Abstract
Background -Fas ligand is a cytotoxic effector molecule of T and NK cells which is characterized by an intracellular N-terminal polyproline region that serves as a docking site for SH3 and WW domain proteins. Several previously described Fas ligand-interacting SH3 domain proteins turned out to be crucial for the regulation of storage, expression and function of the death factor. Recent observations, however, indicate that Fas ligand is also subject to posttranslational modifications including shedding and intramembrane proteolysis. This results in the generation of short intracellular fragments that might either be degraded or translocate to the nucleus to influence transcription. So far, protein-protein interactions that specifically regulate the fate of the intracellular fragments have not been identified.Results -In order to further define the SH3 domain interactome of the intracellular region of Fas ligand, we now screened a human SH3 domain phage display library. In addition to known SH3 domains mediating binding to the Fas ligand proline-rich domain, we were able to identify a number of additional SH3 domains that might also associate with FasL. Potential functional implications of the new binding proteins for the death factor's biology are discussed. For Tec kinases and sorting nexins, the observed interactions were verified in cellular systems by pulldown experiments.Conclusion -We provide an extended list of putative Fas ligand interaction partners, confirming previously identified interactions, but also introducing several novel SH3 domain proteins that might be important regulators of Fas ligand function.
Highlights
Protein-protein interactions link signal transduction pathways from receptors to the nucleus and govern intracellular processes as diverse as organelle targeting, directional transport, cytoskeletal reorganization, membrane positioning, endo- and exocytosis and protein degradation
Conclusion -: We provide an extended list of putative Fas ligand interaction partners, confirming previously identified interactions, and introducing several novel SH3 domain proteins that might be important regulators of Fas ligand function
For less stringent washing conditions (0.15% Tween20), a titer of (2.85 ± 0.15) × 107 PFU/ml was calculated in eluate obtained from the Glutathione S-transferase (GST)-panning, while the titer following more stringent washing was (0.185 ± 0.075) × 107 PFU/ml
Summary
Protein-protein interactions link signal transduction pathways from receptors to the nucleus and govern intracellular processes as diverse as organelle targeting, directional transport, cytoskeletal reorganization, membrane positioning, endo- and exocytosis and protein degradation. SH3 domains are phylogenetically highly conserved protein interaction modules that comprise 50 to 70 amino acids and are found in a variety of functionally unrelated proteins. FasL is employed by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells to kill virusinfected or tumorigenic cells. It is implicated in the downregulation of immune responses by activation-induced cell death, the establishment of immune privilege, and in the modulation of T cell activation [6,7,8]. FasL is stored in so-called secretory lysosomes and is recruited to the immunological synapse in an activation-restricted fashion. The released intracellular domain may translocate to the nucleus or be prepared for degradation [9,10,11]
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