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Protein phosphatase 2A interacts with and directly dephosphorylates RelA.

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.

SUBMITTER: Yang J

PROVIDER: S-EPMC2665282 | biostudies-literature | 2001 Dec

REPOSITORIES: biostudies-literature

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Protein phosphatase 2A interacts with and directly dephosphorylates RelA.

Yang J J Fan G H GH Wadzinski B E BE Sakurai H H Richmond A A

The Journal of biological chemistry 20011008 51

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 fo ...[more]

PMID: 11591705

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Project description:BackgroundInvadopodia are actin-based cell-membrane protrusions associated with the extracellular matrix degradation accompanying cancer invasion. The elucidation of the molecular mechanisms leading to invadopodia formation and activity is central for the prevention of tumor spreading and growth. Protein tyrosine kinases such as Src are known to regulate invadopodia assembly, little is however known on the role of protein tyrosine phosphatases in this process. Among these enzymes, we have selected the tyrosine phosphatase Shp1 to investigate its potential role in invadopodia assembly, due to its involvement in cancer development.MethodsCo-immunoprecipitation and immunofluorescence studies were employed to identify novel substrate/s of Shp1AQ controlling invadopodia activity. The phosphorylation level of cortactin, the Shp1 substrate identified in this study, was assessed by immunoprecipitation, in vitro phosphatase and western blot assays. Short interference RNA and a catalytically-dead mutant of Shp1 expressed in A375MM melanoma cells were used to evaluate the role of the specific Shp1-mediated dephosphorylation of cortactin. The anti-invasive proprieties of glycerophosphoinositol, that directly binds and regulates Shp1, were investigated by extracellular matrix degradation assays and in vivo mouse model of metastasis.ResultsThe data show that Shp1 was recruited to invadopodia and promoted the dephosphorylation of cortactin at tyrosine 421, leading to an attenuated capacity of melanoma cancer cells to degrade the extracellular matrix. Controls included the use of short interference RNA and catalytically-dead mutant that prevented the dephosphorylation of cortactin and hence the decrease the extracellular matrix degradation by melanoma cells. In addition, the phosphoinositide metabolite glycerophosphoinositol facilitated the localization of Shp1 at invadopodia hence promoting cortactin dephosphorylation. This impaired invadopodia function and tumor dissemination both in vitro and in an in vivo model of melanomas.ConclusionThe main finding here reported is that cortactin is a specific substrate of the tyrosine phosphatase Shp1 and that its phosphorylation/dephosphorylation affects invadopodia formation and, as a consequence, the ability of melanoma cells to invade the extracellular matrix. Shp1 can thus be considered as a regulator of melanoma cell invasiveness and a potential target for antimetastatic drugs. Video abstract.

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Protein phosphatase 2A interacts with and directly dephosphorylates RelA.

Publications

Protein phosphatase 2A interacts with and directly dephosphorylates RelA.

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