Phosphorylated ADAP promotes integrin-independent adhesion by stabilizing T cell receptor-induced SLP-76 microclusters.
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 ... read moreenzymatically 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
Thesis (Ph.D.)--Tufts University, 2013.
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