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

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.

Polypyrimidine tract binding protein 1 regulates the activation of mouse CD8 T cells

“…In addition, Ppp3cb has an alternative polyadenylation site in intron‐12. This generates an extended exon‐12 (transcript Ppp3cb‐206 ) encoding a shorter protein termed Aβ1 with an alternaitve C‐terminus with distinctive localization and function and the absence of an inhibitory domain present in the Aβ2 variant [16, 17]. Previous work had indicated this polyadenylation event can be regulated by muscleblind like splicing regulator 1 [18].…”

Calcineurin associates with centrosomes and regulates cilia length maintenance

“…These proteins are enriched for protein-protein interactions (PPI enrichment p-value = 4.02e-05, Fig. S1G) and include several known CN interactors and substrates: AKAP5 (Dell’Acqua et al, 2002), PI4KA and FAM126A (Ulengin-Talkish et al, 2021), PHKA1 (Ingebritsen and Cohen, 1983) and RCAN1 (Mehta et al, 2009). The majority of CN-proximal proteins were PxIxIT-dependent (35/41, Log 2 spectral counts with CNAα WT /CNAα NIRmut ≥ 0.5 for at least one condition), and 17 of these contained a predicted CN-specific SLiM (Figs 1B, S1F and Table S1).…”

“…In silico identification of PxIxIT and LxVP motifs revealed hundreds of putative substrates in humans (Brauer et al, 2019; Sheftic et al, 2016; Wigington et al, 2020). Experimentally, weak affinity SLiM-dependent CN interactions have been captured using proximity-dependent biotinylation coupled to mass spectrometry (PDB-MS) (Ulengin-Talkish et al, 2021; Wigington et al, 2020). Fusion of either WT or mutant CNAs (defective for PxIxIT or LxVP binding) to the promiscuous biotin ligase, BirA*, identified CN-proximal proteins.…”

Polypyrimidine Tract Binding Protein 1 regulates the activation of mouse CD8 T cells

“…In addition, Ppp3cb has an alternative polyadenylation site in intron-12. This generates an extended exon-12 (transcript Ppp3cb-206 ) encoding a shorter protein termed Aβ1 with an alternaitve C-terminus with distinctive localisation and function and the absence of an inhibitory domain present in the Aβ2 variant [16],[17]. Previous work had indicated this polyadenylation event can be can be regulated by Muscleblind Like Splicing Regulator 1 [18].…”