Two independent calcineurin-binding regions in the N-terminal domain of murine NF-ATx1 recruit calcineurin to murine NF-ATx1.
Intracellular calcium regulates events controlling nuclear translocation of nuclear factor of activated T cells (NF-AT). Calcium-dependent phosphatase calcineurin (CN) plays a central role in this process. Structural and functional analyses of the N-terminal domain of murine NF-ATx1, a member of the NF-AT family, have defined two distinct CN binding regions (CNBRs), CNBR1 and CNBR2, which are located in the region preceding the SP boxes of serine/proline-rich sequences and the region between the SP boxes and Rel similarity domain, respectively. The binding of murine NF-ATx1 (mNF-ATx1) to CN was abolished by deletion of these two regions, yet was unaffected by the individual deletion. In contrast, the nuclear translocation of mNF-ATx1 was much reduced when only CNBR2 was removed. Luciferase assay revealed that both regions are required for mNF-ATx1-dependent activation of the murine IL-2 promoter. Most importantly, recombinant CNBR2 bound CN with a higher affinity, and when expressed in Jurkat cells, it functioned as a dominant negative mutant that prevented the transcription driven by exogenous mNF-ATx1, probably by interfering with the function of CN. We propose that activation of mNF-ATx1 can be modulated through two distinct CN target regions. Our findings provide a new opportunity for pharmacological intervention with Ca2+-dependent signaling events.
Liu
J
J
Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Japan. jie.liu@dnax.org
Masuda
E S
ES
Tsuruta
L
L
Arai
N
N
Arai
K
K
D000818:Animals; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D001692:Biological Transport / Q000235:genetics / Q000276:immunology; D019703:Calcineurin / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics / Q000276:immunology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D007376:Interleukin-2 / Q000235:genetics; D051379:Mice; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D017384:Sequence Deletion; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015533:Transcriptional Activation / Q000276:immunology
D004268:DNA-Binding Proteins; D007376:Interleukin-2; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D019703:Calcineurin