title: Functional compartmentalisation of NF-kB-associated proteins  in A431 cells.
abstract: NF-kB proteins belong to a family of ubiquitous transcription factors involved in a number of cellular responses. While the pathways of NF-kB 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-kB proteins RelA/p65 and p50 in relation to the inhibitor IkB-a, using fibronectin (FN) and epidermal growth factor (EGF) for environmental stimulation of epidermoid carcinoma A431 cells. We thus assessed the presence of NF-kB family proteins in the cytosol, membrane, nuclear and cytoskeletal fractions with a special attention to the cytoskeletal fraction to define whether NFkB 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 IkB-a. 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 IkB-a in the cytosol, RelA/p65 and p50 in the nucleus and p50 and IkB-a in the membrane. Furthermore, EGF stimulation for 30 min resulted in a threefold accumulation of RelA/p65 in cytoskeletal fraction. Our results suggest that nuclear-, membrane- and cytoskeleton-associated NF-kB are dynamic and comprise active pools, whereas the cytoplasmic is more constant and likely non-active due to the presence of IkB-a. Moreover, we discovered the existence of a dynamic, IkB-a-free pool of RelA/p65 associated with cytoskeletal fraction, what argues for a special regulatory role of the cytoskeleton in NF-kB stimulation.
authors: Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Ave. 4, St. Petersburg 194064, Russian Federation
authors: Anastasia
authors: A
authors: Bolshakova
authors: Karl-Eric
authors: KE
authors: Magnusson
authors: George
authors: G
authors: Pinaev
authors: Olga
authors: O
authors: Petukhova
mesh_terms: D000199:Actins / Q000378:metabolism; D045744:Cell Line, Tumor; D003599:Cytoskeleton / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000502:physiology; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D021381:Protein Transport; D051996:Transcription Factor RelA / Q000378:metabolism*
chemical_list: D000199:Actins; D005353:Fibronectins; C498143:RELA protein, human; D051996:Transcription Factor RelA; D004815:Epidermal Growth Factor