No abstract

Transcriptional activity of the TATA-binding protein (TBP) is controlled by a variety of proteins. The BTAF1 protein (formerly known as TAF II 170/TAF-172 and the human ortholog of Saccharomyces cerevisiae Mot1p) and the NC2 complex composed of NC2␣ (DRAP1) and NC2␤ (Dr1) are able to bind to TBP directly and regulate RNA polymerase II transcription both positively and negatively. Here, we present evidence that the NC2␣ subunit interacts with BTAF1. In contrast, the NC2␤ subunit is not able to associate with BTAF1 and seems to interfere with the BTAF1-TBP interaction. Addition of NC2␣ or the NC2 complex can stimulate the ability of BTAF1 to interact with TBP. This function is dependent on the presence of ATP in cell extracts but does not involve the ATPase activity of BTAF1 nor phosphorylation of NC2␣. Together, our results constitute the first evidence of the physical cooperation between BTAF1 and NC2␣ in TBP regulation and provide a framework to understand transcription functions of NC2␣ and NC2␤ in vivo.Initiation of gene transcription by eukaryotic RNA polymerase II (pol II) is tightly controlled by a multitude of regulatory factors. The concerted action of these factors results in the formation of the pol II preinitiation complex (32, 33). Recent studies have deepened our knowledge of the regulation of the steps leading to this. It is also becoming clear that the mode and sequence of the recruitment of the basal transcription factors vary among promoters (7). The preinitiation complex consists of several basal transcription factors, including TATAbinding protein (TBP), which plays a central role in the assembly process. This is underscored by the fact that transcription of the majority of cellular genes in vivo requires TBP (11). In human cells several factors were shown to bind directly to and regulate the activity of TBP in pol II transcription. The beststudied factors are TBP-associated factors (TAFs), which together with TBP form the TFIID complex (25,40). Others include the BTAF1 protein (TAF II 170/TAF-172) and the NC2 (Dr1-DRAP1) complex.BTAF1 and its Saccharomyces cerevisiae ortholog Mot1p form stable complexes with TBP in cell extracts (37,38,43,44; for a review, see reference 35). The observation that a large proportion of TBP is complexed with BTAF1 as the B-TFIID complex gives rise to the notion that BTAF1 is an important regulator of TBP function (44). Indeed, in vitro studies show that the B-TFIID complex is able to bind promoter DNA and support transcription (18,34,44). On the other hand, BTAF1 and Mot1p proteins contain dATPase activity, which is involved in the dissociation of TBP from DNA in an ATPdependent stroke. This activity can explain their negative effect on transcription (5,34,36). In accordance with a dual role of Mot1p in transcription, mRNA expression profiling and mutational analyses indicate that Mot1p affects transcription both positively and negatively (1,6,10,12,15,26,27,39). The positive role of Mot1p is strengthened by observations that it is present at the sites of certai...

The aim of the present study was to examine the potential antitumor activity of lovastatin and other statins together with pamidronate, a second generation bisphosphonate (BP), against tumor cell lines. Cytostatic/cytotoxic effects were measured using crystal violet assay. Regulation of the cell cycle and induction of apoptosis were evaluated using flow cytometry and Western blotting, migration of tumor cells was measured in a scratch wound assay and their invasiveness was measured with a Matrigel-invasion assay. Antitumor effects of the combination treatment were evaluated in a murine PANC 02 pancreatic adenocarcinoma model. Combination of pamidronate and lovastatin produced potentiated cytostatic/ cytotoxic effects against breast and pancreatic cancer cell lines. The combination was also effective in inhibition of tumor cell adhesion to collagen IV and fibronectin and interfered with migration and invasiveness of tumor cells. Neither pamidronate nor lovastatin alone affected tumor growth in mice but the combination treatment resulted in retardation of tumor growth and prolongation of mouse survival. The combination of statins and pamidronate, a second generation bisphosphonate, demonstrates promising antitumor effects at doses readily achievable in patients. This combination holds promise for future clinical studies.

