- Authors: Wigington CP. et al.
- Year: 2020
- Journal: Mol Cell 79 342-358
- Applications: in vitro / DNA / jetOPTIMUS
- Cell type: HeLa
Description: Human cervix epitheloid carcinoma cells
- For GFP-peptide analyses, 7.5×10^5 HeLa cells were seeded onto coverslips in a 60-mm dish and transfected with 4 μg of plasmid DNA using JetOPTIMUS. - 24 hours after transfection, cells were subjected to Proximity ligation assay (PLA) analyses. - Due to very high transfection efficiency and GFP-peptide expression, all cells were considered for nuclear PLA puncta analyses.
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.