Functional compartmentalisation of NF-B-associated proteins in A431 cells

NF-B proteins belong to a family of ubiquitous transcription factors involved in a number of cellular responses. While the pathways of NF-B activation and input into the regulation of gene activity have been comprehensively investigated, its cytoplasmic functions are poorly understood. In this study we addressed effects of the compartmentalisation of NF-B proteins RelA/p65 and p50 in relation to the inhibitor IB-, using fibronectin (FN) and epidermal growth factor (EGF) for environmental stimulation of epidermoid carcinoma A431 cells. We thus assessed the presence of NF-B family proteins in the cytosol, membrane, nuclear and cytoskeletal fractions with a special attention to the cytoskeletal fraction to define whether NF-B was active or not. Sub-cellular fractionation demonstrated that the proportion of RelA/p65 differed in diverse sub-cellular fractions, and that the cytoskeleton harboured about 7% thereof. Neither the nuclear nor the cytoskeleton fraction did contain IB-. The cytoskeleton binding of RelA/p65 and p50 was further confirmed by co-localisation and electron microscopy data. During 30-min EGF stimulation similar dynamics were found for RelA/p65 and IB- in the cytosol, RelA/p65 and p50 in the nucleus and p50 and IB- in the membrane. Furthermore, EGF stimulation for 30min resulted in a threefold accumulation of RelA/p65 in cytoskeletal fraction. Our results suggest that nuclear-, membrane- and cytoskeleton-associated NF-B are dynamic and comprise active pools, whereas the cytoplasmic is more constant and likely non-active due to the presence of IB-. Moreover, we discovered the existence of a dynamic, IB--free pool of RelA/p65 associated with cytoskeletal fraction, what argues for a special regulatory role of the cytoskeleton in NF-B stimulation.