ABSTRACT
BACKGROUND: The chemokine CXCL12/SDF-1alpha interacts with its G-protein coupled receptor CXCR4 to induce migration of lymphoid and endothelial cells. T cell specific adapter protein (TSAd) has been found to promote migration of Jurkat T cells through interaction with the G protein beta subunit. However, the molecular mechanisms for how TSAd influences cellular migration have not been characterized in detail. PRINCIPAL FINDINGS: We show that TSAd is required for tyrosine phosphorylation of the Lck substrate IL2-inducible T cell kinase (Itk). Presence of Itk Y511 was necessary to boost TSAd's effect on CXCL12 induced migration of Jurkat T cells. In addition, TSAd's ability to promote CXCL12-induced actin polymerization and migration of Jurkat T lymphocytes was dependent on the Itk-interaction site in the proline-rich region of TSAd. Furthermore, TSAd-deficient murine thymocytes failed to respond to CXCL12 with increased Itk phosphorylation, and displayed reduced actin polymerization and cell migration responses. CONCLUSION: We propose that TSAd, through its interaction with both Itk and Lck, primes Itk for Lck mediated phosphorylation and thereby regulates CXCL12 induced T cell migration and actin cytoskeleton rearrangements.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Chemokine CXCL12/metabolism , Protein-Tyrosine Kinases/metabolism , Animals , Cell Movement , Humans , Jurkat Cells , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Binding , src Homology DomainsABSTRACT
T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, is expressed in activated T cells. The function of TSAd is as yet unknown. We previously showed that TSAd may modulate T cell receptor-triggered signaling events. TSAd contains a Src homology (SH)2 domain, ten tyrosines and a C-terminal proline-rich region. Here, we show that human TSAd interacts with Lck through the Lck SH2 and SH3 domains and is a substrate for Lck. The TSAd C terminus, including the proline-rich region and five tyrosines, is both necessary and sufficient for TSAd interaction with and phosphorylation by Lck. Expression of TSAd in Jurkat TAg cells results in hyperphosphorylation of endogenous Lck on Y394 and to an even larger extent on Y505, resulting in a reduced Y394/Y505 phosphorylation ratio in these cells. Furthermore, full-length TSAd, but not TSAd lacking the C terminus, inhibits the hyperactive Lck Y505F mutant when both are expressed in Jurkat T cells. In contrast, expression of the TSAd C terminus alone is sufficient to inhibit Lck Y505F in phosphorylating its substrates in Jurkat T cells. Our results indicate that the TSAd C terminus is essential for inhibition of Lck activity by TSAd, and suggest a mechanism for how TSAd may inhibit early T cell activation events.
