NF-kappaB-inducing kinase (NIK) is involved in the signal transduction pathway leading to the NF-kappaB activation. In this report, we demonstrate that the NIK-mediated NF-kappaB activation involves the transactivation (TA) domain of p65 subunit of NF-kappaB and the nuclear translocation of IKKalpha. By using luciferase assay, we found that both IKKalpha and IKKbeta could activate NF-kappaB in synergy with NIK. Interestingly, although IKKbeta stimulated the NIK-mediated IkappaB degradation, IKKalpha stimulated the action of NF-kappaB without enhancing the IkappaB degradation. By using heterologous transactivation system with Ga14 DNA-binding domain in fusion with various portions of p65 TA domain, we found that the transactivation domain 1 (TA1) of p65 serves as the direct target for the NIK-IKKalpha cascade and that the serine residue at 536 within p65 TA1 is indispensable for this action. Furthermore, we found that this action of NIK depends on the energy-dependent action of Ras-related protein (Ran) since the dominant negative mutant of Ran (RanQ69L) inhibited the transcriptional activity of p65 by preventing the nuclear import of IKKalpha. (C) 2003 Elsevier Science (USA). All rights reserved.