MAD1 plays an important role in antagonizing MYC-mediated transcription activation by competing with the same target genes' promoter and recruiting transcription repressors. Differential expression has been well-documented using different cells and tissues [6
]. During cell differentiation, MAD1 RNA expression is elevated, while MYC RNA decreases [22
]. This unique expression pattern is the cause of the switch between MYC and MAD1 in the normal cell condition. However, the expression of MAD1 was found in various cancers in which cells are continuously proliferating [10
]. Current concepts of cancer stem cells also suggest that cancer cells may also still undergo a differentiation process, although the proliferation status is preserved due to the dysregulation of oncogenes [23
]. This might provide an explanation as to why MAD1 can be detected in cancer cells.
Since MAD1 is a tumor suppressor gene, what mechanism could cancer cells use to suppress MAD1 function? We hypothesized that post-translation modification is responsible for the functional alteration and identified that AKT-mediated MAD1 phosphorylation caused the abrogation of target promoter binding ability. AKT is frequently activated in cancer cells and inhibits various tumor suppressors such as Forkhead, p21, p27, p53, and GSK3β through post-translation phosphorylation modification [12
]. Upon stimulation by growth factors, AKT detaches from the inner surface of the plasma membrane and re-localizes to the nucleus, suggesting that nuclear proteins may also be targets of AKT [25
]. In this study, we identified that the function of the nuclear protein MAD1 is suppressed by AKT by physical interaction and phosphorylation. In vitro and in vivo assays identified Ser145 as the phosphorylation site on MAD1, and this Ser is also located in the AKT consensus phosphorylation motif (RXRXXS-145). Mutation of MAD1 Ser145 to Ala abolished AKT-mediated phosphorylation. Although the phosphorylation site is located at the C-terminal of MAD1 and is close to the leucine-zipper domain, the phosphorylation does not significantly affect the interaction with MAX. However, the phosphorylation somehow affects the interaction between MAD1 and its target genes' promoter. Consequently, AKT causes the molecules to switch from a MAD/MAX complex to a MYC/MAX complex to activate the expression of target genes.
It has been reported that MAD1 C-terminal Ser182 and Ser184 are phosphorylated by Casein Kinase II (CKII), and that phosphorylation also inhibits MAD1 DNA binding [26
]. Additionally, a recent study reported that Ser145 of MAD1 is phosphorylated by S6K and RSK and that this phosphorylation mediates protein degradation [16
]. Although in such study mentioned that MAD1 Ser145 phosphorylation is blocked by rapamycin and PD98059 under serum stimulation condition, which implies that AKT doesn't involved directly in the phosphorylation of MAD1 Ser145. However, we demonstrated that AKT physically interacts with MAD1 and phosphorylates MAD1 both in vitro and in vivo. In addition, in the presence of constitutive active AKT, rapamycin and PD98059 can only partially reduce MAD1 Ser145 phosphorylation. The possibilities for the discrepancy results could be due to the different cell lines and experimental conditions used. For instance, the cells were serum starved and followed by serum stimulation in their study. However, serum stimulation is able to trigger multiple pathways which might caused more complicated results; thus, in this study, we tried to identify the role of AKT in mediating MAD1 phosphorylation directly by presence of AKT under normal serum containing culture condition. Taken together, our results indicated that AKT is also directly involved in MAD1 Ser145 phosphorylation under our experimental conditions. Moreover, in addition to MAD1 degradation, AKT-mediated MAD1 phosphorylation and inhibition of function seem to occur mainly through the suppression of DNA binding. It is not clear why the same Ser145, which can be phosphorylated by different enzymes, results in a different outcome. One possibility is that other unknown molecules are involving the complex, which may affect the recruitment of different enzymes and lead to different outcomes. In this regards, it is worthwhile to mention that p53 has similar effects in which same serine or threonine sites were phosphorylated by different enzymes and followed by different consequences [27
]. Therefore, the detailed mechanisms of phosphorylated MAD1 in reducing interaction with promoter will need further investigation.
MYC/MAX and MAD1/MAX complexes are well regulated by the status of proliferation and differentiation under normal development. However, in cancer cells, dysregulation of oncogenic pathways may alter that status by inhibiting differentiation-related proteins such as MAD1. Thus, based on our findings, we propose a model of AKT for regulating MAD1 function. AKT-mediated phosphorylation on MAD1 affects its DNA-binding property and subsequently induces MAD1 target gene expression. Taken together, our results provide a model of AKT-mediated MAD1/MAX/MYC network regulation.