Fig. 4.
Ligand-induced PI3K recruitment to the JAML ICD. Anti-PI3K Western blots of JAML immunoprecipitated with anti-JAML HL4E10 IgG from epithelial γδ T cell lysates after stimulation with ligands (A–E) or from JAML mutant-transfected CHO cell lysates (F). (A) Time course of stimulation with CAR-Fc. After 1 min, PI3K association to JAML is significantly elevated over the basal level. (B) PI3K-recruitment is rapidly switched off once CAR is released from the JAML ectodomain. γδ T cells were incubated with buffer (DPBS), Adenovirus F5 protein (F5), or stimulated with CAR (CAR-Fc) followed by an excess of F5 (which competes with JAML for CAR-binding). F5 alone does not alter basal PI3K levels (DPBS), while a 1 min addition of F5 (CAR-Fc+1min F5) significantly decreases PI3K recruitment in CAR-stimulated γδ T cells (CAR-Fc+1min). (C) Functional consequences of mutations in the CAR-JAML interface on downstream signalling by JAML. wt CAR-Fc and a Asp49Ala mutant induce significant PI3K recruitment, consistent with their comparable JAML-binding characteristics (Fig. S9) and location of CAR Asp49 at the periphery of the JAML-binding site (Fig. 2E), where mutations would be expected to cause smaller effects on binding. Mutation of CAR Asp35 induces less, but does not entirely abolish, PI3K-binding to JAML, consistent with its interaction with JAML Arg37, for which mutations are well tolerated. In contrast, CAR mutants Glu37Ala, Lys102Ala, and Lys104Ala, all of which interact with JAML hot spot residues (Fig. 2C), do not induce significant PI3K recruitment. (D+E) Dimeric, but not monomeric, ligands induce PI3K-recruitment to JAML. PI3K levels are increased by JAML binding of bivalent HL4E10 IgG, bivalent HL4E10 Fab’2 fragment, and dimeric CAR (CAR-Fc fusion protein), but not by monovalent HL4E10 Fab or monomeric CAR (CAR-His). Fc- or His-tagged control proteins (CD62L-Fc, CD1d-His) do not induce PI3K recruitment. (F) Dissection of the role of conserved JAML intracellular signaling motifs in PI3K binding. PI3K constitutively binds to wild-type and Tyr314Phe JAML, while binding is abolished to any Tyr336Phe mutants in the YMxM PI3K-binding motif. PI3K association is greatly reduced in JAML polyproline motif mutants Pro340,343,346Ala and Tyr314Phe/Pro340,343,346Ala even although they still exhibit an intact YMxM motif. Data are representative of at least three independent experiments.