Peptide affinity analysis of proteins that bind to an unstructured region containing the transactivating domain of the osmoprotective transcription factor NFAT5

Abstract

NFAT5 is a transcription factor originally identified because it is activated by hypertonicity and that activation increases expression of genes that protect against the adverse effects of the hypertonicity. However, its targets also include genes not obviously related to tonicity. The transactivating domain of NFAT5 is contained in its COOH-terminal region, which is predicted to be unstructured. Unstructured regions are common in transcription factors particularly in transactivating domains where they can bind co-regulatory proteins essential to their function. To identify potential binding partners of NFAT5 from either cytoplasmic or nuclear HEK293 cell extracts, we used peptide affinity chromatography followed by mass spectrometry. Peptide aptamer-baits consisted of overlapping 20 amino acid peptides within the predicted COOH-terminal unstructured region of NFAT5. We identify a total of 351 unique protein preys that associate with at least one COOH-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from cells incubated at various tonicities (NaCl varied). In addition to finding many proteins already known to associate with NFAT5, we found many new ones whose function suggest novel aspects of NFAT5 regulation, interaction, and function. Relatively few of the proteins pulled down by peptide baits from NFAT5 are generally involved in transcription, and most, therefore, are likely to be specifically related to the regulation of NFAT5 or its function. The novel associated proteins are involved with cancer, effects of hypertonicity on chromatin, development, splicing of mRNA, transcription, and vesicle trafficking.

NFAT5, which protects against hypertonicity, is activated by that stress via structuring of its intrinsically disordered domain

“…3C). HMGI-C has previously been shown to be present in N-terminal NFAT5 peptide affinity pulldown from nuclear extracts (20,22).…”

“…The NTD contains an auxiliary export region, involved in nuclear and cytoplasmic localization, a monopartite nuclear localization signal, and transcription activation function AD1 (20,62). The CTD contains transactivation domains AD2 and AD3 (18,19,22). Since several predictors of protein structure agreed that all these domains are ID, we tested the hypothesis that protective organic osmolytes and/or NaCl could promote their folding.…”

“…NFAT5 modulates cellular response to osmotic changes by enhancing the expression of target genes, including those responsible for synthesis and/or transport of multiple organic osmolytes, such that the intracellular concentration of compatible protective osmolytes is increased and compensates for increased extracellular tonicity (13)(14)(15). NFAT5 is a multidomain transcription factor (TF) in which the ID N-terminal domain (NTD) includes a tonicity-dependent auxiliary export region responsible for nuclear and cytoplasmic localization and AD1 (amino acids 1-76), one of NFAT5's three activation domains (16)(17)(18)(19)(20)(21)(22). NFAT5 ID C-terminal domain (CTD) contains two tonicity-dependent transactivation domains, AD2 and AD3.…”