Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells

While the adaptor SKAP-55 mediates LFA-1 adhesion on T-cells, it is not known whether the adaptor regulates other aspects of signaling. SKAP-55 could potentially act as a node to coordinate the modulation of adhesion with downstream signaling. In this regard, the GTPase p21ras and the extracellular signal-regulated kinase (ERK) pathway play central roles in T-cell function. In this study, we report that SKAP-55 has opposing effects on adhesion and the activation of the p21ras -ERK pathway in T-cells. SKAP-55 deficient primary T-cells showed a defect in LFA-1 adhesion concurrent with the hyper-activation of the ERK pathway relative to wild-type cells. RNAi knock down (KD) of SKAP-55 in T-cell lines also showed an increase in p21ras activation, while over-expression of SKAP-55 inhibited activation of ERK and its transcriptional target ELK. Three observations implicated the p21ras activating exchange factor RasGRP1 in the process. Firstly, SKAP-55 bound to RasGRP1 via its C-terminus, while secondly, the loss of binding abrogated SKAP-55 inhibition of ERK and ELK activation. Thirdly, SKAP-55−/− primary T-cells showed an increased presence of RasGRP1 in the trans-Golgi network (TGN) following TCR activation, the site where p21ras becomes activated. Our findings indicate that SKAP-55 has a dual role in regulating p21ras-ERK pathway via RasGRP1, as a possible mechanism to restrict activation during T-cell adhesion

SKAP-55 deficient T-cells show enhanced anti-CD3 induced ERK activation.

<p>Panel A: FACS profile of T-cells from SKAP-55+/+, +/− and −/− mice stained with AlexaFluor647 labeled anti-pERK. T-cells from lymph-nodes were left unstimulated, or stimulated with anti-CD3-biotin (10 µg/ml) and streptavidin for 5 min. Upper panel: SKAP-55+/+; upper middle panel: <i>SKAP-55−/+;</i> lower middle panel: <i>SKAP-55−/−;</i> lower panel: comparison of anti-CD3 stimulated SKAP-55+/+, <i>SKAP-55+/−</i> and <i>SKAP-55−/−</i> cells vs. pervanadate treated cells. Right upper panel: histogram showing the difference in MFI values for SKAP-55+/+, <i>SKAP-55+/−</i> and <i>SKAP-55−/−</i> cells. Right lower panel: histogram showing the difference in the percentage of SKAP-55+/+, <i>SKAP-55+/−</i> and <i>SKAP-55−/−</i> cells staining for pERK. Panel B: Anti-pERK immunoblotting of anti-CD3 activated SKAP-55+/+ versus <i>SKAP-55−/−</i> T-cells. T-cells were activated with 5 µg/ml anti-CD3 for 1–30 minutes. Upper panel: anti-pERK blot; middle panel: anti-ERK blotting; lower panel: anti-SKAP-55 blot. SKAP-55+/+: lanes 1–7; <i>SKAP-55−/−:</i> lanes 8–14; Resting: lanes 1, 8; Stimulated: 1 min: lanes 2,9; 2 min: lanes 3, 10; 5 min: lanes 4,11; 10 min: lanes 5,12; 15 min: lanes 6, 13; 30 min: lane 7, 14. Panel C: SKAP-55 deficient T-cells have impaired adhesion to ICAM-1. Cells were stimulated with anti-CD3 followed by a measurement of binding to immobilized ICAM-1 on plates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001718#s4" target="_blank"><i>Material and Methods</i></a>.</p

Loss of the RasGRP1 binding region in SKAP-55 abrogates its ability to inhibit ERK activation and ELK transcriptional activity.

