S-acylation-dependent membrane microdomain localization of the regulatory Kvβ2.1 subunit

Figures

• 

  Fig. 1. Cytoplasmic Kvβ1.1 and Kvβ2.1 also target the plasma membrane. HEK 293 cells were transfected with Kvβ1.1CFP and Kvβ2.1CFP, and the cellular expression was studied. A Kvβ1.1 (a-c) and Kvβ2.1 (e–g) distribution. (Ad and Ah) Pixel-by-pixel analysis of Kvβs and the membrane marker. Histograms derived from arrow sections in c and g. a and e, Kvβ in red; b and f, membrane in blue; c and g, merge; purple indicates colocalization. Scale bars represent 10 µm. B Kvβ1.1CFP and Kvβ2.1CFP expression in whole lysates from HEK cells. C Purified membranes from HEK cells expressing Kvβ1.1CFP and Kvβ2.1CFP. SN, supernatants; Conc, concentrated membranes. D, E Coimmunoprecipitation of Kvβ1.1CFP and Kvβ2.1CFP with β-actin. D Coimmunoprecipitation of Kvβ1.1CFP with β-actin. E Absence of coimmunoprecipitation of Kvβ2.1CFP with β-actin. Top panels: immunoblot (IB) against CFP (Kvβs). Bottom panels: immunoblot (IB) against β-actin. SM, starting material. SN+ , supernatants in presence of antibody. SN-, supernatants in absence of antibody. IP+ , immunoprecipitation in the presence of antibody. IP-, immunoprecipitation in the absence of antibody

• 

  Fig. 2. Kvβ expression in cell-unroofing preparations (CUPs) and lipid raft microdomains. A–C Kvβ1.1CFP and the membrane marker in CUPs. D–F Kvβ2.1CFP and the membrane marker in CUPs. A and D Kvβs-CFP in red; B and E Membrane marker in blue; C and F merged channel; purple indicates colocalization. Scale bars represent 10 µm. G and H Lipid raft isolation of HEK cells transfected with Kvβ1.1CFP G and Kvβ2.1CFP H. Sequential sucrose fractions were extracted from the top (1, lowest density) to the bottom (12, highest density) of the tube and the Kvβ expression analyzed by western blot

• 

  Fig. 3. Palmitoylation of Kvβ1.1 and Kvβ2.1. A Amino acid sequence alignment of murine Kvβ1.1 and Kvβ2.1. The UniProt (https://www.uniprot.org/) identification number is indicated in brackets. Sequences were analyzed for cysteines. Cysteines are in red. Conserved cysteines are highlighted in yellow. B, C ABE palmitoilation assay on HEK cells transfected with Kvβ1.1CFP (B) and Kvβ2.1CFP (C). + HA, presence of hydroxylamine. −HA, absence of hydroxylamine. Top panels, immunoblot against Kvβ1.1 and Kvβ2.1. Bottom panels, immunoblot against flotillin. SM, starting material. PD, pulldown of the palmitoylated proteins. D Palmitoylated Kvβ2.1 targets the cell surface. Proximity-ligation-assay (PLA). Palmitic acid 15-hexadecynoic acid was used for Alk-C16 protein palmitoylation. (a) Total Kvβ2.1CFP in green; (b) membrane marker staining in blue; (c) Kvβ2.1CFP Alk-C16 palmitoylation in red; (d) merge panel; (e) colocalization of total Kvβ2.1CFP, in green, with the membrane marker (Mb) in blue; (f) colocalization of Kvβ2.1CFP Alk-C16, in red, with the membrane marker (Mb) in blue. Arrowheads highlight Alk-C16 palmitoylation colocalizing with the cell surface in purple. Scale bars represent 10 µm…

