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Results: 7

1.
Figure 4

Figure 4. The SKAP55 PH domain is required for TCR-dependent recruitment to β2 integrins. From: THE PLECKSTRIN HOMOLOGY DOMAIN IN THE SKAP55 ADAPTER PROTEIN DEFINES THE ABILITY OF THE ADAPTER PROTEIN ADAP TO REGULATE INTEGRIN FUNCTION AND NF-?B ACTIVATION.

(A) hCAR/DO11.10 (WT) CD4 T cell blasts were stimulated for the indicated times with anti-CD3/CD28. The cells were lysed and subjected to anti-β2 (CD18) integrin immunoprecipitation. Western blots were probed for ADAP, SKAP55 and for the β2 integrin. Whole cell lysate inputs (lower 2 panels) are shown to demonstrate ADAP and SKAP55 expression. (B,C) hCAR/DO11.10/ADAP−/− (ADAP KO) T cell blasts were transduced with the indicated adenovirus constructs and stimulated and immunoprecipitated for β2 integrin as in (A). Western blots were probed with antibodies against ADAP and SKAP55 to detect ADAP and SKAP55 (B) or the SKAP/ADAP chimera (C), and with antibodies against the β2 integrin to detect the immunoprecipitated CD18 integrin subunit. Whole cell lysate inputs (lower panels) are shown to demonstrate expression of ADAP, SKAP55, and the SKAP/ADAP chimera, and activation was confirmed by blotting for phosphoERK. Similar results were observed in 4 independent experiments.

Brandon J. Burbach, et al. J Immunol. ;186(11):6227-6237.
2.
Figure 1

Figure 1. Impaired conjugate efficiency in the absence of ADAP and SKAP55 interaction. From: THE PLECKSTRIN HOMOLOGY DOMAIN IN THE SKAP55 ADAPTER PROTEIN DEFINES THE ABILITY OF THE ADAPTER PROTEIN ADAP TO REGULATE INTEGRIN FUNCTION AND NF-?B ACTIVATION.

(A) Naïve hCAR/DO11.10 (WT) or hCAR/DO11.10/ADAP−/− (KO) CD4 T cells were transduced with control (Ctrl) Thy1.1 adenovirus or the indicated ADAP or SKAP55 expression construct(s). After 3d, the transduced cells were incubated with OVAp-pulsed splenic B cells and analyzed by flow cytometry for efficiency of T:APC conjugate formation as described in Material and Methods. (B) Intracellular staining for ADAP or SKAP55 was performed on the cells transduced in (A), and plotted versus Thy1.1 expression. Conjugates depicted in A were gated on the top one-third of the Thy1.1 gate to ensure that SKAP55 and/or ADAP levels were comparable to endogenous SKAP55 and/or ADAP expression in wild-type control cells. Results for both the conjugate assays and the intracellular staining are representative of at least 3 independent experiments.

Brandon J. Burbach, et al. J Immunol. ;186(11):6227-6237.
3.
Figure 6

Figure 6. The SKAP55 PH domain suppresses endogenous NF-κB activation. From: THE PLECKSTRIN HOMOLOGY DOMAIN IN THE SKAP55 ADAPTER PROTEIN DEFINES THE ABILITY OF THE ADAPTER PROTEIN ADAP TO REGULATE INTEGRIN FUNCTION AND NF-?B ACTIVATION.

(A) Naïve wild-type hCAR T cells were transduced with adenoviruses to overexpress SKAP55 (F-SK55), GFP-SKAP55 (GFP SK55), or GFP-SKAP55-R131M (GFP R131M). Upper 2 panels, Western blots were performed as in A and B to detect total IκBα or phosphorylated IκBα (p-IκB). Lower 2 panels, Western blots for SKAP55 and ADAP are depicted showing expression of endogenous SKAP55 (Endog. SK55), FLAG-SKAP55 (F-SKAP55), or GFP-SKAP55 (GFP-SKAP55) and equal expression of ADAP in all samples. Results are representative of 3 independent assays performed. (B) Quantification of total IκBα degradation following CD3/CD28 stimulation as described in A. Results are normalized to IκBα densitometry values from the unstimulated samples, and averaged between 3 independent experiments performed. Asterisk (*) indicates P < 0.05 compared to the wild-type control condition within each time point using Bonferroni’s multiple comparison test of the one-way ANOVA. n.s., P>0.05.

