CD25 and CD69 induction by α4β1 outside-in signalling requires TCR early signalling complex proteins

Abstract

Distinct signalling pathways producing diverse cellular outcomes can utilize similar subsets of proteins. For example, proteins from the TCR (T-cell receptor) ESC (early signalling complex) are also involved in interferon-α receptor signalling. Defining the mechanism for how these proteins function within a given pathway is important in understanding the integration and communication of signalling networks with one another. We investigated the contributions of the TCR ESC proteins Lck (lymphocyte-specific kinase), ZAP-70 (ζ-chain-associated protein of 70 kDa), Vav1, SLP-76 [SH2 (Src homology 2)-domain-containing leukocyte protein of 76 kDa] and LAT (linker for activation of T-cells) to integrin outside-in signalling in human T-cells. Lck, ZAP-70, SLP-76, Vav1 and LAT were activated by α4β1 outside-in signalling, but in a manner different from TCR signalling. TCR stimulation recruits ESC proteins to activate the mitogen-activated protein kinase ERK (extracellular-signal-regulated kinase). α4β1 outside-in-mediated ERK activation did not require TCR ESC proteins. However, α4β1 outside-in signalling induced CD25 and co-stimulated CD69 and this was dependent on TCR ESC proteins. TCR and α4β1 outside-in signalling are integrated through the common use of TCR ESC proteins; however, these proteins display functionally distinct roles in these pathways. These novel insights into the cross-talk between integrin outside-in and TCR signalling pathways are highly relevant to the development of therapeutic strategies to overcome disease associated with T-cell deregulation.

Figure 1. ZAP-70 phosphorylation at Tyr³¹⁹ is Lck dependent in α4β1 outside-in and TCR signalling pathways

(A) Jurkat cells were stimulated with the anti-α4β1 antibody 19H8 followed by the addition of a secondary cross-linker (2′) for the indicated time points. Expression of phospho-Tyr³⁹⁴-Lck, phospho-Tyr³¹⁹-ZAP-70, phospho-Tyr⁴⁹³-ZAP-70, Lck and ZAP-70 was determined by Western blot analysis. Non-stimulated cells (NT) and cells treated with either 2′ or 19H8 alone were used as controls. (B) Jurkat cells were added to 24-well fibronectin-coated plates for the indicated time points. Poly-L-lysine was used as a negative control. Expression of phospho-Tyr³⁹⁴-Lck, phospho-Tyr³¹⁹-ZAP-70, phospho-Tyr⁴⁹³-ZAP-70, Lck and ZAP-70 was determined by Western blot analysis. (C) Lck-deficient JCAM1.6 and Lck reconstituted JCAM1.6 (JCAM1.6^WTLck) Jurkat cells were stimulated with 19H8 followed by the addition of 2′ for the indicated time points. Expression of phospho-Tyr³¹⁹-ZAP-70, ZAP-70 and Lck was determined by Western blot analysis. (D) JCAM1.6 and JCAM1.6^WTLck cells were stimulated with the anti-CD3 antibody UCHT1 for the indicated time points. Expression of phospho-Tyr³¹⁹-ZAP-70, phospho-Tyr⁴⁹³-ZAP-70 and ZAP-70 was determined by Western blot analysis. (E) Schematic representation of Lck and ZAP-70 activation following stimulation of TCR (left-hand panel) and α4β1 outside-in (right-hand panel) signalling pathways. (P), phosphorylation; NT, not treated.

