Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

Short linear motifs (SLiMs) form dynamic protein-protein interactions essential for signaling; however, sequence degeneracy and low binding affinities make them difficult to identify. In this study, we employed multiple, unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN), the Ca2+-regulated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in situ SLiM-dependent proximity labeling, as well as in silico modeling of motif determinants uncovered unanticipated CN interactors, including centrosomal and nuclear pore complex (NPC) proteins – structures where Ca2+ signaling is largely uncharacterized. Here, we show that CN binds to and dephosphorylates NPC proteins (nucleoporins) in vitro and promotes the accumulation of a nuclear transport reporter in HeLa cells. Furthermore, IP/MS analyses on HeLa cells treated with DMSO or the CN inhibitor, Cyclosporin A (CsA), reveal multiple CN-dependent phosphosites on key transport nucleoporins in vivo. Together, these data suggest that CN positively regulates nuclear transport by dephosphorylating key transport nucleoporins. In summary, the CN network assembled here provides a resource to investigate Ca2+ and CN signaling and the demonstrated synergy between experimental and computational methods establishes a blueprint for examining SLiM-based networks.