STRUCTURAL ANALYSIS OF THE NFATC CYTOPLASMIC TO NUCLEAR TRANSLOCATION MECHANISM

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Many biological processes are exquisitely sensitive to the level of nuclear occupancy of NFATc proteins. The process of rapid nuclear import and export is regulated by an uncharacterized allosteric switch in the N-terminus of the protein, which regulates the alternate interaction between NFATc proteins and the nuclear import and export machinery. NFAT signaling is initiated by sustained low amplitude Ca2+ signals that activate the heterodimeric phosphatase calcineurin. Calcineurin binds directly to NFATc proteins through a conserved motif and dephosphorylates serines within the SP repeats and serine rich motifs (SRR and SP-repeats) in the N-terminus of NFATc family members. This dephosphorylation unmasks the nuclear localization sequence, allowing interaction with the importin complex and rapid cytoplasmic-to-nuclear translocation. In the nucleus NFAT proteins combine with other transcription factors (NFATn) to form specific transcription complexes. Dyrk1a and GSK3 then act sequentially to mediate export of the proteins from the nucleus. Rephosphorylation in the nucleus induces another conformational change, which exposes the nuclear export sequence (NES) and allows interaction with the nuclear export receptor Crm1. The mechanism of this allosteric switch in the N-terminus has not been defined structurally. The N-terminal region is predicted to be unfolded, while NMR spectra and SAXS data reveal a partially disordered protein. A number of intrinsically disordered regions in eukaryotic transcription factors have been reported to adopt structure upon binding to their partners. We are investigating NFAT in complex with a variety of binding partners in both the phosphorylated (cytoplasmic) and dephosphorylated (nuclear) forms to characterize the conformational change that occurs in the N-terminus of the protein by Small Angle X-ray Scattering.