Hsp90 is a ubiquitous, ATP-dependent chaperone, essential for eukaryotes. It possesses a broad spectrum of substrates, among which is the p53 transcription factor, encoded by a tumor-suppressor gene. Here, we elucidate the role of the adenine nucleotide in the Hsp90 chaperone cycle, by taking advantage of a unique in vitro assay measuring Hsp90-dependent p53 binding to the promoter sequence. E42A and D88N Hsp90␤ variants bind but do not hydrolyze ATP, whereas E42A has increased and D88N decreased ATP affinity, compared with WT Hsp90␤. Nevertheless, both of these mutants interact with WT p53 with a similar affinity. Surprisingly, in the case of WT, but also E42A Hsp90␤, the presence of ATP stimulates dissociation of Hsp90-p53 complexes and results in p53 binding to the promoter sequence. D88N Hsp90␤ is not efficient in both of these reactions. Using a trap version of the chaperonin GroEL, which irreversibly captures unfolded proteins, we show that Hsp90 chaperone action on WT p53 results in a partial unfolding of the substrate. The ATP-dependent dissociation of p53-Hsp90 complex allows further folding of p53 protein to an active conformation, able to bind to the promoter sequence. Furthermore, in support of these results, the overproduction of WT or E42A Hsp90␤ stimulates transcription from the WAF1 gene promoter in H1299 cells. Altogether, our research indicates that ATP binding to Hsp90␤ is a sufficient step for effective WT p53 client protein chaperoning.Hsp90 is an abundant protein in cells of all known organisms, with the exception of the Archea kingdom. Although in bacteria its presence is not required for survival (1), yeast and higher eukaryotes are fully dependent on its activity (2, 3). In multicellular organisms Hsp90 plays a key role in the activation and stabilization of various protein substrates. Among these are kinases (Raf1, Akt, and Src), telomerase, Rab GDP dissociation inhibitors, glucocorticoid hormone receptors (GR), 3 and transcription factors such as the p53 tumor suppressor protein (4 -6). These Hsp90 substrates belong to different protein families and do not share common sequence or structural motifs. Hence, modes of interaction and chaperoning may possess both common features and specific differences.Hsp90 is functional as a dimer, each monomer consisting of three domains connected with flexible linkers of different length and sequence depending on organism and isoform. The main substrate binding region is proposed to be localized in the middle domain (7), however structural (8) and biochemical studies (9 -11) suggest that at least two distinct surfaces of interaction should exist on Hsp90 chaperone while binding its protein substrate. Repositioning of the domains of the Hsp90 may be translated into conformational rearrangements of a protein substrate, changing its tertiary structure and exposing buried residues, thus enabling interaction with other proteins and ligands, such as hormones or nucleic acids.Despite the initial controversy on the ATP dependence of Hsp90 (12), it was unambig...

The BTAF1 transcription factor interacts with TATA-binding protein (TBP) to form the B–TFIID complex, which is involved in RNA polymerase II transcription. Here, we present an extensive mapping study of TBP residues involved in BTAF1 interaction. This shows that residues in the concave, DNA-binding surface of TBP are important for BTAF1 binding. In addition, BTAF1 interacts with residues in helix 2 on the convex side of TBP as assayed in protein–protein and in DNA-binding assays. BTAF1 drastically changes the TATA-box binding specificity of TBP, as it is able to recruit DNA-binding defective TBP mutants to both TATA-containing and TATA-less DNA. Interestingly, other helix 2 interacting factors, such as TFIIA and NC2, can also stabilize mutant TBP binding to DNA. In contrast, TFIIB which interacts with a distinct surface of TBP does not display this activity. Since many proteins contact helix 2 of TBP, this provides a molecular basis for mutually exclusive TBP interactions and stresses the importance of this structural element for eukaryotic transcription.