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NF-kappa B p100 (Lyt-10) is a component of H2TF1 and can function as an I kappa B-like molecule
R I Scheinman1, A A Beg, A S Baldwin
1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 27599.
Insights
Nuclear Factor kappa B (NF-kappa B) activity is regulated by p100, which acts as an inhibitor by sequestering Rel family members in the cytoplasm. This mechanism offers a novel pathway for controlling NF-kappa B-dependent gene expression distinct from the canonical I kappa B pathway.
Area of Science:
- Molecular Biology
- Immunology
- Cell Biology
Background:
- Nuclear Factor kappa B (NF-kappa B) is a crucial transcription factor regulating immune responses, inflammation, and cell growth.
- NF-kappa B activation typically involves its release from cytoplasmic inhibitors (I kappa B) and translocation to the nucleus.
- The Rel multigene family includes subunits like p50 and p52, derived from precursors p105 and p100, respectively.
Purpose of the Study:
- To investigate the role of NF-kappa B p100 in regulating NF-kappa B activity.
- To determine if p100 functions as an inhibitor of NF-kappa B signaling.
- To elucidate the mechanism by which p100 affects Rel family member localization and DNA binding.
Main Methods:
- Identification of p100 as a component of the H2TF1 DNA-binding activity.
- Cytoplasmic localization studies of p100 in HeLa cells using association assays with c-Rel, p50, and p65 (RelA).
- Transient-transfection assays to assess p100's effect on NF-kappa B p65-mediated reporter gene activation.
- Electrophoretic mobility shift assays (EMSA) and immunoblotting to measure nuclear and cytoplasmic DNA-binding and immunoreactivity of p65.
Main Results:
- p100 was found to be localized in the cytoplasm, associated with Rel family members including p65 (RelA).
- Transfection with p100 repressed p65's ability to activate a kappa B reporter construct.
- p100 expression led to decreased nuclear p65 DNA binding and immunoreactivity, with a concomitant increase in cytoplasmic p65 DNA binding and immunoreactivity.
Conclusions:
- p100 functions as an I kappa B-like molecule, sequestering Rel family members in the cytoplasm.
- Proteolytic processing of p100 to p52 represents a regulatory mechanism for NF-kappa B activity.
- This pathway provides a distinct mode of NF-kappa B regulation separate from the canonical I kappa B pathway.
Abstract:
NF-kappa B is an important transcription factor regulating expression of genes involved in immune function, inflammation, and cellular growth control. NF-kappa B 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-kappa B activity involves the release of the transcription factor from a cytoplasmic inhibitor termed I kappa B, allowing translocation of NF-kappa B to the nucleus. I kappa B contains multiple copies of the so-called ankyrin repeat, which are apparently necessary for its function. Subunits comprising NF-kappa B 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-kappa B 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-kappa B p65 to activate a kappa B-containing reporter construct. Transfection of p100 also results in a loss of nuclear p65 DNA binding to a kappa B 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 I kappa B-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-kappa B activity distinct from the classic I kappa B pathway.