ABSTRACT
Antigen recognition by the T cell receptor (TCR) directs the assembly of essential signaling complexes known as SLP-76 (also known as LCP2) microclusters. Here, we show that the interaction of the adhesion and degranulation-promoting adaptor protein (ADAP; also known as FYB1) with SLP-76 enables the formation of persistent microclusters and the stabilization of T cell contacts, promotes integrin-independent adhesion and enables the upregulation of CD69. By analyzing point mutants and using a novel phospho-specific antibody, we show that Y595 is essential for normal ADAP function, that virtually all tyrosine phosphorylation of ADAP is restricted to a Y595-phosphorylated (pY595) pool, and that multivalent interactions between the SLP-76 SH2 domain and its binding sites in ADAP are required to sustain ADAP phosphorylation. Although pY595 ADAP enters SLP-76 microclusters, non-phosphorylated ADAP is enriched in protrusive actin-rich structures. The pre-positioning of ADAP at the contact sites generated by these structures favors the retention of nascent SLP-76 oligomers and their assembly into persistent microclusters. Although ADAP is frequently depicted as an effector of SLP-76, our findings reveal that ADAP acts upstream of SLP-76 to convert labile, Ca2+-competent microclusters into stable adhesive junctions with enhanced signaling potential.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Jurkat Cells/metabolism , Phosphoproteins/metabolism , Receptors, Antigen, T-Cell/metabolism , Adaptor Proteins, Signal Transducing/immunology , Cell Adhesion/physiology , Cell Communication/physiology , Cytoskeleton/immunology , Cytoskeleton/metabolism , Humans , Jurkat Cells/cytology , Jurkat Cells/immunology , Lymphocyte Activation , Phosphoproteins/immunology , Phosphorylation , Receptors, Antigen, T-Cell/immunology , Signal Transduction , src Homology DomainsABSTRACT
We investigated adhesion pathways that contribute to engraftment of breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1)-induced chronic myelogenous leukemia (CML)-like myeloproliferative neoplasia in a mouse retroviral transduction/transplantation model. Compared with normal stem/progenitor cells, BCR-ABL1(+) progenitors had similar expression of very late antigen-4 (VLA4), VLA5, leukocyte functional antigen-1, and CXCR4 but lower expression of P-selectin glycoprotein ligand-1 (PSGL-1) and of L-selectin. Whereas vascular cell adhesion molecule-1 and P-selectin were not required, deficiency of E-selectin in the recipient bone marrow endothelium significantly reduced engraftment by BCR-ABL1-expressing stem cells following intravenous injection, with leukemogenesis restored by direct intrafemoral injection. BCR-ABL1-expressing cells deficient for PSGL-1 or the selectin ligand-synthesizing enzymes core-2 ß1,6-N-acetylglucosaminyltransferase or fucosyltransferases IV/VII were impaired for engraftment, and destruction of selectin ligands on leukemic progenitors by neuraminidase reduced engraftment. BCR-ABL1-expressing L-selectin-deficient progenitors were also defective in homing and engraftment, with leukemogenesis rescued by coexpression of chimeric E/L-selectin. Antibody to L-selectin decreased the engraftment of BCR-ABL1-transduced stem cells. These results establish that BCR-ABL1(+) leukemic stem cells rely to a greater extent on selectins and their ligands for homing and engraftment than do normal stem cells. Selectin blockade is a novel strategy to exploit differences between normal and leukemic stem cells that may be beneficial in autologous transplantation for CML and perhaps other leukemias.
