Reduced phosphorylation of p50 is responsible for diminished NF-kappaB binding to the major histocompatibility complex class I enhancer in adenovirus type 12-transformed cells.
(25/147095)
Reduced cell surface levels of major histocompatibility complex class I antigens enable adenovirus type 12 (Ad12)-transformed cells to escape immunosurveillance by cytotoxic T lymphocytes (CTL), contributing to their tumorigenic potential. In contrast, nontumorigenic Ad5-transformed cells harbor significant cell surface levels of class I antigens and are susceptible to CTL lysis. Ad12 E1A mediates down-regulation of class I transcription by increasing COUP-TF repressor binding and decreasing NF-kappaB activator binding to the class I enhancer. The mechanism underlying the decreased binding of nuclear NF-kappaB in Ad12-transformed cells was investigated. Electrophoretic mobility shift assay analysis of hybrid NF-kappaB dimers reconstituted from denatured and renatured p50 and p65 subunits from Ad12- and Ad5-transformed cell nuclear extracts demonstrated that p50, and not p65, is responsible for the decreased ability of NF-kappaB to bind to DNA in Ad12-transformed cells. Hypophosphorylation of p50 was found to correlate with restricted binding of NF-kappaB to DNA in Ad12-transformed cells. The importance of phosphorylation of p50 for NF-kappaB binding was further demonstrated by showing that an NF-kappaB dimer composed of p65 and alkaline phosphatase-treated p50 from Ad5-transformed cell nuclear extracts could not bind to DNA. These results suggest that phosphorylation of p50 is a key step in the nuclear regulation of NF-kappaB in adenovirus-transformed cells. (+info)
Requirement for transcription factor NFAT in interleukin-2 expression.
(26/147095)
The nuclear factor of activated T cells (NFAT) transcription factor is implicated in expression of the cytokine interleukin-2 (IL-2). Binding sites for NFAT are located in the IL-2 promoter. Furthermore, pharmacological studies demonstrate that the drug cyclosporin A inhibits both NFAT activation and IL-2 expression. However, targeted disruption of the NFAT1 and NFAT2 genes in mice does not cause decreased IL-2 secretion. The role of NFAT in IL-2 gene expression is therefore unclear. Here we report the construction of a dominant-negative NFAT mutant (dnNFAT) that selectively inhibits NFAT-mediated gene expression. The inhibitory effect of dnNFAT is mediated by suppression of activation-induced nuclear translocation of NFAT. Expression of dnNFAT in cultured T cells caused inhibition of IL-2 promoter activity and decreased expression of IL-2 protein. Similarly, expression of dnNFAT in transgenic mice also caused decreased IL-2 gene expression. These data demonstrate that NFAT is a critical component of the signaling pathway that regulates IL-2 expression. (+info)
Shp-2 tyrosine phosphatase functions as a negative regulator of the interferon-stimulated Jak/STAT pathway.
(27/147095)
Shp-2 is an SH2 domain-containing protein tyrosine phosphatase. Although the mechanism remains to be defined, substantial experimental data suggest that Shp-2 is primarily a positive regulator in cell growth and development. We present evidence here that Shp-2, while acting to promote mitogenic signals, also functions as a negative effector in interferon (IFN)-induced growth-inhibitory and apoptotic pathways. Treatment of mouse fibroblast cells lacking a functional Shp-2 with IFN-alpha or IFN-gamma resulted in an augmented suppression of cell viability compared to that of wild-type cells. To dissect the molecular mechanism, we examined IFN-induced activation of signal transducers and activators of transcription (STATs) by electrophoretic mobility shift assay, using a specific DNA probe (hSIE). The amounts of STAT proteins bound to hSIE upon IFN-alpha or IFN-gamma stimulation were significantly increased in Shp-2(-/-) cells. Consistently, tyrosine phosphorylation levels of Stat1 upon IFN-gamma treatment and, to a lesser extent, upon IFN-alpha stimulation were markedly elevated in mutant cells. Furthermore, IFN-gamma induced a higher level of caspase 1 expression in Shp-2(-/-) cells than in wild-type cells. Reintroduction of wild-type Shp-2 protein reversed the hypersensitivity of Shp-2(-/-) fibroblasts to the cytotoxic effect of IFN-alpha and IFN-gamma. Excessive activation of STATs by IFNs was also diminished in mutant cells in which Shp-2 had been reintroduced. Together, these results establish that Shp-2 functions as a negative regulator of the Jak/STAT pathway. We propose that Shp-2 acts to promote cell growth and survival through two mechanisms, i.e., the stimulation of growth factor-initiated mitogenic pathways and the suppression of cytotoxic effect elicited by cytokines, such as IFNs. (+info)
The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells.
(28/147095)
PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. We report here that PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. Finally, we have found that PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and have detected clusters of PINCH that are colocalized with the alpha5beta1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways. (+info)
Induction of AT-specific DNA-interstrand crosslinks by bizelesin in genomic and simian virus 40 DNA.