Figure 2. Activation of Vav1 and LAT by α4β1 outside-in signalling is ZAP-70 independent

(A) Jurkat cells were stimulated with the anti-α4β1 antibody 19H8 followed by the addition of a secondary cross-linker (2′) for the indicated time points. Expression of phospho-Tyr¹⁷⁴-Vav1, phospho-Tyr¹⁹¹-LAT, Vav1 and LAT was determined by Western blot analysis. (B) Jurkat cells were added to 24-well fibronectin-coated plates for the indicated time points. Poly-L-lysine was used as a negative control. Expression of phospho-Tyr¹⁷⁴-Vav1, phospho-Tyr¹⁹¹-LAT, Vav1 and LAT was determined by Western blot analysis. (C) Parental and ZAP-70-deficient P116 Jurkat cells were stimulated with 19H8 followed by the addition of 2′ for the indicated time points. Expression of phospho-Tyr¹⁷⁴-Vav1, phospho-Tyr¹⁹¹-LAT, Vav1, LAT and ZAP-70 was determined by Western blot analysis. (D) ZAP-70-deficient P116 and ZAP-70-reconstituted P116 (P116^WTZAP-70) Jurkat cells were stimulated with the anti-CD3 antibody UCHT1 for the indicated time points. Expression of phospho-Tyr¹⁷⁴-Vav1, phospho-Tyr¹⁹¹-LAT, Vav1, LAT, and ZAP-70 was determined by Western blot analysis. (E) Schematic representation of Vav1 and LAT activation following stimulation of TCR (left-hand panel) and α4β1 outside-in (right-hand panel) signalling pathways. (P), phosphorylation; NT, not treated.

Figure 3. SLP-76 is not required for Vav1 phosphorylation in α4β1 outside-in signalling

(A) Jurkat cells were stimulated with the anti-α4β1 antibody 19H8 followed by the addition of a secondary cross-linker (2′) for the indicated time points. Expression of phospho-Tyr¹¹³-SLP-76, phospho-Tyr¹²⁸-SLP-76 and SLP-76 was determined by Western blot analysis. (B) Jurkat cells were added to 24-well fibronectin-coated plates for the indicated time periods. Poly-L-lysine was used as a negative control. Expression of phospho-Tyr¹¹³-SLP-76, phospho-Tyr¹²⁸-SLP-76 and SLP-76 was determined by Western blot analysis. (C) Parental and SLP-76-deficient J14 Jurkat cells were stimulated with 19H8 followed by the addition of 2′ for the indicated time points. Expression of phospho-Tyr¹⁷⁴-Vav1, Vav1 and SLP-76 was determined by Western blot analysis. (D) J14 and SLP-76-reconstituted J14 (J14^WTSLP-76) Jurkat cells were stimulated with the anti-CD3 antibody UCHT1 for the indicated time points. Expression of phospho-Tyr¹⁷⁴-Vav1, Vav1 and SLP-76 was determined by Western blot analysis. (E) Schematic representation of Vav1 recruitment by SLP-76 following stimulation of TCR (left-hand panel) and α4β1 outside-in (right-hand panel) signalling pathways. (P), phosphorylation; NT, not treated.

Figure 4. α4β1 outside-in signalling activates ERK independently of Ras and Raf-1

(A) Jurkat cells were stimulated with the anti-CD3 antibody UCHT1 for 5 min. Jurkat cells were also pre-treated with the FTI B581 (50 μM) for 30 min prior to UCHT1 stimulation for 5 min. Expression of phospho-(Thr²⁰²/Tyr²⁰⁴)-ERK1/2, phospho-Ser³³⁸-Raf-1, phospho-(Ser²¹⁷/Ser²²¹)-MEK1/2, ERK1/2, Raf-1 and MEK1/2 was determined by Western blot analysis. Non-stimulated cells (NT) and cells treated with B581 alone were used as controls. (B) Jurkat cells were stimulated with the anti-α4β1 antibody 19H8 followed by the addition of a secondary cross-linker (2′) for the indicated time points. Expression of phospho-(Thr²⁰²/Tyr²⁰⁴)-ERK1/2, phospho-Ser³³⁸-Raf-1, phospho-(Ser²¹⁷/Ser²²¹)-MEK1/2, ERK1/2, Raf-1 and MEK1/2 was determined by Western blot analysis. NT (not treated) cells and cells treated with either 2′ or 19H8 alone were used as controls. (C) Jurkat cells were stimulated with 19H8 followed by the addition of 2′ for the indicated time points. A GST-tagged Ras-binding domain of Raf-1 fusion protein conjugated to glutathione–agarose beads was used to affinity precipitate Ras-GTP from the cell lysates. Ras-GTP and total Ras levels were determined by Western blot analysis. (D) FTI-mediated inhibition of Ras activity induced by 19H8 cross-linking was confirmed by determining Ras-GTP levels. (E) Jurkat cells were stimulated with 19H8 followed by the addition of 2′ for 5 min. Jurkat cells were also pre-treated with the FTIs manumycin A (M; 10 μM) or B581 (50 μM) for 30 min prior to the addition of 2′ for 5 min. Expression of phospho-(Thr²⁰²/Tyr²⁰⁴)-ERK1/2, phospho-(Ser²¹⁷/Ser²²¹)-MEK1/2, ERK1/2 and MEK1/2 was determined by Western blot analysis. NT and cells treated with either 2′, 19H8, M or B581 alone were used as controls.

