To isolate and characterize a novel protein that interacts with nuclear factor of activated T cells (NFAT) and potentially regulates its activity, we screened a Jurkat cDNA library by using the NFAT regulatory domain as bait in the yeast two-hybrid system. RACK-1, a receptor for activated protein kinase C and a homologue of the G-protein b subunit, was identified as a NFAT-binding protein. Mammalian two hybrid tests in CV-1 cells and a coimmunoprecipitation assay confirmed protein-protein interaction between NFAT and RACK-1. In addition, overexpression of RACK-1 specifically suppressed transcriptional activation derived by NFAT, but not by NF-kB. These results demonstrate RACK-1 as a potent negative modulator of NFAT activation and suggest a novel mechanism in NFAT regulation.

Keywords: NFAT, RACK-1, Transcription, Transcription Factor, Yeast-Two Hybrid