Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp.
BACKGROUND
NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp.


RESULTS
We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain.


CONCLUSIONS
We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.
Department of Chemistry and Biochemistry University of Colorado at Boulder, USA. loree.kim@u.washington.edu
L J
LJ
Kim
H A
HA
Ferguson
A G
AG
Seto
J A
JA
Goodrich
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010455:Peptides / Q000276:immunology / Q000378:metabolism / Q000502:physiology; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000502:physiology; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D015533:Transcriptional Activation / Q000235:genetics*
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D050778:NFATC Transcription Factors; C494006:NFATC2 protein, human; D009687:Nuclear Proteins; D010455:Peptides; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins; D014157:Transcription Factors