Binding of the Intracellular Fas Ligand (FasL) Domain to the Adaptor Protein PSTPIP Results in a Cytoplasmic Localization of FasL

Abstract

The tumor necrosis factor family member Fas ligand (FasL) induces apoptosis in Fas receptor-expressing target cells and is an important cytotoxic effector molecule used by CTL-and NK-cells. In these hematopoietic cells, newly synthesized FasL is stored in specialized secretory lysosomes and only delivered to the cell surface upon activation and target cell recognition. FasL contains an 80-amino acid-long cytoplasmic tail, which includes a proline-rich domain as a bona fide Src homology 3 domain-binding site. This proline-rich domain has been implicated in FasL sorting to secretory lysosomes, and it may also be important for reverse signaling via FasL, which has been described to influence T-cell activation. Here we report the identification of the Src homology 3 domaincontaining adaptor protein PSTPIP as a FasL-interacting partner, which binds to the proline-rich domain. PSTPIP co-expression leads to an increased intracellular localization of Fas ligand, thereby regulating extracellular availability and cytotoxic activity of the molecule. In addition, we demonstrate recruitment of the tyrosine phosphatase PTP-PEST by PSTPIP into FasL⅐PSTPIP⅐PTP-PEST complexes which may contribute to FasL reverse signaling.Fas ligand (FasL 3 ; CD95/APO-1 ligand, CD178, TNFSF6) is a 281-amino acid (aa)-long type II transmembrane molecule belonging to the large TNF family of proteins that bind to and activate members of the TNF receptor family (1, 2). FasL and its corresponding receptor Fas (CD95/APO-1) both interact as oligomers (3), and the activated Fas receptor complex initiates a proapoptotic death signal in the receptorbearing cell (4, 5).Although mainly known for its death-promoting activity, FasL has also been studied in the context of further nonapoptotic functions (1, 2, 6). Such experiments are motivated by the special structure of this type II transmembrane protein. In addition to its hydrophobic transmembrane domain, which anchors the molecule within the plasma membrane, and to its extracellular ectodomain, responsible for binding to its receptor, the FasL protein possesses an 80-aa-long N-terminal intracellular part that is responsible for the transduction of (extracellular) signals into FasL-bearing cells and/or that may fulfill regulatory functions. Such a "reverse signaling" has been described in mouse T-cells, which display an altered proliferative behavior upon triggering of their FasL surface molecules (7-9). Results obtained with mouse Sertoli cells, in which FasL engagement leads to mitogen-activated protein kinase activation, as measured by an increase in Erk phosphorylation, also imply stimulatory FasL reverse signaling (10).The importance of the intracellular domain for FasL function and regulation is underlined by its high homology among different species (7,11,12). Several signaling motives within the FasL intracellular domain are highly conserved, including a tandem casein kinase I phosphorylation site (aa 17-21 (11)), a phosphorylatable tyrosine at position 7, a proline-rich region (aa 40 -70) with bona...

Fas ligand is localized to membrane rafts, where it displays increased cell death–inducing activity

“…In addition, to compare the relative proportion of raftlocalized FasL, we chose HEK293 cell clones with similar cell surface expression levels of full-length, wild-type FasL, FasL delta 1-40, or FasL delta 1-80 ( Figure 3) because deletion of the intracellular domain has been shown to result in increased levels of cell surface FasL. 51 The 293 cell clones were subjected to biochemical raft separation by Brij 98 solubilization and sucrose gradient. Figure 3 illustrates that both deletion mutants were severely impaired in their raft localization.…”

“…We first transfected Fas-deficient Jurkat cells (J16 Rapo ref) with fulllength FasL or delta 1-40 FasL. Because deletion of the intracellular FasL domain increases FasL localization to the cell surface, 51 we again selected these clones for comparable FasL cell surface expression ( Figure 5D, bottom panel). When we compared their behavior in coculture experiments with the Fas-expressing JH6.2 Jurkat target cells, we found that the killing capacity of FasL delta 1-40 was significantly lower than that of full-length FasL ( Figure 5D, upper panel).…”

The Fas ligand intracellular domain is released by ADAM10 and SPPL2a cleavage in T-cells

“…Different Src homology 3 (SH3)-or WW-domain-containing proteins have been reported to bind to the FasL proline-rich domain in various assays, 1 among them members of the Fes/CIP4 homology/SH3 protein family, which regulate subcellular FasL distribution. 14,15 Nonetheless, the functional significance of the reported interactions for FasL reverse signaling remains unclear.…”

Development of novel chimeric transmembrane proteins for multimodality imaging of cancer cells

“…Chimeric receptors based on hFasL did not express well (data not shown) and thus the TM version was designed in an attempt to obtain better expression levels. As indicated in Figure 1A, hFasL-TM is devoid of several putative signaling motifs including a: casein kinase I site (SSASS), 42 a proline motif, which in some cells sequester FasL to a vesicle compartment, 43,44 and MMP-7 site (ELAELR). 45 Figure 1B is a schematic linear representation of the full-length chimeric proteins with the mammalian portion (cytosolic, transmembrane domain, and short extracellular linker) shown fused to the non-mammalian extracellular domain.…”

“…These proteins have been extensively characterized [34][35][36][37][38][39][40][41][42][43][44][45][46][47] and are known to be expressed on cancer cells. [48][49][50] The available detailed amino acid sequence information as well as expression patterns of altered native forms; e.g., truncated and mutated, of these two receptors [37][38][39][43][44][45]51 was crucial to our considerations of which alterations to the native sequences might give the best expression alternatives (Fig. 1).…”