Brandon J. Burbach, et al. J Immunol. ;186(11):6227-6237.
4.
Figure 7

Figure 7. Model for ADAP and SKAP55 control of LFA-1 integrin activation and NF-κB activation. From: THE PLECKSTRIN HOMOLOGY DOMAIN IN THE SKAP55 ADAPTER PROTEIN DEFINES THE ABILITY OF THE ADAPTER PROTEIN ADAP TO REGULATE INTEGRIN FUNCTION AND NF-?B ACTIVATION.

(A) In wild-type T cells, TCR stimulation promotes ADAP phosphorylation (P) and activation of the constitutively associated ADAP:SKAP55 adapter module. Positively charged arginine 131 (R131) in the SKAP55 pleckstrin homology (PH) domain recruits and restricts the complex to negatively charged phosphoinositides (such as PIP3) in the plasma membrane. Downstream integrin activation components including Rap1 and a putative Rap1-GEF are provided through ADAP and the Rap1 effectors RIAM and/or RapL through SKAP55 are recruited to LFA-1 for promotion of integrin activation. Only free excess ADAP is available for concurrent assembly of the CARMA-1/Bcl10/Malt1 (CBM) complex, which initiates IκB phosphorylation/degradation and release of NFκB to the nucleus. (B) The SKAP/ADAP chimera is exclusively recruited to the integrin pathway. As there is no excess ADAP that is not associated with SKAP55 in ADAP−/− T cells expressing the chimera, no free ADAP is available for NF-κB activation. (C) In ADAP−/− cells expressing the R131M mutation in the SKAP/ADAP chimera, SKAP55 PH domain-mediated interaction of the chimera with membrane lipids is attenuated. This allows the chimera (through the ADAP C-terminus) to engage the CBM complex and promote NF-κB activation.

Brandon J. Burbach, et al. J Immunol. ;186(11):6227-6237.
5.
Figure 5

Figure 5. The SKAP55 PH domain inhibits ADAP-dependent NF-κB activation. From: THE PLECKSTRIN HOMOLOGY DOMAIN IN THE SKAP55 ADAPTER PROTEIN DEFINES THE ABILITY OF THE ADAPTER PROTEIN ADAP TO REGULATE INTEGRIN FUNCTION AND NF-?B ACTIVATION.

(A) Naïve hCAR/DO11.10 (WT) and hCAR/DO11.10/ADAP−/− (ADAP KO) T cells were transduced with control Thy1.1 adenovirus or adenovirus expressing wild-type ADAP (WT), ADAPΔCAR (ΔCAR), or SKAP/ADAP chimera (S/A). After 3d, cells were stimulated with anti-CD3/CD28 for the indicated times (minutes) and lysates prepared. Western blots were probed with antibodies to total IκBα and phospho-IκBα to demonstrate IκBα degradation and phosphorylation, respectively. (B) Western blots from hCAR/DO11.10 and hCAR/DO11.10/ADAP−/− T cells expressing the indicated constructs were performed as in A. RM; SKAP/ADAP R131M mutant. (C) T cells were transduced and stimulated for 15 min as in (A-B) and subjected to immunoprecipitation with mouse anti-Bcl10 antibodies. Immune complexes were analyzed by Western blot with rabbit anti-SKAP55 antibody to detect the appearance of the SKAP/ADAP chimera in the Bcl10 complex. Whole cell lysates are shown in the lower 3 panels and were probed with anti-SKAP55 (to detect the SKAP/ADAP chimera), anti-ADAP, or rabbit anti-Bcl10. (D) T cells from hCAR/DO11.10/ADAP−/− mice were transduced with the indicated constructs as in (C) and stimulated for the indicated times followed by Bcl10 immunoprecipitation and western blotting as described for (C). Similar results were obtained in 3 independent experiments for each panel.

