Mad1 function is regulated through elements within the carboxy terminus

Myc and Mad are basic helix-loop-helix leucine zipper (bHLH-LZ) proteins th at heterodimerize with Max to bind DNA and thereby influence the transcript ion of Mac-responsive genes. Myc-Max dimers transactivate whereas Mad-Max-m Sin3 complexes repress Myc-mediated transcriptional activation. We have pre viously shown that the N-terminal mSin3 binding domain and the centrally lo cated bHLH-LZ are required for Mad1 to function during a molecular switch f rom proliferation to differentiation. Here we demonstrate that the carboxy terminus (CT) of Mad1 contains previously unidentified motifs necessary for the regulation of Mad1 function. We show that removal of the last 18 amino acids of Mad1 (region V) abolishes the growth-inhibitory function of the p rotein and the ability to reverse a Myc-imposed differentiation block Moreo ver, deletion of region V results in a protein that binds DNA weakly and no longer represses Myc-dependent transcriptional activation. In contrast, de letion of the preceding 24 amino acids (region IV) together with region V r estores DNA binding and transcriptional repression, suggesting a functional interplay between these two regions. Furthermore, phosphorylation within r egion IV appears to mediate this interplay. These findings indicate that no vel regulatory elements are present in the Mad1 CT.