Binding of the intracellular Fas ligand (FasL) domain to the adaptor protein PSTPIP results in a cytoplasmic localization of FasL

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 domain-containing 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 FasL.PSTPIP.PTP-PEST complexes which may contribute to FasL reverse signaling.     

Palmitoylation of human FasL modulates its cell death-inducing function

Fas ligand (FasL) is a transmembrane protein that regulates cell death in Fas-bearing cells. FasL-mediated cell death is essential for immune system homeostasis and the elimination of viral or transformed cells. Because of its potent cytotoxic activity, FasL expression at the cell surface is tightly regulated, for example, via processing by ADAM10 and SPPL2a generating soluble FasL and the intracellular fragments APL (ADAM10-processed FasL form) and SPA (SPPL2a-processed APL). In this study, we report that FasL processing by ADAM10 counteracts Fas-mediated cell death and is strictly regulated by membrane localization, interactions and modifications of FasL. According to our observations, FasL processing occurs preferentially within cholesterol and sphingolipid-rich nanodomains (rafts) where efficient Fas–FasL contact occurs, Fas receptor and FasL interaction is also required for efficient FasL processing, and FasL palmitoylation, which occurs within its transmembrane domain, is critical for efficient FasL-mediated killing and FasL processing.     

FCH/Cdc15 domain determines distinct subcellular localization of NOSTRIN

NOSTRIN, an NO synthase binding protein, belongs to the PCH family of proteins, exposing a typical domain structure. While its SH3 domain and the C-terminal coiled-coil region cc2 have been studied earlier, the function of the N-terminal half comprising a Cdc15 domain with an FCH (Fes/CIP homology) region followed by a coiled-coil stretch cc1 is unknown. Here, we show that the FCH region is necessary and sufficient for membrane association of NOSTRIN, whereas the Cdc15 domain further specifies subcellular distribution of the protein. Thus, the FCH region and the Cdc15 domain fulfill complementary functions in subcellular targeting of NOSTRIN.     

Insights into the molecular regulation of FasL (CD178) biology

Fas ligand (FasL, CD95L, APO-1L, CD178, TNFSF6, APT1LG1) is the key death factor of receptor-triggered programmed cell death in immune cells. FasL/Fas-dependent apoptosis plays a pivotal role in activation-induced cell death, termination of immune responses, elimination of autoreactive cells, cytotoxic effector function of T and NK cells, and the establishment of immune privilege. Deregulation or functional impairment of FasL threatens the maintenance of immune homeostasis and defense and results in severe autoimmunity. In addition, FasL has been implicated as an accessory or costimulatory receptor in T cell activation. The molecular mechanisms underlying this reverse signaling capacity are, however, poorly understood and still controversially discussed. Many aspects of FasL biology have been ascribed to selective protein-protein interactions mediated by a unique polyproline region located in the membrane-proximal intracellular part of FasL. Over the past decade, we and others identified a large number of putative FasL-interacting molecules that bind to this polyproline stretch via Src homology 3 or WW domains. Individual interactions were analyzed in more detail and turned out to be crucial for the lysosomal storage, the transport and the surface appearance of the death factor and potentially also for reverse signaling. This review summarizes the work in the framework of the Collaborative Research Consortium 415 (CRC 415) and provides facts and hypotheses about FasL-interacting proteins and their potential role in FasL biology.     

Fibroblast Growth Factor Receptor Like-1 (FGFRL1) Interacts with SHP-1 Phosphatase at Insulin Secretory Granules and Induces Beta-cell ERK1/2 Protein Activation

FGFRL1 is a newly identified member of the fibroblast growth factor receptor (FGFR) family expressed in adult pancreas. Unlike canonical FGFRs that initiate signaling via tyrosine kinase domains, the short intracellular sequence of FGFRL1 consists of a putative Src homology domain-2 (SH2)-binding motif adjacent to a histidine-rich C terminus. As a consequence of nonexistent kinase domains, FGFRL1 has been postulated to act as a decoy receptor to inhibit canonical FGFR ligand-induced signaling. In pancreatic islet beta-cells, canonical FGFR1 signaling affects metabolism and insulin processing. This study determined beta-cell expression of FGFRL1 as well as consequent effects on FGFR1 signaling and biological responses. We confirmed FGFRL1 expression at the plasma membrane and within distinct intracellular granules of both primary beta-cells and βTC3 cells. Fluorescent protein-tagged FGFRL1 (RL1) induced a significant ligand-independent increase in MAPK signaling. Removal of the histidine-rich domain (RL1-ΔHis) or entire intracellular sequence (RL1-ΔC) resulted in greater retention at the plasma membrane and significantly reduced ligand-independent ERK1/2 responses. The SHP-1 phosphatase was identified as an RL1-binding substrate. Point mutation of the SH2-binding motif reduced the ability of FGFRL1 to bind SHP-1 and activate ERK1/2 but did not affect receptor localization to insulin secretory granules. Finally, overexpression of RL1 increased cellular insulin content and matrix adhesion. Overall, these data suggest that FGFRL1 does not function as a decoy receptor in beta-cells, but rather it enhances ERK1/2 signaling through association of SHP-1 with the receptor''s intracellular SH2-binding motif.     

