NF-KAPPA-B P100 (LYT-10) IS A COMPONENT OF H2TF1 AND CAN FUNCTION AS AN I-KAPPA-B-LIKE MOLECULE

NF-kappaB is an important transcription factor regulating expression of genes involved in immune function, inflammation, and cellular growth control. NF-kappaB activity is induced by numerous stimuli, such as phorbol esters, B- and T-cell mitogens, the cytokines tumor necrosis factor and interleukin-1, and serum growth factors. The standard model for the induction of NF-kappaB activity involves the release of the transcription factor from a cytoplasmic inhibitor termed IkappaB, allowing translocation of NF-kappaB to the nucleus. IkappaB contains multiple copies of the so-called ankyrin repeat, which are apparently necessary for its function. Subunits comprising NF-kappaB and related binding activities are members of the Rel multigene family. Two such subunits, p50 and p52 (also called p50B), are proteolytically processed from precursors of 105 kDa (also called p105 and NFKB1) and 100 kDa (also called p100, NFKB2, and Lyt-10), respectively. Both contain N-terminal Rel-homologous domains as well as multiple copies of C-terminal ankyrin repeats. We show here that NF-kappaB p100 is a component of the previously identified DNA-binding activity H2TF1. In addition, we show that p100 is localized in the cytoplasm in HeLa cells, where it is associated with c-Rel, p50, or p65 (RelA). In transient-transfection assays, p100 represses the ability of NF-kappaB p65 to activate a kappaB-containing reporter construct. Transfection of p100 also results in a loss of nuclear p65 DNA binding to a kappaB probe, as measured by an electrophoretic mobility shift assay, and a loss of nuclear p65 immunoreactivity, as measured by immunoblotting. This loss of nuclear p65 is paralleled by a gain of p65 DNA-binding activity and immunoreactivity in the cytoplasm. We interpret these data as demonstrating that p100 functions as an IkappaB-like molecule to sequester Rel family members in the cytoplasm. Proteolytic processing of p100 to the activator p52 is predicted to generate several new forms of Rel family heterodimers and therefore represents a form of regulation of NF-kappaB activity distinct from the classic IkappaB pathway.