Mining the receptorome.
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Computational Biology↗
Biomedical subjects
Blaine N Armbruster
Publications and source records attributed to Blaine N Armbruster.
Inositol diphosphate signaling regulates telomere length.
Activation of phospholipase C-dependent inositol polyphosphate signaling pathways generates distinct messengers derived from inositol 1,4,5-trisphosphate that control gene expression and mRNA export. Here we report the regulation of telomere length by production of a diphosphorylinositol tetrakisphosphate, PP-IP4, synthesized by the KCS1 gene product. Loss of PP-IP4 production results in lengthening of telomeres, whereas overproduction leads to their shortening. This effect requires the presence of Tel1, the yeast homologue of ATM, the protein mutated in the human disease ataxia telangiectasia. Our data provide in vivo evidence of a regulatory link between inositol polyphosphate signaling and the checkpoint kinase family and describe a third nuclear process modulated by phospholipase C activation.
Ataxia Telangiectasia Mutated Proteins↗
Rescue of an hTERT mutant defective in telomere elongation by fusion with hPot1.
The protein hPot1 shares homology with telomere-binding proteins in lower eukaryotes and associates with single-stranded telomeric DNA in vitro as well as colocalizing with telomere-binding proteins in vivo. We now show that hPot1 is coimmunoprecipitated with telomeric DNA and that stable expression of this protein in telomerase-positive cells results in telomere elongation, supporting the idea that hPot1 is a bona fide mammalian telomere-binding protein. We previously found that mutations in the N-terminal DAT domain of the hTERT catalytic subunit of telomerase rendered the enzyme catalytically active but unable to elongate telomeres in vivo. This phenotype could be partially rescued by fusion with the double-stranded telomeric protein hTRF2. Given that hPot1 binds to single-stranded DNA in vitro (at the same site that hTERT binds to in vivo), we addressed whether fusion of hPot1 can rescue the DAT mutations more efficiently than that of hTRF2. We now report that a DAT mutant of hTERT is indeed efficiently rescued upon fusion to hPot1. However, this rescue depended on the ability of hPot1 to localize to telomeres rather than binding to DNA per se. These data support a model whereby the DAT domain of hTERT is implicated in telomere-telomerase associations.
Amino Acid Sequence↗
In vivo regulation of hTERT expression and telomerase activity by androgen.
PURPOSE: The most effective therapy for metastatic prostate cancer is androgen deprivation. Genes activated directly or possibly even indirectly by this steroid hormone represent potential targets for anticancer therapy. One such gene may be hTERT, which encodes the catalytic subunit of telomerase. In prostate cancer cells telomerase is activated, permitting sustained proliferation. Therefore, we tested whether hTERT gene expression is modulated in prostate cancer cells in vitro and in vivo by androgens. MATERIALS AND METHODS: Transcriptional activation of hTERT during androgen stimulation was assayed by luciferase assays using the hTERT promoter fused to the luciferase gene and by reverse transcriptase-polymerase chain reaction to detect endogenous hTERT mRNA in LNCaP cells. hTERT mRNA levels and telomerase activity were also measured in CWR22 prostate cancer cells implanted in mice that were subsequently castrated and left untreated or administered androgen. RESULTS: We report that the endogenous hTERT promoter is activated during the administration of androgen to androgen sensitive LNCaP prostate cancer cells. However, this effect was indirect since an hTERT promoter construct was not activated by androgens and transcription of the endogenous gene was not stimulated early enough in cultured cells to be considered a direct target of this steroid hormone. Importantly in an in vivo model of human prostate cancer androgen deprivation led to a decrease in hTERT expression, followed by a decrease in telomerase activity, which was reversed by a single administration of androgen. CONCLUSIONS: The hTERT gene is regulated in human prostate cancer cells in vivo by androgens.
Androgens↗
Putative telomere-recruiting domain in the catalytic subunit of human telomerase.
Telomerase, the enzyme that elongates telomeres, is essential to maintain telomere length and to immortalize most cancer cells. However, little is known about the regulation of this enzyme in higher eukaryotes. We previously described a domain in the hTERT telomerase catalytic subunit that is essential for telomere elongation and cell immortalization in vivo but dispensable for catalytic activity in vitro. Here, we show that fusions of hTERT containing different mutations in this domain to the telomere binding protein hTRF2 redirected the mutated hTERT to telomeres and rescued its in vivo functions. We suggest that this domain posttranscriptionally regulates telomerase function by targeting the enzyme to telomeres.
Catalytic Domain↗
The nucleolar localization domain of the catalytic subunit of human telomerase.
Telomerase is the enzyme essential to complete the replication of the terminal DNA of most eukaryotic chromosomes. In humans, this enzyme is composed of the telomerase reverse transcriptase (hTERT) and telomerase RNA (hTR) subunits. hTR has been found in the nucleolus, a site of assembly of ribosomes as well as other ribonucleoproteins (RNPs). We therefore tested whether the hTERT component is also found in the nucleolus, where it could complex with the hTR RNA to form a functional enzyme. We report here that hTERT does indeed localize to the nucleolus, and we mapped the domain responsible for this localization to the hTR-binding region of the protein by deletion analysis. Substitution mutations in two of the three conserved hTR-binding domains in this nucleolar localization domain (NoLD) abolished nucleolar localization. However, another mutation that impeded hTR binding did not alter this subcellular localization. Additionally, wild type hTERT was detected in the nucleolus of cells that failed to express hTR. Taken together, we propose that the nucleolar localization of hTERT involves more than just the association with the hTR subunit. Furthermore, the coincidental targeting of both the hTR and hTERT subunits to the nucleolus supports the premise that the assembly of telomerase occurs in the nucleolus.
Adenocarcinoma↗