Apoptosis-antagonizing transcription factor (AATF), also termed Che-1, was identified as interacting protein of Dlk/ZIP kinase and RNA polymerase II, respectively. Che-1 has additionally been shown to bind Rb, thereby activating transcription factor E2F and promoting cell cycle progression. Moreover, AATF enhances steroid receptor-mediated transactivation in a hormone-and dose-dependent manner (Leister, P., Burgdorf, S., and Scheidtmann, K. H., (2003) Signal Transduction 3, 18 -25). These data suggest that AATF exerts its functions through interaction with different transcription factors. In search of novel interaction partners of AATF, we identified the tumor susceptibility gene product TSG101, which had also been recognized as a co-regulator of nuclear hormone receptors. Interestingly, TSG101 and AATF functioned as cooperative coactivators in androgen receptor-mediated transcription. Because TSG101 was also shown to play a role in regulation of ubiquitin conjugation, we asked whether its coactivating function might be linked to ubiquitination. Indeed, TSG101 enhanced monoubiquitination of the androgen receptor in a ligand-dependent manner, and this correlated with enhanced transactivating capacity. Furthermore, a dominant-negative mutant of ubiquitin preventing polyubiquitination also stimulated androgen receptor-mediated transcription, which in this case could not be enhanced by TSG101. We propose that TSG101 activates androgen receptor-induced transcription by transient stabilization of the monoubiquitinated state, thus revealing a novel regulatory mechanism for nuclear receptors. Apoptosis-antagonizing transcription factor (AATF)1 was first identified as a protein interaction partner of Dlk/ZIP kinase (1). Dlk/ZIP kinase, in turn, is a member of the deathassociated protein kinase family that plays a role in different processes like apoptosis (reviewed in Ref.2) and mitosis (3). AATF was shown to interfere with Dlk-induced apoptosis, thus explaining its name (1). AATF is conserved from yeast to man, suggesting that it fulfills an important function. Indeed, the mouse ortholog (termed traube) has been shown to play an essential role in early embryogenesis (4). Moreover, the human ortholog of AATF (termed Che-1) was independently identified as an interacting protein of the RNA polymerase II, subunit 11, and the tumor-suppressor protein Rb (5). This latter interaction releases the repression function of Rb on E2F-mediated transcription, presumably by displacing histone deacetylase-1 from the Rb⅐E2F complex, thereby promoting cell cycle progression (6). Thus, AATF/Che-1 appears to play a role in cell cycle control. Consistent with this assumption is its down-regulation upon TGF-␤-induced differentiation (7).The AATF protein has a modular structure. At its N terminus it contains an acidic region that is characteristic of several transcription factors, such as VP16 or BRCA-1. In its C-terminal half, it contains three highly conserved regions, the significance of which is not known. Furthermore, AATF contains a leu...

Apoptosis antagonizing transcription factor AATF was identified as interaction partner of Dlk/ZIP kinase and shown to exhibit transactivation activity in Gal4‐based reporter gene expression assays. The human ortholog Che‐1 was independently isolated as interacting protein of RNA polymerase II suggesting that AATF might be a transcriptional adapter or mediator which serves to link transcriptional activators to the general transcription machinery. Consistent with this assumption, AATF contains several interaction motifs including a leucine zipper and three putative binding motifs for nuclear hormone receptors. In this study, we investigated a possible functional relationship of AATF to nuclear receptors. Indeed, AATF enhanced transactivation by several steroid hormone receptors including androgen, estrogen and glucocorticoid receptors in a hormone‐dependent manner. This enhancing effect was further increased by p300 but not by a mutant of p300 lacking acetyl transferase activity. AATF bound directly to the receptors in vitro. Co‐activation was impaired with AATF mutants bearing mutations in the LXXLL NR binding motifs. Our data suggest that AATF is a novel coactivator of nuclear hormone receptors.

The androgen receptor (AR) is a ligand-dependent transcription factor that plays a crucial role in the development and homeostasis of the prostate and in prostate cancer. The transcriptional activity of AR is mediated by interaction with multiple co-activators, which serve in chromatin modification or remodeling, or provide a link between specific and general transcription factors. We have identified zipper interacting protein (ZIP) kinase as a novel transcriptional co-activator of the AR. ZIP kinase enhanced expression of AR-responsive promotor/ luciferase reporter constructs in a hormone-and kinasedependent manner. Similar results were obtained for glucocorticoid receptor but not for progesterone receptor and estrogen receptor. Following hormone treatment, AR and ZIP kinase formed physical complexes and associated with the promoter and enhancer of the prostate-specific antigen gene, as revealed by chromatin immunoprecipitation. Strikingly, depletion of ZIP kinase by siRNA led to significant reduction of AR-mediated transactivation. The interaction of ZIP kinase with AR seems to be mediated in part by apoptosis antagonizing transcription factor and in part by direct binding. Interestingly, AR was not phosphorylated by ZIP kinase in vitro, suggesting that it phosphorylates other co-activators or chromatin proteins.

AATF/Che-1 is a coactivator of several transcription factors, including steroid hormone receptors. In search of novel interaction partners of AATF, we identified BLOS2 (BLOC1S2, also termed Ceap) from a rat cDNA library. BLOS2 is extremely conserved with a high degree of homology to yeast She3p. The clone isolated represents a splice variant encoding a polypeptide of 168 residues. Rat BLOS2 mRNA is highly expressed in brain and testis and at lower levels in other tissues, but not in skeletal or smooth muscle. Expression as a tagged fusion protein revealed predominant cytoplasmic, but also nuclear localization. In the cytoplasm, BLOS2 fusion proteins exhibit diffuse, filamentous, or dotted distribution, with the latter partially co-localizing with recycling endosomes. In addition, BLOS2 localizes to centrosomes or the pericentrosomal region. Moreover, BLOS2 co-localizes with myosin V globular tail domains in vesicle-like structures. However, a direct interaction could not be demonstrated. In transactivation assays, BLOS2 enhanced transcription from androgen receptor and p53-responsive promoters. However, this enhancement correlated with accumulation of both androgen receptor and p53, suggesting that BLOS2 has a stabilizing effect on these transcription factors. We propose that BLOS2 functions as an adapter in processes such as protein and vesicle processing and transport, and perhaps transcription.

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