(a) Jurkat T cells were cotransfected with 1500 ng Igκ₂-IFN-LUC, 200 ng pCSK-LacZ, and 100 ng of pc-myc-CARD11ΔID in the absence and presence of 1000 ng of pEBB-GAKIN-HA. The bars represent the mean values of triplicate samples; error bars indicate the standard deviation. (b) HEK293T cells were transfected with either 300 ng WT pc-myc-CARD11 or 400 ng pc-myc-CARD11ΔID in the absence or presence of 500 ng of pc-GAKIN-HA and anti-HA IPs were performed. (c) Jurkat T cells were stimulated with 50 ng/ml PMA and 1 μM ionomycin, lysed and immunoprecipitated with either anti-FLAG or anti-GAKIN antibodies. The arrow indicates the specific CARD11 band.

(a) Representative live cell imaging of conjugate formation between a Jurkat T cell stably expressing GAKIN-eGFP and mCherry-α–tubulin and a Raji B cell in the presence of SEE. The top panel shows the observed fluorescence prior to conjugate formation (Pre). The second panel from the top captures the first observed contact between T cell and B cell (t=0s), while subsequent panels, from top to bottom, represent images captured at 60 seconds and 600 seconds after first contact. The white arrowhead indicates the GAKIN-eGFP signal that colocalizes with the mCherry-α–tubulin signal at the MTOC, as indicated by the white arrow. The yellow arrowhead indicates GAKIN-eGFP signal at the distal pole. (b) Representative live cell imaging of conjugate formation between a Jurkat T cell stably expressing ΔMotor-GAKIN-eGFP and mCherry-α–tubulin and a Raji B cell in the presence of SEE. Timestamps are shown in the upper right corner of each panel. (c) The timepoint of initial recruitment to the APC:T cell contact was determined for conjugates expressing either GAKIN-eGFP or ΔMotor-GAKIN-eGFP. Each dot represents an individual conjugate. Lines indicate the average time of recruitment of 37.7±7.4 seconds (SEM, n=20) for GAKIN-eGFP and 202.5±40.8 seconds (SEM, n=15) for ΔMotor-GAKIN-eGFP. Scale bar, 10μm; Pre, before cell conjugation; s, seconds; SEM, standard error mean. See also Movies S1 and S2.

The kinetics of CARD11-mCherry and PKCθ-mCherry localization were quantitated for Jurkat T cells stably expressing either a GAKIN-specific (sh2b) hairpin, or a non-target control (NTsh) hairpin in the context of the GFPpLKO.1 lentivirus. A Jurkat T cell line expressing no hairpin was also analyzed as a control. All conjugates were formed with Raji B cells in the presence of SEE. The timepoint of initial CARD11-mCherry recruitment (a), the timepoint of CARD11 concentration at the distal synapse region (b), and the duration of CARD11 occupancy in the contact center (c) are shown for all three lines. (d) The timepoint of PKCθ-eGFP recruitment to the cSMAC region is shown for all three lines. (e) Representative live cell imaging of a conjugated Jurkat T cell co-expressing CARD11-mCherry and the control virus, GFPpLKO.1+NTsh. The top and bottom panels display the timepoint of initial T cell:B cell contact and the timepoint of CARD11 concentration at the distal region, respectively. (f) Representative live cell imaging of a conjugated T cell co-expressing CARD11-mCherry and the GAKIN-specific hairpin-expressing virus, GFPpLKO.1+sh2b. Panels selected as in (e). Scale bar, 10μm.

(a) HEK293T cells were transfected with 20 ng Igκ₂-IFN-LUC, 6 ng pCSK-LacZ and the indicated ng amounts of pEBB-GAKIN-HA in the absence or presence of 50 ng pc-mycCARD11. (b) Jurkat T cells were transfected with 1500 ng Igκ₂-IFN-LUC, 200 ng pCSK-LacZ in the absence or presence of 1000 ng of pEBB-GAKIN-HA and stimulated with SEE-pulsed Raji B cells or TNFα for 4.5 hours. (c) Stable Jurkat T cells lines expressing the control (siGFP) or GAKIN-specific (shRNA2b or 5a) hairpins were transfected with 1500 ng Igκ₂-IFN-LUC and 200 ng pCSK-LacZ and stimulated with anti-CD3/anti-CD28 for 4.5 hours. (d) Western blot analysis of samples analyzed in (c). (e) The shRNA2b stable Jurkat T cell line was transfected with 2000 ng Igκ₂-IFN-LUC, 200 ng pCSK-LacZ, and the indicated ng amounts of a hairpin-resistant expression vector for GAKIN (2bRGAKIN-HA), then stimulated with anti-CD3/anti-CD28 for 5.5 hours. The control siGFP Jurkat T cell line was assayed in parallel. (f) ELISA analysis of IL-2 production from the stable RNAi Jurkats lines, siGFP and shRNA2b, after anti-CD3/anti-CD28 coligation for 24 hours. (g) ELISA analysis of IL-2 production from human CD4⁺ T cells that were incubated for 72 hours with either an Accell siRNA duplex that targets GAKIN (siGAKIN-A) or a non-target control siRNA (NTsi) and then stimulated with anti-CD3/anti-CD28 coligation for 24 hours. (h) Western blot analysis of samples analyzed in (g). The bars represent the mean values of triplicate samples; error bars indicate the standard deviation. α, anti; ND, not detectable.

