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.

Fig. 1. CNAβ1 localizes to intracellular membranes via palmitoylation at two conserved cysteines unique to its C-terminal tail.

a Schematic of CNAβ isoforms. CNB and calmodulin binding domains (CNB and CaM); autoinhibitory domain, (AID, blue); LAVP autoinhibitory sequence (green); palmitoylated cysteines (red). b CNAβ1 C-terminal (a.a. 456-496) sequence alignment; autoinhibitory LAVP (green) and palmitoylated cysteines (red, C483 and C493), are boxed. c Representative immunoblot demonstrating subcellular fractionation of COS-7 cells transfected with FLAG-CNAβ2, -CNAβ1 (WT or cysteine mutants), or EFR3B-FLAG using anti-FLAG. GM130 and Gapdh define membrane and cytosol fractions, respectively (n = 4 independent experiments). d Quantification of four independent experiments as in c. Data show the mean ± SEM. n.s. not significant, ***p < 0.001: cytosol, WT vs C483S p = 0.0004; membrane, WT vs C483S p = 0.0004, WT vs C2S p = 0.0002; ****p < 0.0001 using two-way ANOVA with Holm-Sidaks multiple comparison tests. e Representative images of COS-7 cells expressing FLAG-tagged CNAβ2, CNAβ1 or CNAβ1^C2S with Venus-Rit (PM, green). Fixed cells immunostained with anti-FLAG (red) and anti-GM130 (blue). Scale bar = 15 µm. f Top graph: Co-localization of FLAG signal (as in e) with Golgi marker, GM130. Data show median of Pearson’s coefficients with 95% confidence intervals (CI), from ≥100 cells analyzed in three independent experiments (see Statistical Analysis). n.s not significant; ****p < 0.0001, using one-way ANOVA followed by Kruskal–Wallis test. Bottom graph: PM localization quantified as anti-FLAG signal intensity at cell periphery (defined in Supplementary Fig. 1d) over total cell intensity (see Methods section). Data show median with 95% CI from ≥70 cells imaged in three independent experiments. ****p < 0.0001 using one-way ANOVA followed by Kruskal–Wallis test. g Representative immunoblot of Acyl-PEG exchange performed on lysates of COS-7 cells transfected with FLAG-CNAβ2, FLAG -CNAβ1 (WT or cysteine mutants) or EFR3B-FLAG. The number of PEGylation (reflecting S-palmitoylation) events are indicated by asterisks. Arrowhead indicates non-specific antibody band. n ≥ 3 independent experiments for all constructs (see Statistical Analysis).

Fig. 2. CNAβ1 palmitoylation is dynamic: ABHD17A expression promotes CNAβ1 depalmitoylation and alters CNAβ1 subcellular localization.

a Schematic diagram of the pulse-chase experiment using analogs of palmitate (17-ODYA) and methionine (L-AHA) coupled to CLICK chemistry used in this study. b Pulse-chase analysis of palmitate turnover on FLAG-CNAβ1 by dual-click chemistry as described in a in the presence of DMSO or pan-depalmitoylase inhibitor Palm B. Representative in-gel fluorescence scans showing dual detection of 17-ODYA and L-AHA using Alexa Fluor 647 and Alexa Fluor 488, respectively. c Time course of FLAG-CNAβ1 depalmitoylation in DMSO- and Palm B-treated cells after normalizing 17-ODYA to L-AHA signals at each chase time. Data shown are mean of each time point from two independent experiments. d Analysis of GFP-CNAβ1 palmitoylation co-expressed with vector, ABHD17A-FLAG (WT or S190A) or FLAG-APT2, using metabolic labeling with 17-ODYA. Representative immunoblot illustrates total CNAβ1 using anti-GFP and 17-ODYA detected using streptavidin following CLICK chemistry with Biotin-Azide. Anti-FLAG shows amount of ABHD17A and APT2 expression (n = 4 independent experiments). e GFP-CNAβ1 palmitoylation (as in d) is quantified by the streptavidin signal (17-ODYA)/total protein signal (GFP) and normalized to vector control. Data are mean ± SEM. (n = 4 independent experiments) n.s. not significant, ****p < 0.0001 using one-way ANOVA with Dunnett’s multiple comparison tests. f Representative images of fixed, COS-7 cells co-expressing GFP-CNAβ1 with vector, ABHD17A-FLAG (WT or S190A) immunostained with anti-FLAG and anti-GM130 (Golgi). Scale bar = 15 µm. g Images (as in f) quantified as GFP signal at the PM relative to total GFP signal intensity; data show median with 95% confidence intervals. ≥75 cells quantified per condition from four independent experiments. (see Statistical Analysis). n.s. not significant, ****p < 0.0001 using one-way ANOVA followed by Kruskal–Wallis test.

