Menin interacts with activator of S-phase kinase and regulates TGF-beta signaling

Introduction: MEN1 is the tumor suppressor gene responsible for the multiple endocrine neoplasia type 1 (MEN1) syndrome. Menin, the protein product of the MEN1 gene, is thought to function in the regulation of gene transcription and DNA repair pathways. One of key growth regulatory pathways dependent upon menin expression is TGF-beta. In the absence of menin, TGF-beta signaling is significantly reduced. Through yeast two hybrid studies, we identified an important link between menin and ASK, a key protein involved in starting DNA replication. We show for the first time that ASK and menin are important for regulating TGF-beta signaling. Hypothesis: The MEN1 gene tumor suppressor protein, menin, binds the activator of S-phase kinase protein (ASK) and regulates the TGF-beta signaling pathway. Methods: Protein interaction and cell signaling studies were performed with a human embryonic kidney cell line (HEK293T) and a rodent islet cell line (TGP61). HEK293 cells were transiently transfected with mammalian expression constructs to overexpress menin and ASK in HEK293T cells. Menin protein expression was knocked down with siRNA menin transfection. Protein expression was confirmed by western blot analysis. Coimmunoprecipitation was performed to confirm interaction between ASK and menin. BrdU assay was performed to measure DNA synthesis. Flow cytometry was used to determine the cell cycle profile of the transfected cells. Promoter assays were performed in TGP61 cells with a transfected luciferase reporter, p3TP-Lux, that is responsive to TGF-beta. Results: ASK regulates DNA replication through the binding and activation of CDC7 kinase. ASK-CDC7 phosphorylates and releases the DNA helicase inhibitor MCM2 to initiate origin firing and the start of DNA synthesis. Coimmunoprecipitation studies confirmed that menin bound ASK and decreased ASK phosphorylation (slower migration in western blot), but menin did not affect the phosphorylation and chromatin release of MCM2. Similarly, DNA synthesis as measured by BrdU incorporation and cell cycle profile as measured by flow cytometry were not affected by transient overexpression of menin and ASK alone or in combination. These data suggest that menin does not associate with ASK to primarily regulate the initiation of DNA replication during the cell cycle. Since menin is essential for TGF-beta signaling, we sought to determine whether menin and ASK might regulate transcription in response to TGF-beta signaling. Overexpression of menin augmented p3TP-Lux luciferase reporter activity in response to TGFbeta, whereas siRNA menin significantly inhibited TGF-beta signaling. Unexpectedly, ASK overexpression decreased the TGF-beta response. Coexpression of ASK and menin resulted in intermediate TGF-beta response. Conclusion: Menin binds to ASK and regulates ASK phosphorylation, but does not directly affect DNA synthesis. Instead, this interaction is important for regulation of gene transcription. In our model, menin is essential for TGF-beta signaling which is antagonized by ASK overexpression. ASK expression is cell-cycle regulated with maximal expression at late G1. Therefore, loss of menin or overexpression of ASK in endocrine tumors could enable tumor cells to lose their TGF-beta responsiveness.