p53 is a transcriptional activator with a sequence-specific DNA binding domain. In response to genotoxic stress, p53 induces cell cycle arrest or apoptosis, presumably to prevent the genome from accumulating mutations (9
). A number of p53-responsive genes have been identified (for a review, see references 38
). p53 activity is controlled through several mechanisms (for a review, see references 49
, and 69
). Cellular proteins such as Mdm2 (53
) and ARF (62
) have been shown to regulate p53 protein stability. p300 (5
) and JMY (63
), on the other hand, enhance p53 transcriptional activity. The data presented here demonstrate that AMF1 has a significant effect on the transcriptional activity of p53. The facts that AMF1 stimulates reporter gene expression from p53-dependent promoters and that U2OS/AMF1 cells contain a higher basal level of p21WAF1/CIP1
and are partially G1
arrested imply that AMF1 could be an important modulator in p53 transactivation.
Increasing evidence suggests that coactivators play a significant role in regulating eukaryotic transcription (11
). We previously reported that AMF1 activates papillomavirus E2-dependent transcription and directly interacts with both E2 and p300 (8
). Complex formation among E2, AMF1, and p300 may function by bringing p300 (with histone acetyltransferase activity) close to initiation sites of transcription and viral DNA replication. Histone acetylation of nearby nucleosomes is thought to enhance access of the transcriptional or replication machinery to DNA (8
). Since p300 proteins have been shown to act as coactivators in p53-dependent transcription, participation of AMF1 may further strengthen the connection between p300 and p53. AMF1 did not stimulate a p53-dependent promoter in Soas-2 cells (p53 null, Fig. B) or in C33A cells that express mutant p53 (data not shown).
This report shows that there is direct interact between AMF1 and p53. First, AMF1 and p53 coprecipitated in U2OS/AMF1 cells (Fig. B); second, AMF1 bound p53 in different in vitro binding assays. We also performed coimmunoprecipitations of AMF1 and p53 from untreated U2OS cells. Using anti-p53 antibody pAb421, native AMF1 was coimmunoprecipitated as detected by Western blotting (data not shown), however, this result was not reproduced in all experiments. This can be explained by the following: (i) the endogenous AMF1 bound to p53 is below detectable amounts or (ii) the AMF1-p53 complex may be transient. In addition, we attempted unsuccessfully to coprecipitate p53 from untreated U2OS cell lysates using polyclonal rabbit antisera against AMF1. It is possible that the antiserum blocks p53 binding. The AMF1 binding domain on p53 was mapped to aa 161 to 333, and the region on AMF1 responsible for binding to p53 was localized to aa 103 to 250. In contrast to what was shown for Mdm2 (21
), interaction between AMF1 and p53 does not seem to alter intracellular p53 protein levels before or after etoposide treatment (Fig. B). Cotransfection of wild-type p53 with or without AMF1 into Saos-2 cells showed equal levels of p53 (data not shown).
To better understand the mechanisms of how AMF1 functionally interacts with p53, we identified a dominant-negative version of AMF1, AMF1(1–103), which did not bind p53 (Fig. ) but formed complexes with wild-type AMF1 (Fig. B). The observation that AMF1(1–103) can compromise the effects of wild-type AMF1 and inhibit p53-dependent transcriptional activation (Fig. A) argue that proper oligomerization of AMF1 is necessary for its activation of p53 transcription. AMF1(1–103) probably forms a nonfunctional complex with wild-type AMF1 in vivo, resulting in a decreased amount of functional oligomers formed between wild-type molecules.
A significant implication of the present study relates to the importance of transcriptional activation in the p53 response. Previous studies have established that increased p53 transactivation can lead to either cell growth arrest or apoptosis, depending on the cell type and DNA-damaging agent used (18
). Our studies showed that etoposide treatment of U2OS cells results in the same outcome (growth arrest) as X-ray treatment (2
). However, overexpression of AMF1 in U2OS/AMF1 cells delays but does not block apoptosis, probably by raising the p21WAF1/CIP1
protein level. A similar effect was observed with ectopic expression of p21WAF1/CIP1
). In contrast, U2OS/AMF1 cells are more sensitive to UV irradiation-mediated apoptosis. It was previously shown that the UV-mediated apoptosis in U2OS cells is p53 dependent (p53-dominant negative U2OS clones barely undergo apoptosis under UV irradiation), but not via a p21WAF1/CIP1
-activated pathway, as p21WAF1/CIP1
expression was suppressed upon exposure to UV irradiation (2
). Our results confirmed that p21WAF1/CIP1
protein in U2OS/AMF1 cells was undetectable 2 h after UV irradiation (data not shown), indicating that other p53-dependent genes are responsible for the augmented apoptosis in U2OS/AMF1 cells. Ongoing projects will examine the effects of AMF1 on the expression of apoptotic targets of p53.
It is possible for a coactivator to exert variable effects on different classes of transcription activator (11
), and in this respect, it will be interesting to determine the specificity of AMF1-mediated coactivation. It was reported that binding of AMF1 to the HTLV-1 oncoprotein Tax can potently suppress its activation of Jun N-terminal protein kinase 1 (JNK1) (35
). Interestingly, HTLV-1 Tax oncoprotein also repressed the p53-mediated transactivation through coactivator CREB-binding protein sequestration (3
). Like other cell cycle regulators, AMF1 may play multiple roles in different signal transduction pathways. Genetic analysis in yeast indicates that AMF1 functions downstream of STE4-encoded Gβ
subunit and upstream of STE20 (35
). In NIH 3T3 cells, overexpression of AMF1 can suppress G-protein (RAS)-mediated and mitogen-activated protein kinase-mediated signaling and interfere with JNK1 activity (64
). We also found that AMF1 can stimulate the transcription function of a c-Jun AD-GAL4 DBD fusion that is subject to regulation by JNK1 (8
). These data imply that AMF1 may play important roles in signal transduction as well as being a coactivator in E2- or p53-dependent transcription. Given the multiple and complex signaling effects of AMF1, it will be of interest to establish a mouse knockout model to further explore its physiological roles.