Src Homology 2-Domain Containing Leukocyte-Specific Phosphoprotein of 76 kDa Is Mandatory for TCR-Mediated Inside-Out Signaling, but Dispensable for CXCR4-Mediated LFA-1 Activation, Adhesion, and Migration of T Cells

Abstract

Engagement of the TCR or of chemokine receptors such as CXCR4 induces adhesion and migration of T cells via so-called inside-out signaling pathways. The molecular processes underlying inside-out signaling events are as yet not completely understood. In this study, we show that TCR- and CXCR4-mediated activation of integrins critically depends on the membrane recruitment of the adhesion- and degranulation-promoting adapter protein (ADAP)/Src kinase-associated phosphoprotein of 55 kDa (SKAP55)/Rap1-interacting adapter protein (RIAM)/Rap1 module. We further demonstrate that the Src homology 2 domain containing leukocyte-specific phosphoprotein of 76 kDa (SLP76) is crucial for TCR-mediated inside-out signaling and T cell/APC interaction. Besides facilitating membrane recruitment of ADAP, SKAP55, and RIAM, SLP76 regulates TCR-mediated inside-out signaling by controlling the activation of Rap1 as well as Rac-mediated actin polymerization. Surprisingly, however, SLP76 is not mandatory for CXCR4-mediated inside-out signaling. Indeed, both CXCR4-induced T cell adhesion and migration are not affected by loss of SLP76. Moreover, after CXCR4 stimulation, the ADAP/SKAP55/RIAM/Rap1 module is recruited to the plasma membrane independently of SLP76. Collectively, our data indicate a differential requirement for SLP76 in TCR- vs CXCR4-mediated inside-out signaling pathways regulating T cell adhesion and migration.

Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration

“…Notably, T cells expressing the Y571F mutant showed comparable levels of adhesion as cells reexpressing wild type ADAP under both stimulation conditions, whereas a significant reduction of adhesion was observed in the absence of endogenous ADAP, as previously reported (Fig. 5B, 5C) (5,39,51). In accordance with published results, PMA bypassed the requirement for ADAP and Mn 2ϩ treatment served as a positive control of the adhesive capacity of these cells.…”

“…S5) (49). In contrast to TCR stimulation it was previously shown that this supramolecular assembly is not formed upon chemokine stimulation (39). Thus, the phosphorylation of Y571 of ADAP seems mandatory for the interaction between ADAP and ZAP70 only after CXCR4 stimulation.…”

“…Adhesion Assay, Conjugate Formation, and Migration-Transfected Jurkat T cells with the indicated suppression/re-expression vectors were used to address TCR/CXCR4-mediated adhesion, conjugate formation and migration after 48 h as previously described (38,39).…”

“…Immunoprecipitation and Western Blot Analysis-Jurkat T cells were left untreated or stimulated for various time points with anti-CD3 antibodies (OKT3 10 g/ml) or CXCL12 (100 ng/ml) and lysed as previously described (11,38,39). Equivalent amounts of protein (determined by Bradford assay (Roth)) were used in precipitation studies (500 g of total protein from Jurkat T cells).…”

The LAT Story: A Tale of Cooperativity, Coordination, and Choreography

“…Phosphorylated ADAP binds the SH2 domain of SLP-76 (da Silva et al 1997;Musci et al 1997). This interaction is crucial for adhesion and integrin function in T cells (Baker et al 2009;Horn et al 2009;Wang et al 2009). Tyrosine phosphorylated Shb also binds the SH2 domain of SLP-76 and promotes LAT, SLP-76, Vav, and PLC-g1 phosphorylation (Lindholm et al 1999;Lindholm et al 2002).…”