(a) Schematic of GAKIN. Numbers indicate amino acid positions and bracketed segments show the domain constructs used in (b). (b) HEK293T were transfected with 150 ng of pc-myc-CARD11ΔID and the indicated FLAG-tagged GAKIN domain expression constructs and anti-FLAG IPs were performed. The following DNA amounts (ng) were used for GAKIN constructs: Motor, 800; L1-FHA-L2, 300; MBS, 1000; TR1, 400 and TR2, 1850. (c) Jurkat T cells were transfected with 750 ng Igκ₂-IFN-LUC, 200 ng pCSK-LacZ and expression vectors for the indicated FLAG-tagged GAKIN domains in the absence or presence of 100 ng of pc-myc-CARD11ΔID as indicated. The following DNA amounts (ng) were used for FLAG-tagged GAKIN constructs: Motor, 600; L1-FHA-L2, 50; MBS, 1000; TR1, 600 and TR2, 1000. The bars represent the mean values of triplicate samples; error bars indicate the standard deviation. (d) Western analysis of HEK293T cells transfected with the same DNA concentrations of constructs transfected in (c). Equivalent numbers of transfected cells, as determined by β-gal activity, were evaluated for each construct. (e) HEK293T cells were transfected with 500 ng of pc-CARD11ΔID-FLAG and the indicated HA-tagged domain deletion constructs of GAKIN and anti-FLAG IPs were performed. The following DNA amounts (ng) were used for the GAKIN constructs: ΔMotor, 800; ΔFHA, 300; ΔMBS, 1000; and ΔCAP-Gly, 400. (f) HEK293T cells were cotransfected with 500 ng of pcDNA3-GAKIN-HA expression vector and the following amounts (ng) of each CARD11 variant and anti-HA IPs were performed: wt (150); ΔID (150); ΔIDΔCARD (1500); ΔIDΔL1 (175); ΔIDΔCC (125); ΔIDΔPDZ (225); ΔIDΔL3 (260); ΔIDΔSH3 (250); ΔIDΔL4 (150); ΔIDΔGUK (150). (g) HEK293T cells were transfected with expression vectors for myc-CARD11ΔID (10 ng), FLAG-Bcl10 (80-200 ng), and a titration of ΔMotor-GAKIN-HA (900 or 1800 ng) as indicated, and anti-FLAG IPs were performed. (h) HEK293T cells were transfected with expression vectors for myc-CARD11ΔID (10-12 ng), ΔMotor-GAKIN-HA (1600 ng), and FLAG-Bcl10 (80-140 ng), FLAG-TAK1 (200 ng), FLAG-TRAF6 (400 ng), FLAG-IKKγ (400 ng), or FLAG-Caspase-8 C360S (400 ng), as indicated, and anti-FLAG IPs were performed. (i) GAKIN-deficient (sh2b) or control (siGFP) Jurkat T cells were stimulated with 50 ng/ml PMA and 1 μM ionomycin, lysed and immunoprecipitated with anti-Bcl10 antibodies. (j) Quantitation of the amount of CARD11 present in the immunoprecipitates in (i), normalized to the amount of Bcl10 in the immunoprecipitates for each sample. CC, Coiled-coil; L1 and L2, linker 1 and 2; FHA, Forkhead associated; MBS, MAGUK-binding Stalk; TR, Tail region; CAP-Gly, Cytoskeletal-associated protein-glycine-rich.

(a) Representative live cell imaging of conjugate formation between a Jurkat T cell stably expressing CARD11-mCherry and PKCθ-eGFP and a Raji B cell in the presence of SEE. The top panel shows the observed fluorescence before conjugation (Pre). The middle and lower panels display the observed fluorescence at 60 and 80 seconds after initial B cell:T cell contact, respectively. (b) Line intensity graphs representing the average pixel intensity along a 5 pixel wide line drawn from B to E as shown in (a). (c) En face view (X-Z plane) of the IS for the conjugates shown in (a) at 60 and 80 seconds after initial B cell:T cell contact. Scale bar, 10μm.

(a) In unstimulated T cells, CARD11 is kept in a closed, inactive conformation in the cytoplasm through intramolecular interactions between the ID and the CARD and Coiled-coil domains. PKCθ, GAKIN, and the depicted pathway components are cytoplasmic and unbound to CARD11. (b) Upon TCR engagement, PKCθ clusters at the membrane and phosphorylates the ID of CARD11, leading to the conversion of CARD11 to an active conformation that can associate with several signaling proteins including TRAF6, IKKγ, and Bcl10. (c) Subsequent to the activation of CARD11, GAKIN and CARD11 interact in a manner that can compete Bcl10 off of a fraction of CARD11 molecules and that results in the redistribution of CARD11 away from the synapse center to a distal region. GAKIN is depicted here as directly transporting CARD11 along microtubules in a plus-end directed fashion, but other mechanisms by which GAKIN can influence CARD11 localization are possible. Since intramolecular interactions within GAKIN can regulate the association with CARD11, GAKIN is depicted as undergoing a conformational change that is influenced by TCR signaling.