Figure 5. α4β1 outside-in signalling induces ERK activation via a Rap1/B-Raf/MEK pathway

(A) Jurkat cells were stimulated with the anti-α4β1 antibody 19H8 followed by the addition of a secondary cross-linker (2′) for the indicated time points. Expression of phospho-(Thr⁵⁹⁸/Ser⁶⁰¹)-B-Raf, phospho-(Thr²⁰²/Tyr²⁰⁴)-ERK1/2, B-Raf and ERK1/2 was determined by Western blot analysis. NT (not-treated) cells and cells treated with either 2′ or 19H8 alone were used as controls. (B) Jurkat cells were stimulated with 19H8 followed by the addition of 2′ for the indicated time periods. A GST-fusion protein containing the RBD of human Ra1GDS was used to affinity precipitate Rap1-GTP from the cell lysates. Rap1-GTP and total Rap1 levels were determined by Western blot analysis. (C) Jurkat cells transfected with specific B-Raf siRNA or a non-targeting control siRNA (CsiRNA) were stimulated with 19H8 followed by the addition of 2′ for 5 min. Expression of phospho-(Thr²⁰²/Tyr²⁰⁴)-ERK1/2, phospho-(Ser²¹⁷/Ser²²¹)-MEK1/2, B-Raf, ERK1/2 and MEK1/2 was determined by Western blotting. To demonstrate that siRNA-mediated knockdown of B-Raf expression was not due to differences in protein loading, β-actin was used as a loading control. (D) Jurkat cells transfected with specific Rap1 siRNA or CsiRNA were stimulated with 19H8 followed by the addition of 2′ for 5 min. Expression of phospho-(Thr²⁰²/Tyr²⁰⁴)-ERK1/2, phospho-(Ser^217/221)-MEK1/2, phospho-(Thr⁵⁹⁸/Ser⁶⁰¹)-B-Raf, Rap1, ERK1/2, MEK1/2, B-Raf and β-actin was determined by Western blot analysis.

Figure 6. A functional TCR and TCR ESC proteins are not required for ERK activation induced by α4β1 outside-in signalling

Parental, TCRβ-deficient JRT3-T3.5, Lck-deficient JCAM1.6, Lck-reconstituted JCAM1.6 (JCAM1.6^WTLck), ZAP-70-deficient P116, Vav1-deficient J.Vav1, SLP-76-deficient J14 and LAT-deficient JCAM2.5 Jurkat cells were stimulated with the anti-α4β1 antibody 19H8 followed by the addition of a secondary cross-linker (2′) for the indicated time points. Expression of phospho-(Thr²⁰²/Tyr²⁰⁴)-ERK1/2 and ERK1/2 was determined by Western blot analysis. NT, not treated.