(29/147095)
Bizelesin is a bifunctional AT-specific DNA alkylating drug. Our study characterized the ability of bizelesin to induce interstrand crosslinks, a potential lethal lesion. In genomic DNA of BSC-1 cells, bizelesin formed from approx. 0.3 to 6.03+/-0.85 interstrand crosslinks per 106 base pairs, at 5-100 nM drug concentration, respectively, comparable to the number of total adducts previously determined in the same system (J.M. Woynarowski, M.M. McHugh, L.S. Gawron, T.A. Beerman, Biochemistry 34 (1995) 13042-13050). Bizelesin did not induce DNA-protein crosslinks or strand breaks. A model defined target, intracellular simian virus 40 (SV40) DNA, was employed to map at the nucleotide level sites of bizelesin adducts, including potential interstrand crosslinks. Preferential adduct formation was observed at AT tracts which are abundant in the SV40 matrix associated region and the origin of replication. Many sites, including each occurrence of 5'-T(A/T)4A-3', co-mapped on both DNA strands suggesting interstrand crosslinks, although monoadducts were also formed. Bizelesin adducts in naked SV40 DNA were found at similar sites. The localization of bizelesin-induced crosslinks in AT-rich tracts of replication-related regions is consistent with the potent anti-replicative properties of bizelesin. Given the apparent lack of other types of lesions in genomic DNA, interstrand crosslinks localized in AT-rich tracts, and to some extent perhaps also monoadducts, are likely to be lethal effects of bizelesin. (+info)
Functional studies by site-directed mutagenesis on the role of Sp1 in the expression of the pyruvate kinase M and aldolase A genes.
(30/147095)
During the cell cycle of mitogen stimulated rat thymocytes, an 8-10-fold induction of glycolytic enzymes and a corresponding increase in the mRNA levels has been observed. This prompted us to study the transcriptional regulation of the rat aldolase A and pyruvate kinase M genes. cis-Regulatory elements of both promoters were evaluated by site-directed mutagenesis of promoter/luciferase constructs and transient transfections of rat hepatoma FTO2B cells. Furthermore, the binding proteins were identified by mobility shift assays in the presence of specific antibodies. In the aldolase AH1 promoter, five binding sites for Sp1 and Sp3 and a TPA responsive element were identified as essential for transcriptional regulation. Most of the promoter activity can be attributed to these regulatory elements. In the pyruvate kinase M promoter three out of five binding sites of Sp1 and Sp3 (B box and GC boxes 1 and 3) turned out to be functional in the transfection assays whereas the disruption of GC box 2 had no effect, and the disruption of the GC box 4 had only a minor effect on the promoter activity. Both promoters are stimulated by Sp1 as well as Sp3, as judged by cotransfection experiments of Drosophila SL2 cells. Therefore, the Sp1- and Sp3-directed transcription provides a means for common regulatory mechanism of the aldolase A and the pyruvate kinase M genes. (+info)
Cloning and characterization of the promoters of the maxiK channel alpha and beta subunits.
(31/147095)
Large conductance, calcium-activated potassium (maxiK) channels are expressed in nerve, muscle, and other cell types and are important determinants of smooth muscle tone. To determine the mechanisms involved in the transcriptional regulation of maxiK channels, we characterized the promoter regions of the pore forming (alpha) and regulatory (beta) subunits of the human channel complex. Maximum promoter activity (up to 12.3-fold over control) occurred between nucleotides -567 and -220 for the alpha subunit (hSlo) gene. The minimal promoter is GC-rich with 5 Sp-1 binding sites and several TCC repeats. Other transcription factor-binding motifs, including c/EBP, NF-kB, PU.1, PEA-3, Myo-D, and E2A, were observed in the 5'-flanking sequence. Additionally, a CCTCCC sequence, which increases the transcriptional activity of the SM1/2 gene in smooth muscle, is located 27 bp upstream of the TATA-like sequence, a location identical to that found in the SM1/2 5'-flanking region. However, the promoter directed equivalent expression when transfected into smooth muscle and other cell types. Analysis of the hSlo beta subunit 5'-flanking region revealed a TATA box at position -77 and maximum promoter activity (up to 11.0-fold) in a 200 bp region upstream from the cap site. Binding sites for GATA-1, Myo-D, c-myb, Ets-1/Elk-1, Ap-1, and Ik-2 were identified within this sequence. Two CCTCCC elements are present in the hSlo beta subunit promoter, but tissue-specific transcriptional activity was not observed. The lack of tissue-specific promoter activity, particularly the finding of promoter activity in cells from tissues in which the maxiK gene is not expressed, suggests a complex channel regulatory mechanism for hSlo genes. Moreover, the lack of similarity of the promoters of the two genes suggests that regulation of coordinate expression of the subunits does not occur through equivalent cis-acting sequences. (+info)
Cloning of a novel gene specifically expressed in clonal mouse chondroprogenitor-like EC cells, ATDC5.
(32/147095)
We cloned a full-length cDNA encoding a novel mouse protein, A-C2, by differential display method using mouse embryonic fibroblast C3H10T1/2 cells and mouse chondroprogenitor-like EC cells, ATDC5. The deduced amino acid sequence of A-C2 consisted of 106 amino acids with no significant homology to the sequences previously reported. Northern blot analysis showed two major bands of 2.1 and 1.8 kb sizes. Expression of A-C2 mRNA was exclusive to ATDC5 cells at their undifferentiated stage. None of ATDC5 cells at their differentiated stage and adult mice tissues examined expressed A-C2 gene. (+info)