Uncovering Novel Substrates and Functions for the Calcineurin Phosphatase in Human Cells
Authors: Wigington Callie P , Roy Jagoree , Damle Nikhil P , Cho Shein Ei , Davey Norman , Ivarsson Ylva , Wong Cassandra , Gingras Anne‐Claude , Cyert Martha S 
Published: November 2023

Protein phosphatases play essential roles in every signaling pathway; however, systems‐level understanding of phosphatase signaling networks is lacking due to the inherent challenges associated with proteome‐wide identification of their substrates. Calcineurin (CN) is the conserved Ca2+/calmodulin‐activated protein phosphatase and target of the widely prescribed immunosuppressant drugs, FK506 and Cyclosporin A. CN is ubiquitously expressed and plays critical roles in the immune, nervous, skeletal and cardiovascular systems, as well as during development. However, only 50 substrates are currently attributed to this phosphatase. CN utilizes conserved docking surfaces to interact with substrates via Short Linear Motifs (SLiMs) termed PxIxIT and LxVP, which occur preferentially in intrinsically disordered domains and are challenging to identify due to sequence degeneracy and low affinity for CN. We are applying novel experimental and computational approaches to systematically identify CN‐interacting SLiMs within the human proteome with the goal of ultimately establishing the human CN signaling network. We directly identified novel CN‐binding sequences by performing unbiased peptide phage display selections with human CN using a library containing all predicted disordered regions in the human proteome. These SLiM sequences directly identified many novel candidate CN substrates, including the nucleoporin, NUP153. We have shown that NUP153 is directly dephosphorylated by CN in vitro and contains a conserved PxIxIT sequence that is required for interaction with CN in vivo. Furthermore, a NUP153 PxIxIT mutant is dephosphorylated less efficiently by CN in vitro and shows altered dephosphorylation in vivo. To further expand our identification of novel CN substrates, we employed two additional approaches: 1. A Position‐Specific Scoring Matrix (PSSM) generated using the novel CN‐binding SLiMs identified by phage display and 2. Proximity‐dependent biotinylation (BioID) followed by MS analysis in HEK293 cells. Both of these methods identified several nuclear pore components in addition to NUP153 as high confidence CN interactors, indicating a previously uncharacterized role for CN in regulating nuclear pore structure and/or function. In addition to nuclear pore proteins, these combined experimental and computational approaches have identified a host of novel candidate substrates for CN including ion channels, kinases, transcription factors and receptors. The significant overlap between these two datasets underscores the strength of these independent approaches in identifying novel CN substrates in human cells. Together, these studies suggest new points of cross‐talk between CN and other signaling pathways in human cells and will ultimately allow us to establish the first comprehensive signaling network for this critical Ca2+‐dependent regulator of human health.Support or Funding InformationF32GM120916‐01 to CP WigingtonR01GM119336‐01 to MS Cyert