Abstract
BackgroundThe Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. The molecular details as to how TSAd regulates this process remain to be elucidated.ResultsTo identify novel interaction partners for TSAd, we used a scoring matrix-assisted ligand algorithm (SMALI), and found that the Src homology 2 (SH2) domain of the actin regulator Non-catalytic region of tyrosine kinase adaptor protein (Nck) potentially binds to TSAd phosphorylated on Tyr280 (pTyr280) and pTyr305. These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr280 and pTyr305 on TSAd. In addition, the SH3 domains of Nck interacted with the proline rich region (PRR) of TSAd. Pull-down and immunoprecipitation experiments further confirmed the Nck-TSAd interactions through Nck SH2 and SH3 domains. In line with this Nck and TSAd co-localized in Jurkat cells as assessed by confocal microscopy and imaging flow cytometry. Co-immunoprecipitation experiments in Jurkat TAg cells lacking TSAd revealed that TSAd promotes interaction of Nck with Lck and SLP-76, but not Vav1. TSAd expressing Jurkat cells contained more polymerized actin, an effect dependent on TSAd exon 7, which includes interactions sites for both Nck and Lck.ConclusionsTSAd binds to and co-localizes with Nck. Expression of TSAd increases both Nck-Lck and Nck-SLP-76 interaction in T cells. Recruitment of Lck and SLP-76 to Nck by TSAd could be one mechanism by which TSAd promotes actin polymerization in activated T cells.
Highlights
The Lymphocyte specific protein tyrosine kinase (Lck) and Src binding adaptor protein T cell specific adaptor protein (TSAd) (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells
The Non-catalytic region of tyrosine kinase adaptor protein (Nck) Src homology 2 (SH2) domain interacts with TSAd-phosphorylated on Tyr280 (pTyr280) and -pTyr305 TSAd possesses several protein interaction motifs, including an N-terminally located SH2 domain, and a Cterminal part consisting of a proline rich region (PRR) and several tyrosine phosphorylation sites
TSAd is tyrosine phosphorylated in non-stimulated Jurkat cells [4, 18] and in peripheral blood mononuclear cells [3] while increased amount of tyrosine phosphorylated TSAd may be seen upon T cell receptor (TCR) stimulation [18]
Summary
The Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. Regulation of actin dynamics is important for several aspects of T cell function, including differentiation, migration through tissues and proliferation. T cell activation initiates multiple molecular events including activation of protein tyrosine kinases, the formation of protein signaling complexes, and cytoskeletal actin reorganization leading to establishment of the immunological synapse (IS) at the T cell-antigen presenting cell (APC) interface [1, 2]. The molecular aspects concerning actin dynamics in T cells are complex, and many questions remain to be solved. We have shown that TSAd regulates CXCL12-induced migration and actin cytoskeletal rearrangements in T cells by promoting Lck dependent tyrosine phosphorylation of IL2-inducible T-cell kinase (Itk) [8]
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