Dynamic equilibrium between calcineurin and kinase activities regulates the phosphorylation state and localization of the nuclear factor of activated T-cells.
The nuclear factor of activated T-cells (NFATp) is a phosphorylated transcription factor that resides in the cytoplasm of unactivated T-cells. T-cell activation results in the activation of the phosphatase calcineurin (CaN), which leads to the dephosphorylation and subsequent nuclear localization of NFATp. We have investigated the role of kinases in the phosphorylation state and subcellular localization of NFATp. The phosphorylation state and nuclear/cytoplasmic location of NFATp were determined in unstimulated murine HT-2 cells treated with a panel of kinase inhibitors. Two of the seven kinase inhibitors, staurosporine (St) and bisindolylmaleimide I (BI), resulted in the dephosphorylation and nuclear localization of NFATp. These St-induced effects were inhibited by pretreatment with FK506, indicating that CaN activity was required for the observed effects on NFATp. Treatment of cells with ionomycin resulted in NFATp dephosphorylation and nuclear localization. Removal of ionomycin from the cells resulted in the reappearance of phosphorylated NFATp in the cytosol. St and BI also inhibited the re-accumulation of NFATp in the cytoplasm and its re-phosphorylation after ionomycin removal. The re-accumulation of NFATp in the cytosol after ionomycin withdrawal was shown to be energy- and temperature-dependent. Taken together, these results suggest that in unstimulated cells NFATp is actively maintained in the cytoplasm by kinases acting in opposition to basal CaN activity.
Scott
J E
JE
Department of Cell Biology and Inflammation Research, Pharmacia & Upjohn, 7000 Portage Road, Kalamazoo, MI 49001, USA.
Ruff
V A
VA
Leach
K L
KL
D000818:Animals; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003080:Cold Temperature; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D008213:Lymphocyte Activation; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D019311:Staurosporine / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D016559:Tacrolimus / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*
D002148:Calmodulin-Binding Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007166:Immunosuppressive Agents; D007211:Indoles; D007476:Ionophores; D008301:Maleimides; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D014157:Transcription Factors; D015759:Ionomycin; D011494:Protein Kinases; D011493:Protein Kinase C; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D019311:Staurosporine; C088060:bisindolylmaleimide; D013755:Tetradecanoylphorbol Acetate; D016559:Tacrolimus