Direct demonstration of NFATp dephosphorylation and nuclear localization in activated HT-2 cells using a specific NFATp polyclonal antibody.
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 NFATp, Western blot analysis of various mouse tissues demonstrated that the 110-130-kDa NFATp protein was highly expressed in thymus and spleen. Treatment of immunoprecipitated NFATp from untreated HT-2 cells with calcineurin resulted in the dephosphorylation of NFATp, demonstrating that NFATp is an in vitro substrate for calcineurin. NFATp immunoprecipitated from 32P-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 NFATp and a loss of 32P, consistent with NFATp dephosphorylation. The dephosphorylation of NFATp was accompanied by localization of the protein to the nuclear fraction. Both of these events were blocked by preincubation of the cells with FK506, a calcineurin inhibitor, consistent with the hypothesis that NFATp is a calcineurin substrate in cells.
Ruff
V A
VA
Department of Cell Biology and Inflammation Research, Upjohn Company, Kalamazoo, Michigan 49001, USA.
Leach
K L
KL
D000595:Amino Acid Sequence; D000818:Animals; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D013601:T-Lymphocytes / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010455:Peptides; D010750:Phosphoproteins; D014157:Transcription Factors; D019000:Phosphotyrosine; D014443:Tyrosine; D015759:Ionomycin; D016559:Tacrolimus