1. The Dynamic Distribution of CARD11 at the Immunological Synapse Is Regulated by the Inhibitory Kinesin GAKIN 
Rebecca L. Lamason, Abraham Kupfer, Joel L. Pomerantz 
Molecular Cell 
Volume 40, Issue 5, Pages (December 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1 GAKIN Inhibits CARD11 and Attenuates TCR Signaling to NF-κB
(A) HEK293T cells were transfected with 20 ng Igκ2-IFN-LUC, 6 ng pCSK-LacZ, and the indicated nanogram 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 and 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 hr.
(C) Stable Jurkat T cell 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 hr.
(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 nanogram amounts of a hairpin-resistant expression vector for GAKIN (2bRGAKIN-HA), then stimulated with anti-CD3/anti-CD28 for 5.5 hr. The control siGFP Jurkat T cell line was assayed in parallel.
(F) ELISA analysis of IL-2 production from the stable RNAi Jurkat lines siGFP and shRNA2b after anti-CD3/anti-CD28 coligation for 24 hr.
(G) ELISA analysis of IL-2 production from human CD4+ T cells that were incubated for 72 hr with either an Accell siRNA duplex that targets GAKIN (siGAKIN-A) or a nontarget control siRNA (NTsi) and then stimulated with anti-CD3/anti--CD28 coligation for 24 hr.
(H) Western blot analysis of samples analyzed in (G).
The bars indicate fold stimulation (A–C and E) or IL-2 concentration (F and G) and represent the mean values of triplicate samples; the error bars indicate the standard deviation. The following abbreviations are used: α, anti; ND, not detectable.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 2
GAKIN Inhibits CARD11 after ID Neutralization and Associates with CARD11 in a Signal-Inducible Manner
(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 indicate fold stimulation and 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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

4. Figure 3
GAKIN Associates with CARD11 through Multiple Domains, Requires the CARD and Coiled-Coil of CARD11 for Association, and Can Compete with Bcl10
(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, The bars indicate fold stimulation and represent the mean values of triplicate samples; the 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.
The following abbreviations are used: 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.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 GAKIN Dynamically Localizes to the IS
(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. Top: The observed fluorescence prior to conjugate formation (Pre). Second row: Capture of the first observed contact between T cell and B cell (t = 0 s); (third and fourth rows) images captured at 60 s and 600 s after first contact, respectively. The white arrowhead indicates the GAKIN-eGFP signal that colocalizes with the mCherry-α-tubulin signal at the MTOC, 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 time point 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 s (standard error of the mean [SEM], n = 20) for GAKIN-eGFP and ± 40.8 s (SEM, n = 15) for ΔMotor-GAKIN-eGFP.
The scale bars in (A) and (B) represent 10 μm. Pre indicates before cell conjugation. See also Movies S1 and S2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

6. Figure 5 Two Phases of CARD11 Localization at the IS
(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. Top: The observed fluorescence before conjugation (Pre). Middle and bottom: The observed fluorescence at 60 and 80 s after initial B cell-T cell contact, respectively. The scale bar represents 10 μm.
(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 s after initial B cell-T cell contact.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

7. Figure 6
GAKIN Regulates the Duration of CARD11 Occupancy in the IS Center
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 nontarget 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 time point of initial CARD11-mCherry recruitment (A), the time point 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 time point 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 coexpressing CARD11-mCherry and the control virus, GFPpLKO.1+NTsh. Top and bottom: The time point of initial T cell-B cell contact and the time point of CARD11 concentration at the distal region, respectively.
(F) Representative live-cell imaging of a conjugated T cell coexpressing CARD11-mCherry and the GAKIN-specific hairpin-expressing virus, GFPpLKO.1+sh2b. Panels selected as in (E).
The scale bars in (E) and (F) represent 10 μm.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

8. Figure 7 Schematic of GAKIN Action during TCR Signaling
(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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions