds in Bcr-Abl transformed cells. These results suggest that STAT5 contributes to transformation by Bcr-Abl by induction of cyclin D1 and bcl-xL expression.

Acylphosphatase (AcP) is a low-molecular-weight protein widely distributed in many vertebrate tissues with a yet unknown physiologic function. To study the in vivo behavior of AcP, HeLa cells were transiently transfected with a vector expressing the AcP/EGFP fusion protein. Analysis of the transfected cells showed a high level of cellular death in cells expressing the AcP/EGFP fusion protein with respect to control cells expressing EGFP alone. Flow cytometry and time lapse analysis of AcP/EGFP transfected cells evidenced a typical pattern of apoptosis. Surprisingly, cells transfected with a mutated form of AcP, with negligible in vitro acylphosphatase activity, undergo apoptosis as well as cells transfected with wild-type protein, suggesting that the physiologic role of AcP could be not related to this catalytic activity.

Paracentrotus lividus embryos, at post-blastular stage, when subjected to a rise in temperature from physiologic (20°C) to 31°C, synthesize a large group of heat shock proteins (hsps), and show a severe inhibition of bulk protein synthesis. We show, by mono- and two-dimensional electrophoresis, that also EGTA (ethylene glycol-bis[β-aminoethyl ether] tetraacetic acid) treatment induces in sea urchin embryos both marked inhibition of bulk protein synthesis and the synthesis of the entire set of hsps. Furthermore, EGTA-treated sea urchin embryos are able to survive at a temperature otherwise lethal (35°C) becoming thermotolerant. Because incubation with a different calcium-chelator, EDTA (ethylenediaminetetraacetic acid), or in calcium-free medium did not induce hsps synthesis we conclude that the stress response caused by EGTA is not related to its calcium chelator function.

c-fos is the prototypic member of a family of transcription factors that regulate many cellular processes, including proliferation. c-fos heterodimerizes with jun family members to form the AP-1 transcription factor complex which binds specific DNA recognition elements in the promoters of many genes. Following rapid induction in response to serum or growth factors, c-fos regulates expression of downstream target genes involved in cellular proliferation. Although much work has focused on activation of cell cycle regulatory genes by c-fos, less is known about negative regulation of gene expression by this transcription factor. The cyclin-dependent kinase (cdk) inhibitor p21^Cip1/WAF1 is a negative regulator of cdk activity, thereby impeding cell cycle progression. By sequence analysis, we identified a putative AP-1 element in the p21^Cip1/WAF1 promoter. To investigate how this site regulated p21^Cip1/WAF1 expression and mitigate external effects on c-fos expression, we used a c-fos/estrogen receptor (c-fosER) fusion construct in which this transcription factor is conditionally activated by estradiol. In the presence of estradiol, c-fosER downregulated p21^Cip1/WAF1 promoter activity. This inhibition was dependent on the putative AP-1 site. Activation of c-fosER induced cell cycle progression and proliferation in a manner similar to serum stimulation. We concluded that activation of c-fosER mediated transcriptional inhibition of p21^Cip1/WAF1 through a previously uncharacterized AP-1 site, revealing an important role for c-fos in negative control of cell cycle regulatory genes.

The paired box and homeodomain containing transcription factors Pax4 and Pax6 are known to be essential for development of the pancreatic endocrine cells. In this report we demonstrate that stable expression of Pax4 in a rat glucagon-producing cell line inhibits the endogenously expressed glucagon gene completely. Furthermore, Pax4 represses Pax6 independent transcription of the insulin promoter, suggesting that Pax4 can actively repress transcription in addition to acting by competition with the transcriptional activator Pax6.

Phosphorylation of Ser 209 is thought to modulate the activity of the cap-binding factor eIF-4E which is a crucial component in the initiation complex for cap-dependent translation of mRNA. We report here the full reconstitution of the p38 Map kinase cascade leading to phosphorylation of eIF-4E in vitro and the generation of antibodies specific for phospho-serine 209 in eIF-4E. These antibodies were used to probe the phosphorylation of eIF-4E in mammalian cells stimulated with mitogens and pro-inflammatory cytokines. Treatment of human dermal fibroblasts with FCS led to a transient hyperphosphorylation, followed by hypophosphorylation and return to normal state phosphorylation at 16 h after the initial stimulation. By using a potent small molecular weight inhibitor of Mnk1, the upstream kinase for eIF-4E, we observed a rapid dephosphorylation of eIF-4E within 45 min after addition of the inhibitor, suggesting a high turnover of phosphate on eIF-4E mediated by Mnk1 and a yet unidentified phosphatase.

We have identified a mammalian homologue of yeast Ump1p by searching for similar proteins in human and mouse expressed sequence tag (EST) databases. Ump1p is an accessory protein that is required for normal proteasome assembly in yeast (1). A mammalian homologue, which we refer to as “proteassemblin,” is a constituent of proteasome assembly intermediates (preproteasomes), but not fully assembled 20S proteasomes, as is Ump1p in yeast. We also provide evidence that proteassemblin is a constituent of pre-immunoproteasomes that contain the precursor of the interferon-γ-inducible subunit LMP2. By analogy with Ump1p, we hypothesize that proteassemblin is required for normal mammalian proteasome assembly.

The cytosolic factor p47-phox (NCF1) is a key component of the phagocyte NADPH-oxidase system, critical for microbicidal activity. The human p47-phox gene has been well characterized and resides on chromosome 7q11. Here we describe the molecular characterization of the mouse ortholog (Ncf1), which maps to distal chromosome 5, and compare the structure of the genes, commenting on the degree of homology. The mouse and human genes contain the same number of exons and introns, but the mouse gene is more compact (7.8 kb versus 15.2 kb). A percentage identity plot analysis comparing the human and mouse genes indicates that sequence homology is generally restricted to exons and does not include any large segment of introns or the 5′ flanking sequence. The mouse gene also contains notably fewer repetitive elements than its human counterpart (34% versus 50%). The start of transcription of the mouse gene has been localized to within 12 nucleotides of the translation start site, similar to the human ortholog. Our findings provide an important foundation for investigating the evolutionary history of the p47-phox gene, particularly as it relates to understanding the molecular basis of the p47-phox-deficient autosomal recessive form of chronic granulomatous disease.