• 

  Fig. 4. Palmitoylation-dependent lipid raft targeting of Kvβ2.1. A–D Lipid raft microdomains were isolated in the absence (A) or presence of (B) 2-bromopalmitate (2-BP), (C) H2O2 or both (D), and lipid raft fractions were analyzed for Kvβ2.1CFP by western blot. Lipid raft fractions were sequentially extracted from the top (1, lowest density and highest buoyancy) to the bottom (12, highest density and lowest buoyancy) of the tube. (E) Quantification of the expression of Kvβ2.1CFP in low-density fractions, defined by the presence of caveolin, relativized by the total amount of expression. A.U., arbitrary units. **p < 0.01; ***p < 0.001 vs. no additions (Student’s t test)

• 

  Fig. 5. Human T lymphocytes express palmitoylated Kvβ2.1, which targets lipid raft microdomains and concentrates in the IS during the immunological response. (A) Human CD4 + lymphocytes express Kvβ2 and Kv1.3. T lymphocytes from 4 different donors (D1-4) were obtained and analyzed. (B) Kvβ2 undergoes palmitoylation in human CD4 + T-cells. SN, supernatant in the absence (−) or the presence (+) of HA. SM, starting material in the absence (−) or the presence (+) of HA. PD, pulldown of palmitoylated proteins in the absence (−) or presence (+) of HA. C Human Jurkat T lymphocytes express Kvβ2 and Kv1.3. D Kvβ2 undergoes palmitoylation in human Jurkat T cells. SM, starting material in the presence of HA. PD, pulldown of palmitoylated proteins in the presence of HA. E Proximity ligation assay (PLA) in Jurkat lymphocytes. Palmitic Alk-C16 Kvβ2 palmitoylation. Total Kvβ2 in green; Alk-C16 Kvβ2 palmitoylation in red; merged panel highlights Alk-C16 palmitoylation at the cell surface. F Kvβ2 targets lipid rafts in Jukat cells. Lipid raft fractions were sequentially extracted from the top (1, lowest density and highest buoyancy) to the bottom (12, highest density and lowest buoyancy) of the tube.…

• 

  Fig. 6. Cellular distribution of different Kvβ2.1 mutants. A Cartoon of the crystal structure of murine Kvβ2.1 (UniProtKB—P62482), highlighting 5 cysteines in blue. B Representative cartoon of Kvβ2.1 mutants. While Ser substitutions are indicated in red, Ala mutations are indicated in green. C Cellular staining of Kvβ2.1 WT (a-d), C(52–212−248)S (e–h), C(52–212−248–301)S (i-l) and Kvβ2.1ClessA (m-p). Panels d, h, l and p show the pixel-by-pixel analysis of white arrow sections in c, g, k and o, respectively. Scale bars represent 10 µm. D Quantification of membrane colocalization using Mander’s overlap coefficient (MOC). **p < 0.01; ***p < 0.001 (Student’s t test) vs. WT. Values are mean ± SE of 20–30 cells

• 

  Fig. 7. Palmitoylation of Kvβ2.1 WT and mutants. HEK 293 cells were transfected with Kvβ2.1CFP WT and several cysteine mutants (C(51–212−248)S, C(51–212−248–301)S and ClessA), and palmitoylation was analyzed. A Representative ABE experiment of Kvβ2.1 WT and C(51–212−248)S, C(51–212−248–301)S and ClessA mutants. Flotillin was the positive control. SM, starting material; PD, palmitoylated pull down. B Quantification of pulldowns normalized to starting materials. Values are mean ± SE of three independent experiments. **p < 0.01; ***p < 0.001 (Student’s t test) vs. WT. White bar, Kvβ2.1 WT; light gray bar, C(51–212−248)S; dark gray bar, C(51–212−248–301)S; black bar, Kvβ2.1ClessA. C, D Localization of Kvβ2.1 (C) and Kvβ2.1 ClessA (D) in lipid rafts. Lipid raft fractions were sequentially extracted from the top (1, low density and high buoyancy) to the bottom (12, high density and low buoyancy) of the tube. (E) Quantification of the abundance of Kvβ2.1CFP in low buoyant fractions, defined by caveolin, relativized by the total amount of expression. ***p < 0.001 (Student’s t test) vs. WT. Values are mean ± SE of three independent experiments. White bar, Kvβ2.1 WT; black bar, Kvβ2.1ClessA

