Biomedical subjects

C Demonacos

Publications and source records attributed to C Demonacos.

5 recordsLinked to original sources

PubMed2001

A TPR motif cofactor contributes to p300 activity in the p53 response.

The transcription of p53 target genes involves p300/CBP coactivators, which are multiprotein complexes that interact with the p53 activation domain. We report a cofactor in the p300 coactivator complex, Strap, which has an unusual structure, being composed almost entirely of a tandem series of six tetratricopeptide repeat (TPR) motifs. The TPR motif functions as a protein interaction domain, and it is consistent with this property that Strap harbors distinct and dedicated domains that allow it to bind and augment the interaction between different components of the p300 complex. Strap facilitates p53 activity in response to stress, in part through the stress-responsive accumulation of Strap protein and interfering with the MDM2-dependent downregulation of p53.

Acetyltransferases↗

PubMed2001

Acetylation control of the retinoblastoma tumour-suppressor protein.

The retinoblastoma tumour-suppressor protein (pRb) and p300/CBP co-activator proteins are important for control of proliferation and in tumour cells these are sequestered by viral oncoproteins such as E1A. pRb is involved in negatively regulating growth, and p300/CBP proteins have histone acetyltransferase (HAT) activity, which influences gene expression. Although it is known that phosphorylation by G1 cyclin-dependent kinases (CDKs) regulates pRb activity, the nature and role of other post-translational modifications is not understood. Here we identify acetylation as a new type of modification and level of control in pRb function. Adenovirus E1A, which binds p300/CBP through an amino-terminal transformation-sensitive domain, stimulates the acetylation of pRb by recruiting p300 and pRb into a multimeric-protein complex. Furthermore, pRb acetylation is under cell-cycle control, and acetylation hinders the phosphorylation of pRb by cyclin-dependent kinases. pRb binds more strongly when acetylated to the MDM2 oncoprotein, which indicates that acetylation may regulate protein-protein interactions in the pRb pathway. The acetylation of pRb defines a new level of cell-cycle control mediated by HAT. Furthermore, our results establish a relationship between p300, pRb and acetylation in which E1A acts to recruit and target a cellular HAT activity to pRb.

Acetylation↗

PubMed1998

Using yeast to study glucocorticoid receptor phosphorylation.

The glucocorticoid receptor (GR) is a phosphoprotein and a member of the steroid/thyroid receptor superfamily of ligand dependent transcription factors. When the glucocorticoid receptor is expressed in yeast (Saccharomyces cerevisiae), it is competent for signal transduction and transcriptional regulation. We have studied the glucocorticoid receptor phosphorylation in yeast and demonstrated that the receptor is phosphorylated in both the absence and presence of hormone, on serine and threonine residues. This phosphorylation occurs within 15 min upon addition of radioactivity in both hormone treated and untreated cells. As reported for mammalian cells, additional phosphorylation occurs upon hormone binding and this phosphorylation is dependent on the type of the ligand. We have followed the hormone dependent receptor phosphorylation by electrophoretic mobility shift assay, and have shown that this mobility change is sensitive to phosphatase treatment. In addition, the appearance of hormone dependent phosphoisoforms of the receptor depends on the potency of the agonist used. Using this method we show that the residues contributing to the hormone dependent mobility shift are localized in one of the transcriptional activation domains, between amino acids 130-247. We altered the phosphorylation sites within this domain that correspond to the amino acids phosphorylated in mouse hormone treated cells. Using phosphopeptide maps we show that hormone changes the peptide pattern of metabolically labelled receptor, and we identify peptides which are phosphorylated in hormone dependent manner. Then we determine that phosphorylation of residues S224 and S232 is increased in the presence of hormone, whereas phosphorylation of residues T171 and S246 is constitutive. Finally, we show that in both yeast and mammalian cells the same residues on the glucocorticoid receptor are phosphorylated. Our results suggest that yeast cells would be a suitable system to study glucocorticoid receptor phosphorylation. The genetic manipulability of yeast cells, together with conservation of the phosphorylation of GR in yeast and mammalian cells and identification of hormone dependent phosphorylation, would facilitate the isolation of molecules involved in the glucocorticoid receptor phosphorylation pathway and further our understanding of this process.

Animals↗

PubMed1995

The mitochondrion as a primary site of action of glucocorticoids: the interaction of the glucocorticoid receptor with mitochondrial DNA sequences showing partial similarity to the nuclear glucocorticoid responsive elements.

Six mitochondrial genome sequences, showing strong similarity to the glucocorticoid responsive element consensus sequence (GRE), four localized within the cytochrome c oxidase (COX) subunit I and II genes (GREs I-IV) and two within the D-loop region (GREs a and b) have been examined as binding sites of glucocorticoid receptor (GR) from rat liver cytosol. Purified GR from rat liver cytosol binds with high specificity to all potential mitochondrial GREs, as shown by filter retention and gel shift assays. Specific binding of protein(s), present in a mitochondrial extract from dexamethasone-induced mice, to all six putative mitochondrial GREs was also documented by the same methodology. Both purified GR and protein(s) from mitochondrial extract give the same band in the gel retardation assay. Using monospecific anti-glucocorticoid receptor polyclonal antibody (EP), a supershift of the gel retarded protein-DNA band was obtained. These results demonstrate that the mitochondrial genome sequences examined have characteristics of GREs, since they show the capacity to specifically bind the respective receptor protein. These findings support the hypothesis that the mitochondrial genome is a primary site of action of steroid and thyroid hormones (Sekeris C.E.: The mitochondrial genome: a possible primary site of action of steroid hormones, In vivo 4 (1990) 317-320).

Animals↗

PubMed1993

Import of the glucocorticoid receptor into rat liver mitochondria in vivo and in vitro.

Administration of inducing doses of dexamethasone (10 microg/100 g) to adrenalectomized rats results, within 2-5 min, in import of the glucocorticoid receptor from liver cytoplasm into mitochondria, as demonstrated by Western blotting and by electron microscopy. Furthermore, glucocorticoid receptor (GR) synthesized in an in vitro reticulocyte system programmed with the respective mRNA, enters within minutes to added rat liver mitochondria in the form of intact GR, as demonstrated by Western blotting using either monoclonal or polyclonal antibodies against different domains of GR. In vitro studies show that the import is dependent on temperature and/or activation of the hormone-GR complex. These results, in connection with the presence in the human and rodent mitochondrial genome of sequences showing partial homology to the nuclear glucocorticoid response elements, support the hypothesis that the well documented effects of glucocorticoids on mitochondrial functions result from a direct interaction of the GR complex with the mitochondrial genome.

Adrenalectomy↗