Fig 2: NKD1 and NKD2 are targets of tankyrase. a Schematic diagram of NKD1/2 showing the N-terminal myristoylation site. The shaded box indicates the conserved EFX (EF-hand containing) domain that binds DVL. Alignment of the tankyrase-binding site in human NKD1 and NKD2. Identical amino acids are in black. b Immunoblot analysis showing NKD2 is stabilized in RNF146 siRNA-treated HEK293T cells. c, d Endogenous tankyrase and GFP-NKD2 coimmunoprecipitate, dependent on the NKD2 tankyrase-binding site. Immunoblot analysis of HEK293T cells transfected with GFP, GFP-NKD2 WT, or GFP-NKD2 Mut, immunoprecipitated with c tankyrase or d GFP antibodies, and probed with the indicated antibodies. e Immunoblot analysis showing that NKD1 is stabilized upon treatment of HepG2 cells with tankyrase inhibitor Ti8. f Immunoblot analysis showing that HA-NKD1 coimmunoprecitates with tankyrase. Immunoblot of HEK293T cells transfected with HA-NKD1, immunoprecipitated with control or TNKS IgG, and probed with the indicated antibodies

Fig 3: Localization of NKD2 in intracellular ORAI1+ vesicles(A) Top: representative confocal images of Jurkat T cells expressing GFP-ORAI1 and NKD2-mCherry (NKD2-mCh) under resting conditions (top two panels) or 10 min after dropping onto anti-CD3 Ab-coated coverslips. Images represent different depths within the cells, as indicated. Data are representative of similar observations from 10 (resting) and 16 (anti-CD3 Ab-stimulated) cells in two independent experiments. Bottom: scatterplot showing Pearson’s correlation coefficient for colocalization between ORAI1 and NKD2 under resting (13 cells) or anti-CD3 Ab-stimulated (13 cells) conditions from two independent experiments (left). Scatterplot showing percentage of Orai1+ NKD2+ vesicles among the Orai1+ vesicles under resting (16 cells) and anti-CD3 Ab-stimulated (16 cells) conditions from two independent experiments (right).(B) Representative confocal images of Jurkat T cells expressing GFP-ORAI1 and NKD2-mCh under resting conditions (top panels) or 30 min after stimulation with staphylococcal enterotoxin E (SEE)-pulsed Raji B cells loaded with CellTracer Blue CMAC (7-amino-4-chloromethylcoumarin) dye. Leftmost panel shows bright-field images overlaid with Raji B cells in blue in the bottom panel. Data are representative of similar observations from 10 (resting) and 14 (stimulated) cells in two independent experiments. Scale bar, 5 µm.(C) Representative TIRF images of a Jurkat T cell expressing GFP-ORAI1 and NKD2-mCh at early (30 s, top panels) and later (200 s, bottom panels) time points after dropping onto anti-CD3 Ab-coated coverslip. The line graph on the right shows normalized (mean ± SEM) fluorescence intensities from indicated cell numbers (N) depicting kinetics of accumulation of ORAI1 and NKD2 at the site of contact between the cell and the coverslip. Scale bar, 5 µm.

Fig 4: NKD2-mediated ORAI1+ vesicle trafficking is crucial for the effector function of primary T cells(A) Representative histograms showing levels of NKD2 protein in primary T cells transduced with lentiviral vectors encoding scrambled sgRNA (control), NKD2 sgRNA #1, and NKD2 sgRNA #3, together with those encoding Cas9. Cells were permeabilized and stained with anti-NKD2 Ab. The bar graph (right) shows average (±SEM) from four independent experiments.(B) Representative traces showing averaged SOCE from primary control (73 cells) and NKD2 KO (KO sgRNA #1, 82 cells; KO sgRNA #3, 71 cells) T cells after TCR stimulation using anti-CD3 Abs, followed by ionomycin treatment in the presence of external solution containing 2 mM Ca2+. Bar graph (right) shows averaged baseline-subtracted SOCE (±SEM) from three independent experiments.(C) Representative histograms showing levels of newly inserted PM-resident ORAI1 protein in primary control and NKD2 KO effector T cells after TCR stimulation. Cells were transduced as described above to induce deletion of NKD2 and stimulated and stained as described in Figure 3B for detection of newly inserted PM-resident ORAI1. Bar graph (right) shows average (±SEM) of pooled technical replicates from three independent donors.(D) Representative flow plots showing expression of IFN-?, IL-2, and TNF in control, NKD2 KO (sgRNA #1), and NKD2 KO (sgRNA #3) cells after re-stimulation with anti-CD3 and anti-CD28 Abs. Bar graph shows means ± SEM of pooled technical replicates from two independent experiments.(E) Representative histograms showing levels of T-bet in control, NKD2 KO (sgRNA #1), and NKD2 KO (sgRNA #3) cells. Bar graph shows means ± SEM of pooled technical replicates from two independent experiments.*p < 0.05; **p < 0.005; ***p < 0.0001.See also Figure S6.

Fig 5: NKD2 is involved in regulation of the Ca2+-NFAT signaling pathway in T cells(A) Representative immunoblot showing expression of NKD2 in lysates from HeLa cells, unstimulated Jurkat T cells, or those stimulated with PMA + ionomycin or anti-CD3 antibody (Ab) for 6 h. ß-Actin, loading control. Data are representative of two independent experiments.(B) Representative immunoblot showing expression of NKD2 in lysates of control or NKD2 knockout (KO) Jurkat T cells generated using two independent sgRNAs (#1 and #3). ß-Actin, loading control. Data are representative of two independent experiments.(C) Representative traces showing averaged SOCE from control (69 cells), NKD2 KO (KO sgRNA #1, 70 cells) Jurkat T cells, or NKD2 KO Jurkat T cells reconstituted for expression of NKD2 (KO sgRNA #1 + NKD2, 68 cells) after TCR stimulation using anti-CD3 antibodies, followed by ionomycin treatment in the presence of external solution containing 2 mM Ca2+. Bar graph (right) shows averaged baseline-subtracted SOCE (±SEM) from three independent experiments with NKD2 KO cells generated using two independent sgRNAs (#1 and #3).(D) Representative immunoblot of NFATc2 levels in nuclear extracts from wild-type (WT) and NKD2 KO (sgRNA #1) Jurkat T cells stimulated with 1 µM ionomycin for indicated times. A nuclear protein, Ku86, was used as a loading control. Bar graph shows the densitometric analysis of relative band intensities of nuclear NFATc2. Data show means ± SEM of pooled technical replicates from two independent nuclear extracts.(E) Representative immunoblot showing expression of NFATc1 in total cellular lysates from WT and NKD2 KO (sgRNA #1) Jurkat T cells stimulated with anti-CD3 and anti-CD28 antibodies for the indicated times. ß-Actin, loading control. Bar graph shows the densitometric analysis of relative band intensities of total NFATc1. Data show means ± SEM of pooled technical replicates from two independent cellular lysates.(F) Representative flow plots showing expression of IL-2 in control, NKD2 KO (sgRNA #1) Jurkat T cells, or NKD2 KO Jurkat T cells expressing NKD2 after stimulation with PMA + ionomycin for 16 h. Bar graph shows means ± SEM from three independent experiments.*p < 0.05; **p < 0.005; ***p < 0.0001.See also Figures S1, S2, and Table S1.