• 

  Fig. 8. FBS-dependent proliferation increases Kvβ2.1 membrane and lipid raft targeting. HEK 293 cells were transfected with Kvβ2.1CFP. After 24 h of FBS deprivation, cells were further cultured for 24 h in the presence (A–D) or absence (E–H) of 10% FBS, and the cellular distribution of Kvβ2.1 was analyzed. A, E Kvβ2.1 CFP in red. B, F WGA membrane staining in blue. C, G merge, purple indicates colocalization. Scale bars represent 10 µm. D and H Pixel-by-pixel analysis of the arrow sections in C and G, respectively. I Quantification of membrane targeting using Mander’s overlap coefficient (MOC). *p < 0.05 vs. + FBS (Student’s t test). Values are mean of 20–30 cells. J, K Lipid raft localization of Kvβ2.1CFP in the presence (J) or absence (K) of FBS. (L) Quantification of Kvβ2.1CFP floatability. Expression of Kvβ2.1CFP in low buoyant fractions (defined by caveolin expression) relative to the total amount. **p < 0.01 vs. + FBS (Student’s t test). Values are mean of 4 independent experiments. Black bars, presence of FBS; white bars, absence of FBS

• 

  Fig. 9. Lipid raft microdomain expression of Kvβ2.1 in the presence of PMA. HEK 293 cells were transfected with Kvβ2.1CFP. Isolation of lipid rafts was performed after 1 μM PMA incubation for 30 min at 37 °C. A Kvβ2.1CFP expression in lipid raft fractions in the absence of PMA. B Kvβ2.1CFP expression in the presence of PMA. C, D HEK 293 cells were cotransfected with Kvβ2.1CFP and Myc-PSD95 in the absence (C) or presence (D) of PMA. Immunoblot against CFP shows Kvβ2.1CFP. Immunoblot against Myc indicates Myc-PSD95. E Quantification of the Kvβ2.1 floatability, indicated by caveolin expression, relativized to the total amount of expression. **p < 0.01 vs. Kvβ2.1 in the absence of PMA (Student’s t test). Black bar, Kvβ2.1 alone; white bar, Kvβ2.1 in the presence of PMA; dark gray bar, Kvβ2.1 in the presence of PSD95 but in the absence of PMA; light gray bar, Kvβ2.1 in the presence of PSD95 and PMA. Values are men ± SE of 4 independent experiments. F Kvβ2.1 does not interact with PSD95. Cells were cotransfected with Kvβ2.1CFP and Myc-PSD95 in the absence (−) or presence (+) of PMA. Total lysates were coimmunoprecipitated against Kvβ2.1CFP (IP: CFP) and immunoblotted (IB) against CFP (Kvβ2.1)…

• 

  Fig. 10. Cartoon representing the structural Kv1.3-associated proteins in the T-lymphocyte immunological synapse. The Kv1.3 channelosome merges a number of proteins modulating channel function at the IS during immunological synapses. PSD95 (also named synapse-associated protein 90; SAP90), which is encoded by the hDLG4 (discs large homolog 4) gene, stabilizes Kv1.3 at the IS. SAP97 (synapse-associated protein 97; hDlg1) plays similar roles. Therefore, SAP peptides bind to the PDZ domain in the C-terminus of Kv1.3, coupling p56lck to CD4. Kvβ2 links the N-terminus of the Kv1.3 channel to the ZIP1/2 protein, which may interact with several partners, such as p56lck and PKC. CD3 and Kv1.3 are in molecular proximity, and the channel interacts with β1-integrins. Our data indicate that ~ 10% of Kvβ2 targets to lipid rafts either associated or not with Kv1.3 and situate palmitoylated Kvβ2 (red sparkline) at the IS, independent of the Kv1.3 interaction, and stabilized by PSD95. Kvβ2 may link cellular metabolic activity and redox state with calcium signaling in lymphocytes. Kvβ2 also serves as a bridge with ZIP-1/2, which also links the complex to p56lck. Other proteins within IS are the T-cell…