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

Authors

  • Anastasia Bolshakova,

    1. Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russian Federation
    2. Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
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  • Karl-Eric Magnusson,

    1. Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
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  • George Pinaev,

    1. Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russian Federation
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  • Olga Petukhova

    Corresponding author
    • Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russian Federation
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Corresponding author: e-mail: petukhova@yandex.ru

Abstract

NF-κB proteins belong to a family of ubiquitous transcription factors involved in a number of cellular responses. While the pathways of NF-κB 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-κB proteins RelA/p65 and p50 in relation to the inhibitor IκB-α, using fibronectin (FN) and epidermal growth factor (EGF) for environmental stimulation of epidermoid carcinoma A431 cells. We thus assessed the presence of NF-κB family proteins in the cytosol, membrane, nuclear and cytoskeletal fractions with a special attention to the cytoskeletal fraction to define whether NF-κB 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 IκB-α. 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 IκB-α in the cytosol, RelA/p65 and p50 in the nucleus and p50 and IκB-α 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-κB are dynamic and comprise active pools, whereas the cytoplasmic is more constant and likely non-active due to the presence of IκB-α. Moreover, we discovered the existence of a dynamic, IκB-α-free pool of RelA/p65 associated with cytoskeletal fraction, what argues for a special regulatory role of the cytoskeleton in NF-κB stimulation.

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