Fig. 3. CNAβ1-enriched interactors are membrane-associated and include all PI4KA complex members.

a Schematic of experimental plan for AP-MS analyses (created using BioRender.com). b Dotplot of AP-MS results including CNAβ1-enriched interactors (those with spectral counts $\ge$ 1.5x more for CNAβ1 than other baits). Node edge color corresponds to bayesian false discovery rate (BFDR), node size displays prey abundance and node darkness represents number of spectral counts. Full results reported in Supplementary Fig. 3c and Supplementary Data 1. c Cartoon representation of the structural organization of the phosphatidylinositol 4-kinase complex containing PI4KA (gray), FAM126A (orange), TTC7B (green) and EFR3B (pink). PI Phosphatidylinositol (white), PI4P phosphatidylinositol 4-phosphate (purple). d Immunoblot analysis of anti-GFP immunoprecipitates from inducible Flp-In-T-REx cells expressing GFP-CNAβ2, CNAβ1, or CNAβ1^C2S, transfected with EFR3B-HA, TTC7B-MYC, FLAG-FAM126A, and GFP-PI4KA. (n = 4 independent experiments) e Amount of GFP-CNAβ2 and GFP-CNAβ1^C2S co-purified with EFR3B-HA, quantified as bound GFP signal/bound HA signal normalized to input. Data are mean ± SEM (n = 4 independent experiments). n.s. not significant (p = 0.7), ***p = 0.0007, ****p < 0.0001 using unpaired, two-tailed t-test.

Fig. 4. CN-PI4KA complex interactions include a PxIxIT motif in FAM126A.

a FAM126A schematic showing PxIxIT motif (PSISIT, bold)-containing peptide and mutations (ASASAA, red); phosphorylated residues (gray circles) including Ser 485 (red circle). b Representative immunoblot showing proximity-dependent biotinylation analysis of the expressed PI4KA complex containing FLAG-FAM126A (WT or ASASAA) with MYC-BirA*-CNAβ1 in HeLa cells; arrow indicates uncut P2A protein (n = 3 independent experiments). c Biotinylation of each (as in b) quantified as respective bound signal/MYC bound signal normalized to respective signal/Actin signal in inputs. Data show mean ± SEM (n = 3 independent experiments). n.s. not significant, *p = 0.0037, **p = 0.0014, ***p = 0.000262 for PI4KA, ***p = 0.000886 for TTC7B using multiple unpaired t-tests using Sidak Method. d, e HDX data for CN (CNA/CNB) and the PI4KA/TTC7B/FAM126A trimer. (n = 3 independent replicates) d Sum of the differences in the number of deuterons incorporated for all analyzed peptides over the HDX time course shown for proteins that differ significantly in apo vs. complex state. Peptides with significant HDX (>5%,  >0.5 Da, and an unpaired, two-tailed t-test p < 0.01) (red); central residue of each peptide is plotted. e Deuterium incorporation differences between selected CNA and CNB peptides in the presence (red) or absence (black) of PI4KA/TTC7B/FAM126A trimer are shown. f Peptides with maximum significant HDX differences in CNA/CNB upon incubation with PI4KA trimer mapped onto the structure of CN (PDB: 6NUC), coloring explained in legend; PxIxIT and LxVP motifs of the NHE1 peptide (green). g Deuterium incorporation in FAM126A and PI4KA peptides displaying significantly decreased amide exchange in the presence (red) vs absence (black) of CN. All error bars in panels d–g show the S.D. (n = 3 independent replicates), with many being smaller than the size of the point. h Maximum significant differences in HDX observed at any timepoint for PI4KA/FAM126A/TTC7B trimer in the presence of CN mapped onto the structure of the PI4KA trimer (PDB: 6BQ1). Dotted lines: unresolved regions in the PI4KA/TTC7/FAM126A structure; colors show differences in exchange as indicated in the legend. i Schematic of PI4KA complex showing putative CN-interacting sites. For complete dataset see Supplementary Fig. 4d, e, Supplementary Table 1 and Source Data file.

