Palmitoylation targets the calcineurin phosphatase to the phosphatidylinositol 4-kinase complex at the plasma membrane.

Nature communications (2021-10-20)
Idil Ulengin-Talkish, Matthew A H Parson, Meredith L Jenkins, Jagoree Roy, Alexis Z L Shih, Nicole St-Denis, Gergo Gulyas, Tamas Balla, Anne-Claude Gingras, Péter Várnai, Elizabeth Conibear, John E Burke, Martha S Cyert

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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Hydroxylamine solution, 50 wt. % in H2O, 99.999%
Tris(2-carboxyethyl)phosphine hydrochloride solution, 0.5 M, pH 7.0(aqueous solution; pH was adjusted with ammonium hydroxide)
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
Trypsin Singles, Proteomics Grade
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, 1 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4)
Anti-HA antibody, Mouse monoclonal, clone HA-7, purified from hybridoma cell culture