RESUMO
Menin acts as contextual a tumor suppressor and a tumor promoter, partly via epigenetic regulation of gene transcription. While menin is phosphorylated, it remains unclear whether wild type menin has other post-translational modifications. Here, we report that menin is SUMOylated by SUMO1 in vivo and in vitro, and the SUMOylation is reduced by a SUMO protease. Lysine 591 of menin was covalently modified by SUMO1 and K591R mutation in menin blocked SUMOylation of the C-terminal part of menin in transfected cells. Full-length menin with K591 mutation was still SUMOylated in vivo, suggesting the existence of multiple SUMOylation sites. Menin K591R mutant or menin-SUMO fusion protein still retains the ability to regulate cell proliferation and the expression of the examined menin target genes.
RESUMO
BACKGROUND: Menin is a ubiquitously expressed protein encoded by the multiple endocrine neoplasia type 1 (MEN1) gene. Besides its importance in endocrine organs, menin has been shown to interact with the mixed lineage leukemia (MLL) protein, a histone H3 lysine 4 methyltransferase, and plays a critical role in hematopoiesis and leukemogenesis. Previous studies have shown that menin promotes transforming growth factor beta (TGF-ß) signaling in endocrine cells. However, little is known regarding the impact of TGF-ß pathway on menin in hematopoietic system. Here, with leukemia cell lines generated from conditional MEN1 or TGF-ß receptor (TßRII) knockout mouse models, we investigated the possible cross-talk of these two pathways in leukemia cells. METHODS: MEN1 or TßRII conditional knockout mice were bred and the bone marrow cells were transduced with retroviruses expressing oncogeneic MLL-AF9 (a mixed lineage leukemia fusion protein) to generate two leukemia cell lines. Cell proliferation assays were performed to investigate the effect of TGF-ß treatment on MLL-AF9 transformed leukemia cells with/without MEN1 or TßRII excision. Menin protein was detected with Western blotting and mRNA levels of cell proliferation-related genes Cyclin A(2) and Cyclin E(2) were examined with real-time RT-PCR for each treated sample. In vivo effect of TGF-ß signal on menin expression was also investigated in mouse liver tissue after TßRII excision. RESULTS: TGF-ß not only inhibited the proliferation of wild type MLL-AF9 transformed mouse bone marrow cells, but also up-regulated menin expression in these cells. Moreover, TGF-ß failed to further inhibit the proliferation of Men1-null cells as compared to Men1-expressing control cells. Furthermore, excision of TßRII, a vital component in TGF-ß signaling pathway, down-regulated menin expression in MLL-AF9 transformed mouse bone marrow cells. In vivo data also confirmed that menin expression was decreased in liver samples of conditional TßRII knockout mice after TßRII excision. CONCLUSION: These results provided the first piece of evidence of cross-talk between menin and TGF-ß signaling pathways in regulating proliferation of leukemia cells, suggesting that manipulating the cross-talk of the two pathways may lead to a novel therapy for leukemia.
