Phosphorylated ADAP promotes integrin-independent adhesion by stabilizing T cell receptor-induced SLP-76 microclusters.
Lewis, Juliana.
2012
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Abstract: T cell activation occurs in the context of the immune synapse, a specialized adhesive junction that forms at the interface between T cells and antigen presenting cells. Signaling complexes that contain the SLP-76 adaptor protein form alongside the TCR at the immune synapse and effectively augment stable T cell contacts by activating integrins. These SLP-76 microclusters also bridge enzym... read moreatically active proteins to their substrates, supporting downstream signals that activate T cells. Individual SLP-76 microclusters originate at the distal actin-rich region of the immune synapse, pass through a peripheral integrin-rich adhesive zone, and accumulate at the center of the contact. The amino-terminus of SLP-76 contributes to microcluster persistence and calcium responses, suggesting that microcluster lifetime affects T cell activation. By contrast, the contribution of the SLP-76 SH2 domain to T cell activation and SLP-76 microclusters is unclear. Here, the SLP-76 SH2 domain and its ligand ADAP were analyzed to clarify their roles in T cell activation and SLP-76 microclusters. ADAP and the SLP-76 SH2 domain contribute to SLP-76 microcluster cohesion, persistence, and movement. Neither ADAP nor the SLP-76 SH2 domain promoted TCR-induced calcium responses, suggesting that the labile microclusters that form in the absence of the interaction between ADAP and SLP-76 support calcium. T cell adhesion to TCR ligands requires a functional SH2 domain and ADAP, suggesting that stable microclusters are required for optimal adhesion to TCR ligands. The recruitment of ADAP to SLP-76 microclusters and to lamellipodia were supported by its N-terminus. Associations between ADAP and SLP-76 require multivalent interactions that are supported by Y595, Y651, and multiple regions in the ADAP N-terminus. SLP-76, SKAP55, and the ADAP N-terminus unexpectedly contribute to ADAP phosphorylation. Phosphorylated ADAP is observed in SLP-76 microclusters, while unphosphorylated ADAP is largely lamellipodial. In the absence of interactions with SLP-76, ADAP is observed in the actin-rich fingerlike projections of landing cells, at the lamellipodia, and at sites that overlap with labile microcluster formation, indicating that the TCR communicates with ADAP through a previously unrealized pathway. These observations realign the placement of ADAP downstream of the TCR at similar hierarchical levels as the transmembrane adaptor molecule LAT. The observations further indicate that the TCR builds labile, calcium competent microclusters that become stable, adhesive structures upon merging the LAT and ADAP pathways.
Thesis (Ph.D.)--Tufts University, 2012.
Submitted to the Dept. of Immunology.
Advisors: Stephen Bunnell, and Henry Wortis.
Committee: Alexander Poltorak, and Naomi Rosenberg.
Keywords: Immunology, Cellular biology, and Biochemistry.read less - ID:
- tm70n681h
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