Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling

Short linear motifs (SLiMs) drive 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)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo 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 transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks. [Display omitted] •Global calcineurin signaling in humans revealed through systematic substrate mapping•Discovery of calcineurin-binding sequences enables robust in silico SLiM predictions•BioID uncovers SLiM-dependent calcineurin proximity to nuclear pores and centrosomes•Calcineurin dephosphorylates nuclear pore proteins and regulates transport in vivo Wigington, Roy, et al. elucidate a signaling network for the Ca2+-activated phosphatase calcineurin by systematically uncovering calcineurin-binding SLiMs throughout the human proteome. Using in vitro, in silico, and in vivo methods, they discover multiple calcineurin substrates, including Notch1 and several nucleoporins, leading to the unexpected finding that calcineurin regulates nuclear transport.