Citation

  • Authors: Ulengin-Talkish I. et al.
  • Year: 2021
  • Journal: Nat Commun 12 6064
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell types:
    1. Name: COS-7
      Description: African green monkey kidney cells
      Known as: COS, COS7
    2. Name: HEK-293T
      Description: Human embryonic kidney Fibroblast
      Known as: HEK293T, 293T
    3. Name: HeLa
      Description: Human cervix epitheloid carcinoma cells

Method

HeLa, COS-7, and HEK 293T cells were grown at 37 °C in a 5% CO2 atmosphere in DMEM supplemented with 10% FBS. Cells were transfected as indicated in each experiment using jetOPTIMUS as per the manufacturer’s instructions. The Acyl-RAC protocol was performed as described previously with minor changes. In brief, COS-7 cells were seeded on 60 mm plates and transfected at 70% confluency with FLAG-CNAβ2, FLAG-CNAβ1 (WT or C483S, C493S, C483/C493S) or EFR3B-FLAG using JetOptimus. In all, 48 h following transfection, cells were harvested in ice-cold PBS and snap frozen in liquid nitrogen.

Abstract

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAβ1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a calcineurin-regulated signaling pathway which highlights the PI4KA complex as a regulatory target and reveals that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAβ1.

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