Characterization of rat BLOS2/Ceap, a putative yeast She3 homolog, as interaction partner of apoptosis antagonizing transcription factor/Che-1.

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.

TSG101 interacts with apoptosis-antagonizing transcription factor and enhances androgen receptor-mediated transcription by promoting its monoubiquitination.

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.