Receptor-interacting protein (RIP) is a serine/threonine protein kinase that is critically involved in tumor necrosis factor receptor-1 (TNF-R1)-induced NF-kappaB activation. In a yeast two-hybrid screening for potential RIP-interacting proteins, we identified a novel protein designated as NKAP. Although NKAP interacts with RIP in yeast, NKAP does not interact with RIP in mammalian cells in co-immunoprecipitation experiments. When overexpressed in 293 cells, NKAP activated NF-kappaB in a dose-dependent manner. Moreover, down-regulation of NKAP by antisense RNA significantly inhibited TNF- and IL-1-induced NF-kappaB activation. Immunofluorescent staining indicated that NKAP was localized in the nucleus. Our findings suggest that NKAP is a novel nuclear regulator of TNF- and IL-1-induced NF-kappaB activation. (C) 2003 Elsevier Inc. All rights reserved.