Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling

Mol Cell. 2020 Jul 16;79(2):342-358.e12. doi: 10.1016/j.molcel.2020.06.029. Epub 2020 Jul 8.


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.

Keywords: NPC; Notch1; ProP-PD; SLiM; Short Linear Motif; calcineurin; calcium signaling; centrosome; in silico motif discovery; nuclear pore complex; protein phosphatase; proteomic peptide phage display; proximity-dependent biotinylation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus
  • Amino Acid Motifs
  • Biotinylation
  • Calcineurin / metabolism*
  • Centrosome / metabolism
  • Computer Simulation
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Mass Spectrometry
  • Nuclear Pore Complex Proteins / metabolism*
  • Phosphoric Monoester Hydrolases / chemistry
  • Phosphoric Monoester Hydrolases / metabolism*
  • Phosphorylation
  • Protein Interaction Maps
  • Proteome / metabolism
  • Receptor, Notch1 / metabolism
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins / metabolism
  • Signal Transduction


  • NOTCH1 protein, human
  • NUP153 protein, human
  • Nuclear Pore Complex Proteins
  • Proteome
  • Receptor, Notch1
  • Saccharomyces cerevisiae Proteins
  • calcineurin phosphatase
  • Calcineurin
  • Phosphoric Monoester Hydrolases