AMF1 (GPS2) Modulates p53 Transactivation

The p53 tumor suppressor is a common target for genetic alteration in human tumors and is one of the most frequently altered genes in cancer cells (38, 43). Under normal circumstances, the p53 protein remains in a latent state. Following DNA damage or other forms of cellular stress, the p53 protein rapidly accumulates and becomes activated through posttranslational mechanisms. Wild-type p53 limits cellular proliferation by inducing either a transient G1 arrest or apoptosis, depending on the cellular context (for a review, see reference 41). Differential regulation of p53 transactivation has been observed between cells undergoing growth arrest and apoptosis. Transactivation is required for the efficient execution of p53-mediated growth arrest, yet its role in apoptosis is equivocal (19, 70). The growth arrest response mediated by p53 relies on its ability to act as a sequence-specific DNA-binding transcription factor (38, 73). A variety of downstream target genes that influence cell growth, including p21WAF1/CIP1, Bax, Mdm2, cyclin G, and Gadd45, have been identified (for a review, see references 38 and 43). The p53-mediated G1 arrest is believed to occur primarily through induction of p21WAF1/CIP1, a general inhibitor of cyclin-dependent kinases whose functions are required for cell cycle progression (18, 28, 75). In contrast, although increases of p21WAF1/CIP1 have been reported to be associated with apoptosis in some cases (51, 68), p21WAF1/CIP1 activity appears to be dispensable for apoptosis (2, 9). The human Mdm2 protein binds to p53 and inhibits its transactivation function by targeting p53 for rapid degradation (29, 40). Transcription of the Mdm2 gene itself is activated by p53, representing a feedback loop to control p53 activity (74). The human p53 protein consists of 393 amino acids (aa) and contains four major functional domains for a review, see reference 49). At the N terminus is a transcriptional activation domain (aa 1 to 50) which recruits the basal transcriptional machinery, including the TATA box binding protein (TBP) and TBP-associated factors (47, 67). The central region of p53 is the sequence-specific DNA-binding domain (DBD) (aa 102 to 292). It is within this central domain that 80 to 90% of the tumor mutations are found (12). The central region also functions as protein binding domain interacting with simian virus 40 large T antigen (34) and the cellular proteins 53BP1 and 53BP2 (20, 33). The C-terminal portion contains an oligomerization domain (aa 323 to 356) and a regulatory domain (aa 360 to 393). It is well established that p53 forms tetramers via the oligomerization domain (39). Tetramerization appears to be required for efficient transactivation in vivo and for p53-mediated suppression of growth of carcinoma cell lines (55). The extreme C-terminal regulatory domain acts as a negative regulator of p53 sequence-specific DNA binding (1, 60). The activation of p53 in cultured mammalian cells has been correlated with phosphorylation (38), acetylation (59), glycosylation (61), and proteolytic removal (52) of the C-terminal domain. These modifications are thought to activate p53 by causing a conformational change of the protein, regulated by an allosteric effect. Although it is widely recognized that transcriptional activation plays a pivotal role in mediating the p53 response, relatively limited information is available on the mechanisms through which p53 stimulates transcription. Several cellular proteins have been shown to play roles in p53 transactivation. The human TBP-associated factor TAFII31, a component of TFIID, binds to p53 via residues in the amino-terminal domain that are essential for transcription. Antibodies directed against TAFII31 inhibit p53-activated transcription in vitro (47). Members of the p300 (also known as CREB-binding protein) transcriptional coactivator family were shown to interact with p53 and stimulate its sequence-specific DNA binding (23). Interaction with p300 modulates p53-mediated activation of the p21WAF1/CIP1 and Bax promoters, as well as p53-induced cell cycle arrest and apoptosis (24, 44). Furthermore, p300-binding proteins, such as PCAF and JMY, were also shown to facilitate the p53 response by augmenting p53-dependent transcription and apoptosis (45, 59, 63). We report here the properties of a novel cellular protein, AMF1 (also called G-protein pathway suppressor 2 or GPS2), and its stimulatory interaction with p53. AMF1 is a human nuclear protein of 327 aa with a predicted molecular size of 37 kDa. It was initially identified as one of two human suppressors of lethal G-protein subunit-activating mutations in the pheromone response pathway of the yeast Saccharomyces cerevisiae and was named GPS2 (64). Meanwhile, this protein was found to interact with the papillomavirus E2 protein activation domain and to be required for the stimulation of E2 transcriptional activity; therefore, it was named AMF1 (for activation-domain modulating factor 1) (8). The human papillomavirus 16 E2 protein was reported to interact with p53 and induce apoptosis (17, 48, 72). Recently, we demonstrated that AMF1 interacts with the transcriptional adapter p300, and both AMF1 and p300 additively increased E2 transactivation (54), suggesting that AMF1 could act as a transcription cofactor. GPS2 also interacts with the human T-cell leukemia virus type 1 (HTLV-1) Tax protein (14, 35) and influences the transcriptional activities of Tax and c-Jun (35, 64). In this study, we investigated the physical and functional relationship between AMF1 and p53.