Abstract
Nuclear factor-kappa B (NF-kappa B)/Rel transcription factors are key regulators of a variety of genes involved in inflammatory responses, growth, differentiation, apoptosis, and development. There are increasing lines of evidence that NF-kappa B/Rel activity is controlled to a great extent by its phosphorylation state. In this study, we demonstrated that RelA physically associated with protein phosphatase 2A (PP2A) subunit A (PR65). Both the N- and C-terminal regions of RelA were responsible for the PP2A binding. RelA co-immunoprecipitated with PP2A in melanocytes in the absence of stimulation, indicating that RelA forms a signaling complex with PP2A in the cells. RelA was dephosphorylated by a purified PP2A core enzyme, a heterodimer formed by the catalytic subunit of PP2A (PP2Ac) and PR65, in a concentration-dependent manner. Okadaic acid, an inhibitor of PP2A at lower concentration, increased the basal phosphorylation of RelA in melanocytes and blocked the dephosphorylation of RelA after interleukin-1 stimulation. Interestingly, PP2A immunoprecipitated from melanocytes was able to dephosphorylate RelA, whereas PP2A immunoprecipitated from melanoma cell lines exhibited decreased capacity to dephosphorylate RelA in vitro. Moreover, in melanoma cells in which I kappa B kinase activity was inhibited by sulindac to a similar level as in melanocytes, the phosphorylation state of RelA and the relative NF-kappa B activity were still higher than those in normal melanocytes. These data suggest that the constitutive activation of RelA in melanoma cells (Yang, J., and Richmond, A. (2001) Cancer Res. 61, 4901-4909) could be due, at least in part, to the deficiency of PP2A, which exhibits decreased dephosphorylation of NF-kappa B/RelA.
Highlights
Nuclear factor-B (NF-B)1/Rel transcription factors are key regulators of immune and inflammatory responses [1,2,3]
Treatment of Jurkat cells with okadaic acid (OA), a specific inhibitor of PP1 and PP2A, induces translocation of NF-B to the nucleus and activation of NF-B [31]. These findings suggest a potential involvement of PP2A in the regulation of NF-B signaling
To assess which subunit of the heterodimeric PP2A core enzyme binds directly to RelA, GST fusion proteins of PR65 and PP2Ac were incubated with the cell lysate from Sk Mel 5 melanoma cells, and coprecipitated RelA was detected by Western blotting
Summary
We demonstrated that RelA physically associated with protein phosphatase 2A (PP2A) subunit A (PR65). In melanoma cells in which IB kinase activity was inhibited by sulindac to a similar level as in melanocytes, the phosphorylation state of RelA and the relative NF-B activity were still higher than those in normal melanocytes. PP1 and PP2A are widely expressed in mammalian cells and are involved in the regulation of signaling pathways by a mechanism of phosphorylation/dephosphorylation with a variety of protein kinases [24]. Treatment of Jurkat cells with okadaic acid (OA), a specific inhibitor of PP1 and PP2A, induces translocation of NF-B to the nucleus and activation of NF-B [31] These findings suggest a potential involvement of PP2A in the regulation of NF-B signaling. The activity of PP2A in melanoma cells is lower than that in melanocytes, which partially contributes to the higher phosphorylation state of RelA and the increased NF-B activity in the melanoma cells
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