In this report, we present the identification of a novel AR-associated protein, ARA67/PAT1, which functions as a repressor of AR. Many coregulators of AR have been identified and characterized. Compared to coactivators, the identified corepressors of AR are relatively fewer and less well characterized. Calreticulin can bind to AR DNA-binding domain and suppress AR transactivation by blocking AR binding to target DNA sequences (3
). Cyclin D1 has been reported to suppress AR function presumably through influencing androgen-dependent transactivation function in ARN (40
). Since androgen action involves dissociation of AR from heat shock protein complex, homodimerization, nuclear translocation, and binding to target genes, all these processes can be influenced by coregulators.
Nuclear localization of androgen bound AR is a prerequisite for its transactivation function. However, relatively little is known about the mechanism of its nucleocytoplasmic trafficking and its potential interacting proteins that may contribute to this process. Our data show that ARA67/PAT1 is able to alter the subcellular distribution pattern of AR in the presence of DHT, indicating that it may play a role in AR trafficking. Early studies showed that ARA67/PAT1 shares homology with kinesin light chain, a molecular motor driving the trafficking of cargos along the microtubule, directly interacts with the microtubule, and is functionally related to amyloid precursor protein trafficking/processing (48
). Our data correlate with these findings, supporting the role of ARA67/PAT1 in protein trafficking. However, the detailed mechanisms need further investigation.
Studies with glucocorticoid receptor suggest that an intact cytoskeleton network is required for the shuttling of glucocorticoid receptor between the cytoplasm and nucleus in physiological conditions (13
). It is not clear whether this represents a common feature in nucleocytoplasmic shuttling among all steroid hormone receptors, since the ligand-dependent translocation of progesterone receptor has been suggested as independent of cytoskeleton integrity (39
). ARA67/PAT1 can bind microtubules and the binding can be enhanced 5- to 10-fold in the presence of Mg-ATP (48
), suggesting the possibility that the microtubule network may be an important component for ARA67/PAT1 to promote cytoplasmic retention of AR. Whether the decreased nuclear AR level in the presence of DHT is due to a decreased nuclear import or a increased nuclear export remains unclear. We observed a decreased interaction between ARA67/PAT1 and AR in the presence of DHT (Fig. ), and also a reverse of ARA67/PAT1's inhibitory effect by a high dose (1,000 nM) of DHT (unpublished data), suggesting a potential competition between ARA67/PAT1 and the ligand in binding to the AR. Since ligands promote the dissociation of AR from heat shock protein in the cytoplasm, and the subsequent nuclear translocation of AR, ARA67/PAT1 may interfere at these steps which may lead to the trapping of AR in the cytoplasm. Besides heat shock proteins, filamin has been reported to be involved in the nuclear translocation of AR (36
). It is also known that nuclear import of steroid receptors is mediated by importins, although the nuclear export mechanism remains largely unknown (8
). Whether ARA67/PAT1 can be associated with these factors and influence their function requires further study.
Although we demonstrate that ARA67/PAT1 can influence the subcellular distribution of liganded AR, which may contribute to its suppression effect on AR transactivation, other mechanisms may still exist. The possession of inherent repression activity has also been examined for ARA67/PAT1 by testing its ability to influence Gal4-DNA-binding domain-ARN transcriptional activity. Results show ARA67/PAT1 can enhance Gal4 DNA-binding domain-ARN transcriptional activity by two- to threefold rather than suppressed it (data not shown), indicating the lack of an inherent repression activity of ARA67/PAT1. Nevertheless, this one-hybrid assay result still provides extra evidence showing that ARA67/PAT1 can interact and influence AR function. However, since ARA67/PAT1 presents in both cytoplasm and nucleus (15
), a direct influence on the nuclear AR transactivation is still possible. Several deletion constructs of ARA67/PAT1 can also sufficiently inhibit AR transactivation (Fig. ), even without effective interaction with AR compared to the full-length ARA67/PAT1 (Fig. ), which may further support this hypothesis. However, we would also like to point out that small fragment constructs may act independently, and may not represent the behavior of its full-length protein. ARA67/PAT11-411
, a deletion construct that remains in the cytoplasm (15
), can mediate a repression on AR transactivation (Fig. ) and decrease both nuclear and total AR protein level (Fig. ). This is distinct from the full-length ARA67/PAT1, which can increase total AR protein level (Fig. ) while suppressing the nuclear AR signal (Fig. ).
It is known that AR N and C termini can directly interact through the LXXLL-like motif present in the AR N terminus and the AF-2 domain in AR C terminus (18
). The N/C interaction may help to stabilize AR protein and modulate the transactivation activity of AR (49
). Coregulators that influence the AR N/C interaction could affect AR transactivation. One of the mechanisms by which coactivators enhance AR transactivation is through facilitating AR N/C interactions as seen in SRC-1 and CBP-mediated coactivation (25
). Since ARA67/PAT1 can interact with both AR N and C termini, it is reasonable to hypothesize that ARA67/PAT1 may influence the AR N/C interaction. Our results show ARA67/PAT1 enhances the interaction between AR N and C termini, and accordingly we also observed a mild increase in AR protein level that may result from an increased AR stability. These seem to be contradictory to the role of ARA67/PAT1 as a repressor. However, we show that ARA67/PAT1 can decrease nuclear localization of AR upon AR ligand binding. This may prevent elevated AR protein from enhancing AR transactivation, leading to the repression of AR transactivation as a net result, since only nuclear AR can exert its influence on its target genes.
The suppression effect of ARA67/PAT1 may differ among various cells, since subcellular environments may vary. It is possible that some unknown modifications on ARA67/PAT1 may weaken its ability to block AR nuclear translocation, while the increased AR protein level may be dominant, in which case ARA67/PAT1 may function as either a weaker repressor or even as a coactivator.
In summary, we demonstrate that ARA67/PAT1 can interact with AR and suppress AR transactivation. The interrupted nuclear localization of AR in the presence of its ligand may contribute to ARA67/PAT1-mediated suppression. In addition, ARA67/PAT1 has the potential to enhance AR transactivation through enhancing the AR N/C interaction and AR stability. Since AR is one of the key players in prostate carcinogenesis, it is possible that subcellular environments in some prostate cancer cells may modify the ability of ARA67/PAT1 to repress AR and favor cancer growth. Furthermore, ARA67/PAT1 can also bind specifically to amyloid precursor protein (48
), a protein that is involved in the pathogenesis of Alzheimer's disease. Testosterone has been reported to protect neurons from neurotoxic damage through the AR-mediated pathway (17
). Whether AR may play a role in neuron-related diseases through the linkage of ARA67/PAT1 remains as an interesting area for further investigation.