Transcriptional activation by estrogen receptor (ERalpha) and steroid receptor coactivator (SRC1) involves distinct mechanisms in yeast and mammalian cells.

Steroid receptors activate transcription in yeast cells via interactions with endogenous coactivators and/or basal factors. We examined the effects of mutations in the ligand binding domain on the transcriptional activity of ERalpha in yeast. Our results show that mutations in Helix 3 (K366A) and Helix 12 (M547A, L548A) disrupt transcriptional activity of ERalpha in yeast, as previously observed in mammalian cells. However, replacement of a conserved tyrosine residue in Helix 12 with alanine or aspartate (Y541A and Y541D), which renders ERalpha constitutively active in mammalian cells, had only a weak stimulatory effect on ligand-independent reporter activation by ERalpha in yeast. Two-hybrid interaction experiments revealed that a Y541A mutant expressed in yeast was capable of ligand-independent binding to a mammalian coactivator, suggesting that there is a subtle difference in how this mutant interacts with mammalian and yeast cofactors. We also show that the ligand-dependent activities of ERalpha and progesterone receptor (PR) in yeast cells were strongly enhanced by the human p160 protein steroid receptor coactivator (SRC1), but not by CREB-Binding Protein (CBP) or the p300/CBP associated factor (P/CAF). Although the SRC1 activation domains AD1 and AD2 are functional in yeast, deletion of these sequences only partially impaired SRC1 coactivator function in this organism; this is in contrast to similar experiments in mammalian cells. Thus SRC1 sequences involved in recruitment of CBP/p300 and Co-Activator-Associated Arginine Methyltransferase (CARM-1) in mammalian cells are not essential for its function in yeast, suggesting that SRC1 operates via distinct mechanisms in yeast and mammalian cells.

p53 Stimulates TFIID-TFIIA-promoter complex assembly, and p53-T antigen complex inhibits TATA binding protein-TATA interaction.

Simian virus 40 large T antigen has been shown to inhibit p53-mediated transcription once tethered to p53-responsive promoters through interaction with p53. In this study we report that p53 stimulates transcription by enhancing the recruitment of the basal transcription factors, TFIIA and TFIID, on the promoter (the DA complex) and by inducing a conformational change in the DA complex. Significantly, we have demonstrated that T antigen inhibits p53-mediated transcription by blocking this ability of p53. We investigated the mechanism for this inhibition and found that DA complex formation was resistant to T-antigen repression when the TFIID-DNA complex was formed prior to addition of p53-T antigen complex, indicating that the T antigen, once tethered to the promoter by p53, targets TFIID. Further, we have shown that the p53-T antigen complex prevents the TATA binding protein from binding to the TATA box. Thus, these data suggest a detailed mechanism by which p53 activates transcription and by which T antigen inhibits p53-mediated transcription.

Analysis of the steroid receptor coactivator 1 (SRC1)-CREB binding protein interaction interface and its importance for the function of SRC1.

The transcriptional activity of nuclear receptors is mediated by coactivator proteins, including steroid receptor coactivator 1 (SRC1) and its homologues and the general coactivators CREB binding protein (CBP) and p300. SRC1 contains an activation domain (AD1) which functions via recruitment of CBP and and p300. In this study, we have used yeast two-hybrid and in vitro interaction-peptide inhibition experiments to map the AD1 domain of SRC1 to a 35-residue sequence potentially containing two alpha-helices. We also define a 72-amino-acid sequence in CBP necessary for SRC1 binding, designated the SRC1 interaction domain (SID). We show that in contrast to SRC1, direct binding of CBP to the estrogen receptor is weak, suggesting that SRC1 functions primarily as an adaptor to recruit CBP and p300. In support of this, we show that the ability of SRC1 to enhance ligand-dependent nuclear receptor activity in transiently transfected cells is dependent upon the integrity of the AD1 region. In contrast, the putative histone acetyltransferase domain, the Per-Arnt-Sim basic helix-loop-helix domain, the glutamine-rich domain, and AD2 can each be removed without loss of ligand-induced activity. Remarkably, a construct corresponding to residues 631 to 970, which contains only the LXXLL motifs and the AD1 region of SRC1, retained strong coactivator activity in our assays.

Transcription by RNA polymerase II in DNA-PK deficient scid mouse cells.

