Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

Short linear motifs (SLiMs) form dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed 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, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including Notch1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins - structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear reporter, suggesting conserved NPC regulation by CN. 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.