Members of the ATM family of proteins function in humans, yeast, and other organisms as critical sensors of genomic integrity or growth conditions. In humans, the ATM family includes ATM, ATR, FRAP, and DNAPKcs, and homologs of these proteins are found in yeast, flies, and other organisms (e.g., Mec1p, Tel1p, RAD3p, TOR1p, and Mei-41;Keith and Schreiber 1995). The defining features of the proteins in the ATM family are their general large size (2500-4000 amino acids) and a 300-amino-acid motif in their C terminus that resembles the catalytic domain of PI3-kinases. However, most of these proteins have been shown to phosphorylate other proteins rather than lipids. More extended alignment of the ATM family members identifies two other conserved domains in addition to the PI3-kinase-like domain (Bosotti et al. 2000). The PI3-kinase-like domain is flanked by the FATC domain of 35 amino acids at the extreme C terminus and the FAT domain, which comprises ∼500 amino acids amino terminal of the catalytic domain. All existing evidence suggests that the integrity of the kinase domain is essential for the function of ATM family proteins, but the functions of the FATC and FAT domains remain unknown. However, our recent identification of the novel ATM-related TRRAP proteins has raised new questions about the function of these conserved domains. The TRRAP protein family was identified as transcriptional cofactors that mediate the recruitment of large multiprotein histone acetyltransferase (HAT) complexes to sequence-specific activators (Grant et al. 1998;McMahon et al. 1998;Saleh et al. 1998;Vassilev et al. 1998). We initially identified TRRAP orthologs in humans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Caenorhabditis elegans, and more recently in Arabidopsis thaliana and Drosophila melanogaster. The TRRAP proteins are true members of the ATM family because they possess the FAT, kinaserelated, and FATC domains. However, the kinase-related domain is conserved in sequence but not in function as the specific residues mediating phosphate transfer are absent ). This lack of catalytic activity has been supported by biochemical studies on both the human and yeast TRRAP proteins (Saleh et al. 1998). The paradox of sequence conservation in the absence of kinase activity prompted us to explore a noncatalytic role for this conserved domain in the recruitment of HAT complexes to transcriptional activation domains. ResultsTo investigate the function of the ATM-related region of TRRAP, a progressive series of C-terminal deletions (1-3760, 1-3713, and 1-3087) was constructed from a FLAG epitope-tagged full-length (1-3830) TRRAP cDNA expression vector (Fig. 1A). An internal deletion (⌬2108-2403) within the TRRAP cDNA was also constructed. Each protein was transiently expressed in HEK293 cells, immunoprecipitated with anti-FLAG antibodies, eluted from the beads with an excess of FLAG peptide, and subjected to histone acetyltransferase assays using core histones as substrates. The reaction products were resolved by graded poro...
The p53 tumor suppressor regulates the cellular response to genetic damage through its function as a sequence-specific transcription factor. Among the most well-characterized transcriptional targets of p53 is the mdm2 oncogene. Activation of mdm2 is critical in the p53 pathway because the mdm2 protein marks p53 for proteosome-mediated degradation, thereby providing a negative-feedback loop. Here we show that the ATMrelated TRRAP protein functionally cooperates with p53 to activate mdm2 transcription. TRRAP is a component of several multiprotein acetyltransferase complexes implicated in both transcriptional regulation and DNA repair. In support of a role for these complexes in mdm2 expression, we show that transactivation of the mdm2 gene is augmented by pharmacological inhibition of cellular deacetylases. In vitro analysis demonstrates that p53 directly binds to a TRRAP domain previously shown to be an activator docking site. Furthermore, transfection of cells with antisense TRRAP blocks p53-dependent transcription of mdm2. Finally, using chromatin immunoprecipitation, we demonstrate direct p53-dependent recruitment of TRRAP to the mdm2 promoter, followed by increased histone acetylation. These findings suggest a model in which p53 directly recruits a TRRAP/acetyltransferase complex to the mdm2 gene to activate transcription. In addition, this study defines a novel biochemical mechanism utilized by the p53 tumor suppressor to regulate gene expression.The gene encoding p53 is the most frequently mutated locus in human cancer (reviewed in reference 64). p53 is a tumor suppressor which induces either cell cycle arrest or apoptosis in response to DNA damage. These effects rely on the ability of p53 to function as a sequence-specific transcription factor. Following DNA damage, multiple signaling pathways result in the stabilization of the normally short-lived p53 protein. Stabilized p53 then activates or represses the transcription of a number of downstream target genes, many of which play critical roles in cell cycle arrest or apoptosis. The biochemical mechanism by which p53 represses certain target loci was recently described in studies showing that p53 recruits histone deacetylase (HDAC) complexes (49). In contrast, the precise biochemical mechanism by which p53 activates transcription is still being elucidated. Interactions between p53 and the TATA-binding protein (TBP) and between p53 and several of the TBP-associated factors have been reported (11,17,37,39,42,(59)(60)(61). These interactions are likely to play an important role in the loading of the RNA polymerase holoenzyme onto target gene promoters. They do not, however, explain how p53 resolves the inhibitory effects of the chromatin structure at target genes prior to polymerase loading. It has recently been learned that sequence-specific transcription factors such as p53 overcome the repressive effects of chromatin structure via the recruitment of multiprotein enzyme complexes (21, 54). In fact, DNA footprinting studies of p53 have demonstrated that the r...
