Integrated phosphoproteome and transcriptome data-driven analysis of major transcription-regulatory phosphosites in...

Nuclear factor interleukin-3-regulated protein (NFIL3) is a transcription factor involved in the regulation of circadian rhythms and immune crosstalk. The transcriptional repressive and activational roles that influence cytokine expressions and tumor microenvironment have made NFIL3 a therapeutic target for several diseases. While protein-level functions of NFIL3 are widely explored, the molecular details regarding phospho-signals regulating NFIL3 and their functional significance are underexplored. To interpret the phosphoregulatory patterns of NFIL3, we conducted an extensive literature survey for mass-spectrometry-based cellular phosphoproteomics datasets with representation of NFIL3 phosphosites. From the compiled list of 656 cellular profiles and 116 datasets with differential expression, Ser301 and Ser353 of NFIL3 were identified as the predominant phosphosites, accounting for the majority of NFIL3 phosphosignals. Further, we explored the coregulated phosphosites in the interacting partners and transcription factors associated with NFIL3. With our integrative phosphoproteomics and the IL-4 dependent transcriptome dataset analysis, we report Ser301 and Ser353 as positively co-occurring phosphorylations in NFIL3 that essentially regulate its transcriptional activity. Further, we also unveil Mitogen-activated protein kinase 14 (MAPK14) as a high-confidence upstream kinase that can phosphorylate Ser301. This study identifies novel coregulation expression patterns and highlights regulatory phosphosites, establishing the foundation for future exploration of the NFIL3-associated dark phosphoproteome.