DNA-dependent protein kinase (DNA-PK) is involved in DNA repair but there is some evidence to suggest that it is also involved in regulating transcription. We used a pair of cell lines, SCVA2 and SC(8)-10, which are DNA-PK negative and positive respectively, in order to examine the effect of DNA-PK upon transcription. Initial experiments were performed using p53 as an activator of transcription because DNA-PK has been proposed as a candidate upstream activator of p53. It was found both in vivo and in vitro that efficient p53-dependent transcription required the presence of DNA-PK. However, phosphorylation of p53 by DNA-PK did not affect the DNA-binding ability of p53 nor its transcriptional activity when tested in vitro. Subsequent in vivo experiments suggested that a number of transcription activators functioned more efficiently in the presence of DNA-PK. Therefore DNA-PK may play a general role in regulation of transcription driven by RNA polymerase II. In addition, DNA-PK is shown to have no specific effect on p53-dependent transcription.

Phosphorylation by DNAPK inhibits the DNA-binding function of p53/T antigen complex in vitro.

Interaction of p53 with Mdm2 is hindered if either protein is phosphorylated by DNA-dependent protein kinase (DNAPK), which may account for the activation of p53 in response to double-stranded DNA breaks. This finding raises the question of whether phosphorylation of p53 by DNAPK may have a general effect on its interaction with other proteins. Here we report that unlike the p53/Mdm2 complex, p53/T antigen complex remains intact following phosphorylation by DNAPK, indicating that the effect of phosphorylation upon p53 interaction is dependent on the protein partner. We have previously shown that a mouse p53/T antigen complex can bind DNA in vitro. This complex, however, was significantly reduced in its ability to bind DNA following treatment with DNAPK. This indicates that although phosphorylation did not disrupt the p53/T antigen complex, it did result in a conformational change leading to an alteration of p53' s ability to bind DNA as a protein complex.

Crystallization of the haptoglobin-hemoglobin complex.

Two orthorhombic forms of crystals of the haptoglobin-hemoglobin complex were obtained using polyethylene glycol as precipitant. These crystals did not diffract well enough for data collection and work on the complex is no longer continued. However, the description of the crystallization conditions may be useful in future endeavors to obtain suitable crystals.

New insights into the mechanism of inhibition of p53 by simian virus 40 large T antigen.

Simian virus 40 (SV40) large tumor antigen (T antigen) has been shown to inhibit p53-dependent transcription by preventing p53 from binding to its cognate cis element. Data presented in this report provide the first direct functional evidence that T antigen, under certain conditions, may also repress p53-dependent transcription by a mechanism in which the transactivation domain of p53 is abrogated while DNA binding is unaffected. Specifically, p53 purified as a complex with T antigen from mouse cells was found to bind DNA as a transcriptionally inactive intact complex, while that purified from human cells was found to bind DNA independently of T antigen and could activate p53-dependent transcription. This difference in activity may be dependent on a different interaction of T antigen with mouse and human p53 and, in addition, on the presence of super T, which is found only in transformed rodent cells. These results suggest that subtle yet important differences exist between the inhibition of p53 by T antigen in mouse and human cells. The implications of this finding with respect to SV40-associated malignancies are discussed.

Mitogen-activated protein kinase is involved in the degradation of p53 protein in the bryostatin-1-induced differentiation of the acute promyelocytic leukemia NB4 cell line.

Overexpression of mutant p53 has been reported to promote tumorigenicity in several cancers. However, despite its potential importance, the signals regulating mutant p53 protein expression are not known. Here we show that a form of p53 that is incapable of binding DNA is overexpressed in the acute promyelocytic leukemia NB4 cell line. Our results demonstrate that treatment of NB4 cells with bryostatin-1, which induces differentiation in this cell line, leads to hyperphosphorylation of this DNA binding-impaired form of p53 via mitogen-activated protein kinase. After this phosphorylation, the p53 protein is degraded by the ubiquitin/proteasome pathway. Furthermore, we show that inhibition of p53 hyperphosphorylation blocks p53 protein degradation and cell differentiation. In addition, inhibition of the ubiquitin/proteasome pathway also blocks p53 protein degradation and cell differentiation. These findings suggest a role for mitogen-activated protein kinase in the degradation of the DNA binding-impaired form of p53 protein and in the bryostatin-induced differentiation observed in this cell line. The implications of these results with respect to the functional significance of p53 phosphorylation and degradation in cell differentiation are discussed.