Mitogene-inducible gene-6 (Mig-6) is involved in the intracellular signaling pathway as an adaptor molecule. Mig-6 expression is rapidly induced upon various kinds of mitogenic and stressful stimulations. We previously demonstrated that Mig-6 expression is induced by activated Ki-ras in human colon cancer cells and the limited proteolytic processed NH2-terminal region containing the Cdc42/Rac interaction and binding (CRIB) domain of Mig-6 is bound to IκBα, resulting in NF-κB activation. In this study, we tried to determine the critical region of Mig-6 for regulating NF-κB activation by using various kinds of deletion-constructs of Mig-6. The CRIB domain-deleted Mig-6 fragment (residues 68 to 462) did not show significant NF-κB activation. Furthermore, NH2-terminal 148, 158 and 180 amino acid regions of Mig-6 did not show NF-κB activation. On the other hand, the NH2-terminal 264 amino acid region of Mig-6 did show NF-κB activation to a similar extent of the full length of Mig-6. Interestingly, the full length and NH2-terminal 264 amino acid region of Mig-6 produced two kinds of cleaved NH2-terminal fragments, N1 and N2 with an approximate molecular size of 5 and 7 kDa, respectively. The NH2-terminal 148 and 158 amino acid regions of Mig-6 did not produce N1 or N2 fragments, while the NH2-terminal 180 amino acid region of Mig-6 only produced the N1 fragment. Furthermore, the N2 fragment was bound with IκBα, but the N1 fragment was not. These results together suggested that the NH2-terminal 264 amino acid region is critical for NF-κB activation and proper limited proteolytic processing of Mig-6.

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