Mapping of the critical region of mitogene-inducible gene-6 for NF-kappaB activation.
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 NH(2)-terminal region containing the Cdc42/Rac interaction and binding (CRIB) domain of Mig-6 is bound to IkappaBalpha, resulting in NF-kappaB activation. In this study, we tried to determine the critical region of Mig-6 for regulating NF-kappaB 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-kappaB activation. Furthermore, NH(2)-terminal 148, 158 and 180 amino acid regions of Mig-6 did not show NF-kappaB activation. On the other hand, the NH(2)-terminal 264 amino acid region of Mig-6 did show NF-kappaB activation to a similar extent of the full length of Mig-6. Interestingly, the full length and NH(2)-terminal 264 amino acid region of Mig-6 produced two kinds of cleaved NH(2)-terminal fragments, N1 and N2 with an approximate molecular size of 5 and 7 kDa, respectively. The NH(2)-terminal 148 and 158 amino acid regions of Mig-6 did not produce N1 or N2 fragments, while the NH(2)-terminal 180 amino acid region of Mig-6 only produced the N1 fragment. Furthermore, the N2 fragment was bound with IkappaBalpha, but the N1 fragment was not. These results together suggested that the NH(2)-terminal 264 amino acid region is critical for NF-kappaB activation and proper limited proteolytic processing of Mig-6.
Mabuchi
Reiko
R
Department of Pathology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan.
Sasazuki
Takehiko
T
Shirasawa
Senji
S
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D003110:Colonic Neoplasms / Q000473:pathology; D006801:Humans; D016328:NF-kappa B / Q000096:biosynthesis* / Q000494:pharmacology; D010957:Plasmids; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D015854:Up-Regulation
D048868:Adaptor Proteins, Signal Transducing; C484017:ERRFI1 protein, human; D016328:NF-kappa B; D025521:Tumor Suppressor Proteins