Figure 7. Induction of CD25 expression by α4β1 outside-in signalling requires TCR ESC proteins

(A) Jurkat cells and (B) naïve CD4⁺ T-cells were stimulated with the anti-CD3 antibody UCHT1, the anti-α4β1 antibody 19H8 plus secondary cross-linker (2′) or UCHT1 in combination with 19H8 plus 2′ for 30 min. CD25 expression was determined by Western blot analysis. β-actin was used as a loading control. (C) Parental, Lck-deficient JCAM1.6, Lck-reconstituted JCAM1.6 (JCAM1.6^WTLck), ZAP-70-deficient P116, ZAP-70-reconstituted P116 (P116^WTZAP-70), Vav1-deficient J.Vav1, Vav1-reconstituted J.Vav1 (J.Vav1^WTVav1), SLP-76-deficient J14 and LAT-deficient JCAM2.5 Jurkat cells were stimulated with 19H8 plus 2′ for 30 min. Expression of CD25 expression was determined by Western blotting. β-actin was used as a loading control. (D) Naïve CD4⁺ T-cells were pre-treated for 2 h with Lck inhibitor II (20 nM) or the (E) ZAP-70 inhibitor piceatannol (50 μg/ml) prior to stimulation with 19H8 plus 2′ for 24 h. Expression of CD25 expression was determined by Western blot analysis. β-actin was used as a loading control. NT, not treated.

Figure 8. α4β1 outside-in signalling requires TCR ESC proteins to co-stimulate CD69 expression

(A) Jurkat cells and (B) naïve CD4⁺ T-cells were stimulated with the anti-CD3 antibody UCHT1, the anti-α4β1 antibody 19H8 plus secondary cross-linker (2′) or UCHT1 in combination with 19H8 plus 2′ for 30 min. CD69 expression was determined by Western blot analysis. β-actin was used as a loading control. (C) Parental, Lck-deficient JCAM1.6, Lck-reconstituted JCAM1.6 (JCAM1.6^WTLck), ZAP-70-deficient P116, ZAP-70-reconstituted P116 (P116^WTZAP-70), Vav1-deficient J.Vav1, Vav1-reconstituted J.Vav1 (J.Vav1^WTVav1), SLP-76-deficient J14, SLP-76-reconstituted J14 (J14^WTSLP-76), LAT-deficient JCAM2.5 and LAT-reconstituted JCAM2.5 (JCAM2.5^WTLAT) Jurkat cells were stimulated with UCHT1, 19H8 plus 2′ or UCHT1 in combination with 19H8 plus 2′ for 30 min. Expression of CD69 expression was determined by Western blot analysis. β-actin was used as a loading control. (D) Naïve CD4⁺ T-cells were stimulated with UCHT1, 19H8 plus 2′, or UCHT1 in combination with 19H8 plus 2′ for 24 h. Cells were also pre-treated for 2 h with Lck inhibitor II (20 nM) or the ZAP-70 inhibitor piceatannol (50 μg/ml) prior to co-stimulation with UCHT1 and 19H8 for 24 h. Expression of CD69 expression was determined by Western blotting. β-actin was used as a loading control. NT, not treated.

Figure 9. α4β1-mediated CD25 and CD69 signalling occur independently of ERK

Jurkat cells were stimulated with the anti-α4β1 antibody 19H8 plus secondary cross-linker (2′) for 30 min. Jurkat cells were also pre-treated with U0126 (10 μM) for 2 h prior to the addition of 2′ for 30 min. CD25 and CD69 expression were determined by Western blot. β-actin was used as a loading control. NT (not treated) cells and cells treated with U0126 alone served as controls.

Figure 10. Schematic representation of the differential requirement of TCR ESC proteins in α4β1 outside-in signalling in T-cells

α4β1 outside-in signalling induced CD25 expression, co-stimulation of CD69 expression and activation of the MAPK ERK pathway. Unlike TCR signalling, ERK activation occurred independently of TCR ESC proteins. In contrast, TCR ESC proteins were required for the induction of CD25 and co-stimulation of CD69 by α4β1 outside-in signalling. Lck, ZAP-70 and Vav1 were required for CD25 induction and CD69 co-stimulation by α4β1 outside-in signalling; however, SLP-76 and LAT were only required for CD69 co-stimulation.