Cln synthetase (CS) catalyzes the ATP-dependent condensation of ammonia with glutamate to yield Cln. I n higher plants CS is an octameric enzyme and the subunits are encoded by members of a small multigene family. I n soybeans (Glycine max), following the onset of N, fixation there is a dramatic increase in CS activity in the root nodules. CS activity staining of native polyacrylamide gels containíng nodule and root extracts showed a common band of activity (CSrs). The nodules also contained a slower-migrating, broad band of enzyme activity (CSns). The GSns activity band i s a complex of many isozymes made up of different proportions of two kinds of CS subunits: GSr and CSn. Root nodules formed following inoculation with an Nif-strain of Bradyrhizobium japonicum showed the presence of CS isoenzymes (CSnsl) with low enzyme activity, which migrated more slowly than CSns. Csnsl is most likely made up predominantly of CSn subunits. Our data suggest that, whereas the class I GS genes encoding the CSr subunits are regulated by the availability of NH3, the class II GS genes coding for the CSn subunits are developmentally regulated. Furthermore, we have demonstrated that the CSnsl isozymes in the Nif-nodules are relatively more labile. Our overall conclusion is that CSns activity in soybean nodules is regulated by N, fixation both at the level of transcription and at the level of holoprotein stability.GS (EC 6.3.1.2) is a key enzyme in the assimilation of NH,, catalyzing the ATP-dependent condensation of NH, with glutamate to yield Gln (Lea et al., 1990). The NH, is derived from symbiotic N, fixation, the reduction of NO,-or NO,-, photorespiration, or amino acid catabolism (Hirel et al., 1993). The reaction is believed to proceed via a two-step process, the first involving the formation of GSbound glutamyl phosphate from ATP and glutamate, followed by the addition of NH, to form a tetrahedral adduct with the subsequent liberation of Gln, ADP, and Pi (Lea and Ridley, 1989).
The T cell receptor (TCR) beta chain transmembrane domain contains two evolutionarily conserved tyrosines (Y). In this study, the functional basis for the evolutionary conservation is addressed by mutation of the residues, expression of the mutants in hybridoma and primary T cells, and examination of TCR signaling function. We find that the phenotype of the mutants, both surface expression and ability to signal for IL-2 production, is highly variable in different mouse T hybridoma lines. Although we have not been able to determine the basis for these differences in the hybridomas, expression of the mutants in primary T cells provides a definitive assessment of mutant phenotype. We show that mutation of the N-terminal Y to either leucine (L) or alanine (A) results in low surface expression in primary T cells, while mutation of both N- and C-terminal Y to A or L abrogates surface expression. However, the more conservative mutation of both transmembrane Y to phenylalanine maintained receptor surface expression and assembly while severely disrupting signaling in primary T cells. Our data demonstrate that TCR beta chain transmembrane Y are essential for TCR signal transduction as well as complex assembly. These findings suggest that protein-protein interactions involving membrane-spanning domains are likely relevant for TCR signal transduction mechanisms.
These studies address the role of PU.1 in T cell development through the analysis of PU.1−/− mice. We show that the majority of PU.1−/− thymocytes are blocked in differentiation prior to T cell commitment, and contain a population of thymocyte progenitors with the cell surface phenotype of CD44+, HSAbright, c-kitint, Thy-1−, CD25−, Sca-1−, CD4−, and CD8−. These cells correspond in both number and cell surface phenotype with uncommitted thymocyte progenitors found in wild-type fetal thymus. RT-PCR analysis demonstrated that PU.1 is normally expressed in this early progenitor population, but is down-regulated during T cell commitment. Rare PU.1−/− thymi, however, contained small numbers of thymocytes expressing markers of T cell commitment. Furthermore, almost 40% of PU.1−/− thymi placed in fetal thymic organ culture are capable of T cell development. Mature PU.1−/− thymocytes generated during organ culture proliferated and produced IL-2 in response to stimulation through the TCR. These data demonstrate that PU.1 is not absolutely required for T cell development, but does play a role in efficient commitment and/or early differentiation of most T progenitors.
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