Direct Demonstration of NFATpDephosphorylation and Nuclear Localization in Activated HT-2 Cells Using a Specific NFATpPolyclonal Antibody
Journal of Biological Chemistry
Nuclear factor of activated T cells (NFAT) regulates transcription of a number of cytokine genes, and NFAT DNA binding activity is stimulated following T cell activation. Several lines of evidence have suggested that NFAT is a substrate for calcineurin, a serine/threonine phosphatase. Using a polyclonal antibody to murine NFAT p , Western blot analysis of various mouse tissues demonstrated that the 110 -130-kDa NFAT p protein was highly expressed in thymus and spleen. Treatment of
... nt of immunoprecipitated NFAT p from untreated HT-2 cells with calcineurin resulted in the dephosphorylation of NFAT p , demonstrating that NFAT p is an in vitro substrate for calcineurin. NFAT p immunoprecipitated from 32 P-labeled HT-2 cells migrated as an approximately 120-kDa protein that was localized to the cytosol of the cells. Treatment of the cells with ionomycin resulted in a decrease in the molecular weight of NFAT p and a loss of 32 * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: The Upjohn Company, 7000 Portage Rd., Kalamazoo, MI 49001. Tel.: 616-385-5390; Fax: 616-384-9308. 1 The abbreviations used are: CsA, cyclosporin A; IL, interleukin; CaN, calcineurin; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; NFAT, nuclear factor of activated T cells.