<p>Panel A: RasGRP1 binding to SKAP-55 depends on the C-terminal domain of SKAP-55. Upper panel: deletion of the SKAP-55 SH3 domain prevents RasGRP1 binding. Lanes 1,2: myc-RasGRP1 expression in Jurkat cells transfected with SKAP-55 and SKAP-55ΔSH3. Lanes 3–5: association of RasGRP1 with SKAP-55 depends on the C-terminal domain of SKAP-55. Cell lysates: Lanes 1,2; anti-SKAP-55 precipitates: lanes 3–5. SKAP-55 transfection: Lanes 1, 4. SKAP-55ΔSH3 transfection: Lanes 2, 5. Lower panel: anti-SKAP-55 blot. Lane 1: transfection with myc-RasGRP1; lane 2: transfection with myc-RasGRP1 and SKAP-55; lane 3: transfection with myc-RasGRP1 and SKAP-55ΔSH3. Panel B: Loss of the RasGRP1 binding region in SKAP-55 abrogates its ability to inhibit ERK and ELK activation. Upper panel: anti-pERK blot of lysates from cells transfected with myc-RasGRP1 plus SKAP-55 or SKAP-55ΔSH3. Resting cells: lanes 1,3,5,7; anti-CD3: lanes 2,4,6,8. Middle panel: anti-ERK blot; Lower panel: anti-pELK blot. Panel C: Loss of the RasGRP1 binding region in SKAP-55 abrogates its ability to inhibit ELK transcriptional activity. Left panel: histogram showing inhibition of ELK transcription activity by SKAP-55 and lack of inhibition by SKAP-55ΔSH3. Right panel: anti-SKAP-55 (upper) and anti-myc (myc-RasGRP1) blot of cell lysates.</p

Hyper-activation of p21^ras in SKAP-55 KD cells.

<p>Upper panel: WT, Z.21 and Z.23 cells were ligated with anti-CD3 for various times and lysates were subjected to a pull-down assay using GST-Raf-1-RBD and blotted with Ras specific antibody. Middle panel: Anti-Ras blotting of cell lysates. Lanes 1–3: WT; lanes 4–6: Z.21; lanes 7–9: Z.23. Untreated: lanes 1,4,7; anti-CD3 ligation: 2 min: lanes 2,5,8; 7.5 min: lanes 3,6,9. Lower panel: Histogram showing the relative intensity of signal in the blot.</p

SKAP-55 associates with a subset of RasGRP1 and co-localizes with RasGRP1 in the TGN.

<p>Panel A: SKAP-55 binds to RasGRP-1. DC27.10 T-cells were stimulated with anti-CD3 or anti-CD18 (LFA-1) for 15 minutes. Cells were then lysed and subjected to precipitation with anti-SKAP-55 followed by anti-RasGRP1 blotting (lanes 1–9). Lanes 1–4: cell lysates; lane 9: rabbit anti-mouse control. Right panel: histogram showing the percent of RasGRP1 associated with SKAP-55 in resting and stimulated cells. Panel B: SKAP-55 partially co-localizes with RasGRP-1 in the TGN of DC27.10 cells. Cells were stained with anti-rabbit RasGRP-1/anti-rabbit AlexaFluor568 and anti-mouse Syntaxin6/anti-mouse AlexaFluor647 or anti-mouse SKAP-55/anti-mouse Alexa647. Upper panels: RasGRP-1 staining occurs in the TGN. Panel a: anti-RasGRP-1; panel b: anti-syntaxin 6; panel c: anti-RasGRP1/Syntaxin. Lower panels: panel d: anti-RasGRP1; panel e: anti-SKAP-55; panel f; anti-SKAP-55/RasGRP-1; panel g: anti-SKAP-55/RasGRP-1 staining of activated C57BL/6 primary T-cells. For analysis, at least 100 cells were counted.</p

SKAP-55 knock-down and knock-up T-cells show altered ERK activation.

<p>Panel A: Upper panel: Reduction in SKAP-55 expression in SKAP-55KD cells. Z.21, Z.22 and Z.23 cell lines were derived from distinct SKAP55 shRNAs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001718#s4" target="_blank"><i>Material and Methods</i></a>. Panel B: Increased pERK in response to anti-CD3 in SKAP-55 KD cells. T8.1 WT and T8.1 SKAP-55 KD cells (Z.21, Z.22, Z.23) were either left untreated (lanes 1, 7,13, 19) or stimulated with anti-CD3 (10 µg/ml) (lanes 2–6; lanes 8–12; lanes 14–18; lanes 20–24). Upper panels: anti-pERK blotting; lower panels: anti-ERK blotting. Panel C: Reduction of ERK activation in DC27.10 cells. DC27.10 cells (lanes 1–4) and cells transfected with SKAP-55 (lanes 5–8) were either left untreated (lanes 1,5) or stimulated with anti-CD3 (5 µg/ml) for the indicated times (lanes 2–4 and 6–8). Upper panels: anti-pERK blotting; middle panels: anti-ERK blotting; lower panels: anti-SKAP-55 blotting.</p