Brandon J. Burbach, et al. J Immunol. ;186(11):6227-6237.
6.
Figure 3

Figure 3. The R131 amino acid residue in the SKAP55 PH domain is critical for efficient ADAP-dependent T:APC conjugate formation. From: THE PLECKSTRIN HOMOLOGY DOMAIN IN THE SKAP55 ADAPTER PROTEIN DEFINES THE ABILITY OF THE ADAPTER PROTEIN ADAP TO REGULATE INTEGRIN FUNCTION AND NF-?B ACTIVATION.

(A) Naïve hCAR/DO11.10 (WT) or hCAR/DO11.10/ADAP−/− (KO) T cells were transduced with the indicated constructs and conjugate assays were performed as described in Figures 1-2. The SKAP/ADAPΔPH construct (S/AΔPH) lacks the entire 100 aa PH domain of SKAP55. (B) Amino acid alignment of SKAP-HOM and SKAP55 depicting the lipid binding region of the PH domain. Arginine 140 in SKAP-HOM corresponds to R131 in SKAP55. (C) Expression of SKAP55, SKAP/ADAP, or SKAP/ADAP R131M (S/A R131M) in naïve hCAR/DO11.10/ADAP−/− T cells. Note that the highest levels of SKAP55 expression achieved in the absence of ADAP (Thy1.1hi cells; boxed gate marked “Hi” in middle panel) approach the levels of endogenous SKAP55 found in naïve wild-type T cells, and correspond to the SKAP55 level found in Thy1.1lo cells expressing the SKAP/ADAP chimera or the SKAP/ADAP R131M mutant. (D) Conjugate assays were performed as described in Figures 1-2. T cells were gated on Thy1 lo cells (leftmost gate on each plot in C) to match endogenous levels of SKAP55 found in wild-type cells. For comparison, the conjugate efficiency of Thy1.1hi cells following SKAP55 expression alone in ADAP−/− is also shown (SKAP55Hi). Similar results were obtained in at least 4 independent experiments.

Brandon J. Burbach, et al. J Immunol. ;186(11):6227-6237.
7.
Figure 2

Figure 2. A SKAP/ADAP chimeric fusion protein restores T:APC conjugate formation in ADAP−/− T cells. From: THE PLECKSTRIN HOMOLOGY DOMAIN IN THE SKAP55 ADAPTER PROTEIN DEFINES THE ABILITY OF THE ADAPTER PROTEIN ADAP TO REGULATE INTEGRIN FUNCTION AND NF-?B ACTIVATION.

(A) Schematic diagram of key expression constructs used in this study. Amino acid numbering is given for the murine ADAP p130kDa isoform and for human SKAP55. PRO, proline-rich domain; E/K, glutamic acid and lysine-rich domain; hSH3N or hSH3C, N-terminal or C-terminal helical SH3 domain; PH, pleckstrin homology domain. Asterisks indicate the positions of key tyrosine residues (547/549/584/615/687) that have been reported for SLP-76 and Fyn binding to ADAP. The SKAP/ADAP chimera is a 692 aa molecule comprised of the N terminal 299 aa of human SKAP55 fused to the C-terminus of murine ADAP beginning at aa426. (B) Western blot analysis of SKAP55 and ADAP expression in whole cell lysates prepared from naïve wild-type (WT) or ADAP−/− (KO) cells expressing the indicated construct. (C) Naïve hCAR/DO11.10 (WT) or hCAR/DO11.10/ADAP−/− (KO) T cells were transduced for 3d with adenovirus for the indicated construct, fixed, and analyzed by intracellular staining for ADAP (N-terminus), SKAP55, or the HA-Tag. (D) Conjugate assays were performed as in Figure 1. The conjugate efficiency was evaluated from the Thy1.1 gated boxes depicted in panel C, to ensure that the level of the SKAP/ADAP chimera and the control constructs matched that of endogenous SKAP55 observed in wild-type control cells. Results are representative of at least 4 independent experiments for each construct.

Brandon J. Burbach, et al. J Immunol. ;186(11):6227-6237.

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