Fig. 5. FAM126A is a CN substrate.

a Representative immunoblot showing electrophoreticmobility shifts observed for FLAG-FAM126A when expressed in HeLa cells. Lysates of cells expressing FLAG-FAM126A (WT, S485A, ASASAA, or ASASAA+S485A) in the presence or absence of EFR3B-HA/ TTC7B-MYC were analyzed using anti-MYC, anti-HA, anti-FLAG (red bands), and phospho-specific pFAM126A S485A (green bands) antibodies. PI and PII phosphorylated states, deP dephoshorylated state (n = 3 independent experiments). b Representative immunoblot showing analysis of FLAG FAM126A (WT or ASASAA) phosphorylation status in HeLa cells co-expressing FLAG-FAM126A, TTC7B-MYC, and EFR3B-HA across indicated treatments: DMSO (vehicle), FK506 (FK, CN-inhibitor), PMA (PKC activator), BIM (PKC inhibitor) using anti-FLAG (red), anti-HA and anti-pFAM126A S485A (green) antibodies. PKC activation was assessed by phosphorylation of the downstream substrate, ERK using anti-p44/42 ERK 1/2 antibody. Arrows denote non-specific antibody background. (n = 5 independent experiments) c FAM126A phosphorylation at Ser485 (from b) was quantified as the ratio of total pFAM126A S485 signal/ total FLAG signal relative to DMSO-treated FLAG-FAM126A WT signal ratio. Data are mean ± SEM (n = 5 independent experiments). **p = 0.0081, ***p < 0.001 (p = 0.0002 for DMSO vs FK506; p = 0.0005 for DMSO vs PMA + FK), ****p < 0.0001, n.s. (non-significant): p = 0.53 for DMSO vs PMA; p > 0.99 for the rest, calculated using one-way ANOVAs with Dunnett’s multiple comparison tests.

Fig. 6. CN regulates PI4P synthesis by the PI4KA complex.

a Representative immunoblot showing analysis of the PI4KA complex components in anti-HA immunoprecipitates of HeLa cells expressing GFP-PI4KA, EFR3B-HA/TTC7B-MYC with WT or ASASAA mutant FLAG-FAM126A (n = 3 independent experiments). Arrow points to the uncut P2A form. Arrowhead denotes non-specific antibody bands. b Co-purification of each component with EFR3B-HA (from a) is quantified as respective signals/EFR3B-HA signal in bound fractions normalized to respective signals/Actin signal in input fractions. Data are the mean ± SEM (n = 3 independent experiments). n.s. not significant using multiple, unpaired, two-tailed t-tests. c Cartoon representation of the BRET pair used in experiments shown in d. PI4P binding domain of the Legionella SidM protein (SidM-P4M, gray) attached to Renilla Luciferase (orange) as the donor and Venus (green), targeted to the PM using the first 10 amino acids of Lck, L10, as the acceptor. d Normalized BRET ratios reflecting changes in PM PI4P levels in response of carbachol stimulation (10⁻⁷ M) in HEK293T cells transiently expressing muscarinic receptor, M₃R, pre-treated with DMSO (blue), or CN inhibitors (red): FK506 (1 µM) (left) and Cyclosporin A, CsA (10 µM) (right) for 1 hr. Data shown are mean ± SD (n = 4 independent experiments). ${}^{**}$p = 0.0063, ${}^{***}$p = 0.0004 using Kolmogorov–Smirnov test.

Fig. 7. Model for CNAβ1 mediated regulation of the PI4KA complex that promotes PI4P synthesis at the PM during GPCR signaling.

Ligand binding and subsequent PLC-mediated cleavage of PI(4,5)P₂ generates DAG and IP3, which causes Ca²⁺ release from the ER. This increase in intracellular Ca²⁺ activates CN (CNAβ1), and PKC, which in turn regulate the PI4KA complex at the PM to promote PI4P and PI(4,5)P₂ replenishment. See text for details. Schematic generated using BioRender.com.