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Notch signaling plays a crucial role in cell development and proliferation. The Notch receptor is an inducible transcription factor found on the cell surface. Upon interaction with its ligand, the Notch receptor is cleaved, releasing the Notch Intra-cellular Domain (NICD), which enters the nucleus and complexes with CBF1 and Mastermind (MAML1) to activate target genes. MAML1 is also involved in many other cell signaling pathways independently of Notch signaling. In this study, we focused on determining whether MAML family members follow the same path as the Notch intra-cellular domain, which undergoes ubiquitination and degradation via the ubiquitin-proteasome pathway. We also examined the regions of MAML1 required for ubiquitination and the kinase(s) that may be involved in phosphorylation of MAML1. We determined that MAML1 was ubiquitinated as the shortest-lived family member and that the (75-300) amino acid region of MAML1 is essential for ubiquitination and degradation of MAML1. We also found MAML2, which is not ubiquitinated, to be the most stable member of the family.

Early studies demonstrated the involvement of ubiquitination of the Notch intracellular domain for rapid turnover of the transcriptional complex at Notch target genes. It was shown that this ubiquitination was promoted by the co-activator Mastermind like 1 (MAML1). MAML1 also contains numerous lysine residues that may also be ubiquitinated and necessary for protein regulation. In this study, we show that over-expressed MAML1 is ubiquitinated and identify eight conserved lysine residues which are required for ubiquitination. We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination. Furthermore, we show that a mutant MAML1 that has decreased ubiquitination shows increased output from a HES1 reporter gene assay. Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation. In summary, this study identifies that MAML1 is ubiquitinated in the absence of Notch signaling to maintain low levels of MAML1 in the cell. Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway.