NF-ATp, a T lymphocyte DNA-binding protein that is a target for calcineurin and immunosuppressive drugs.
The nuclear factor of activated T cells (NF-AT) is essential for transcription of the interleukin-2 gene upon T cell activation. Here we use a technique involving elution and renaturation of proteins from SDS-acrylamide gels to identify a DNA-binding component of NF-AT (NF-ATp) that is present in hypotonic extracts of T cells prior to activation and appears in nuclear extracts when T cells are activated. NF-ATp is present in resting T cells predominantly in a form migrating with an apparent molecular weight of 110,000-140,000, while NF-ATp from nuclear extracts of activated T cells migrates with a lower apparent molecular weight (90,000-125,000). This difference is likely to reflect dephosphorylation of NF-ATp, since treatment of NF-ATp with calf intestinal phosphatase or the calcium- and calmodulin-dependent phosphatase calcineurin in vitro results in a similar decrease in its apparent molecular weight. We show that NF-ATp is dephosphorylated in cell lysates by a calcium-dependent process that is blocked by inclusion of EGTA or a specific peptide inhibitor of calcineurin in the cell lysis buffer. Moreover, dephosphorylation of NF-ATp in cell extracts is inhibited by prior treatment of T cells with the immunosuppressive drugs cyclosporin A or FK506, which inhibit the phosphatase activity of calcineurin when complexed with their specific binding proteins, cyclophilin and FK506-binding protein. This work identifies NF-ATp as a DNA-binding phosphoprotein and a target for the drug/immunophilin/calcineurin complexes thought to mediate the inhibition of interleukin-2 gene induction by cyclosporin A and FK506.
McCaffrey
P G
PG
Division of Tumor Virology, Dana Farber Cancer Institute, Boston, Massachusetts 02115.
Perrino
B A
BA
Soderling
T R
TR
Rao
A
A
D000591:Amino Acid Isomerases / Q000378:metabolism; D000818:Animals; D019703:Calcineurin; D002118:Calcium / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002999:Clone Cells; D016572:Cyclosporine / Q000378:metabolism / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D051379:Mice; D019696:Peptidylprolyl Isomerase; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism*; D016559:Tacrolimus / Q000378:metabolism / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins
D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D007166:Immunosuppressive Agents; D010750:Phosphoproteins; D004533:Egtazic Acid; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D000591:Amino Acid Isomerases; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase; D002118:Calcium; D016559:Tacrolimus