The adaptor protein Grb2 regulates cell surface Fas ligand in Schwann cells

Fas Ligand (FasL, CD178) is a cytokine that may be secreted or expressed as a transmembrane ligand at the cell surface, and induces apoptosis by binding to the “death receptor” Fas (CD95). Here, we show that Grb2, an SH3 domain-containing adaptor protein, binds to the proline-rich domain of FasL and regulates its cell surface expression. We found that knocking down Grb2 expression decreased the amount of FasL at the cell surface and increased the abundance of intracellular vesicles containing FasL. Furthermore, we showed that Grb2 acts as an adaptor for FasL to interact with adaptin β, a molecule known to regulate trafficking. Our data reveal that Grb2 facilitates the association of FasL with adaptinβ, and promotes sorting of FasL to the cell surface. As FasL is a potent regulator of cell death, dynamic regulation of its cell surface localization is critical for controlling local tissue remodeling and inflammation.     

Serine 6 of Lck tyrosine kinase: a critical site for Lck myristoylation, membrane localization, and function in T lymphocytes

Lck is a member of the Src family kinases expressed predominantly in T cells, and plays a pivotal role in TCR-mediated signal transduction. Myristoylation of glysine 2 in the N-terminal Src homology 4 (SH4) domain of Lck is essential for membrane localization and function. In this study, we examined a site within the SH4 domain of Lck regulating myristoylation, membrane localization, and function of Lck. A Lck mutant in which serine 6 (Ser6) was substituted by an alanine was almost completely cytosolic in COS-7 cells, and this change of localization was associated with a drastic inhibition of myristoylation in this mutant. To assess the role of Ser6 of Lck in T cell function, we established stable transfectants expressing various Lck mutants using Lck-negative JCaM1 cells. The Lck mutant of Ser6 to alanine, most of which did not target to the plasma membrane, was not able to reconstitute TCR-mediated signaling events in JCaM1 cells, as analyzed by tyrosine phosphorylation of intracellular proteins and CD69 expression. These results demonstrate that Ser6 is a critical factor for Lck myristoylation, membrane localization, and function in T cells, presumably because the residue is important for N-myristoyl transferase recognition.     

Regulation of the bovine kidney microsomal chloride channel p64 by p59fyn, a Src family tyrosine kinase

p64 is a chloride channel of intracellular membranes which is present in regulated secretory vesicles. Mechanisms by which the p64 channel could be regulated are largely unknown. p59(fyn) is a non-receptor tyrosine kinase of the Src family that has been implicated in a variety of intracellular signaling events. The N-terminal portion of p64 has several potential binding sites for Src family SH2 domains. In this paper, we demonstrate that p64 becomes tyrosine phosphorylated when co-expressed with p59(fyn) in HeLa cells. We show that co-expression of p64 with p59(fyn) renders p64 a ligand for the SH2 domain of p59(fyn) and this SH2 binding is eliminated by treating p64 with alkaline phosphatase. Using site-directed mutagenesis, we find that tyrosine 33 in the p64 sequence is necessary for SH2 binding. We also characterized p64-p59(fyn) interactions using native material from bovine kidney. We found that a small fraction of native kidney p64 can bind Fyn SH2 in vitro. Immunoprecipitation of p64 from solubilized kidney membranes yields a kinase activity with the same mobility by SDS-polyacrylamide gel electrophoresis as authentic bovine p59(fyn). Finally, we demonstrate that co-expression of p64 and p59(fyn) in HeLa cells results in enhanced p64-associated chloride channel activity.     

Attenuation of cell adhesion in lymphocytes is regulated by CYTIP,a protein which mediates signal complex sequestration

An important theme in molecular cell biology is the regulation of protein recruitment to the plasma membrane. Fundamental biological processes such as proliferation, differentiation or leukocyte functions are initiated and controlled through the reversible binding of signaling proteins to phosphorylated membrane components. This is mediated by specialized interaction modules, such as SH2 and PH domains. Cytohesin-1 is an intracellular guanine nucleotide exchange factor, which regulates leukocyte adhesion. The activity of cytohesin-1 is controlled by phospho inositide-dependent membrane recruitment. An interacting protein was identified, the expression of which is upregulated by cytokines in hematopoietic cells. This molecule, CYTIP, is also recruited to the cell cortex by integrin signaling via its PDZ domain. However, stimulation of Jurkat cells with phorbol ester results in re-localization of CYTIP to the cytoplasm, and membrane detachment of cytohesin-1 strictly requires co-expression of CYTIP. Consequently, stimulated adhesion of Jurkat cells to intracellular adhesion molecule-1 is repressed by CYTIP. These findings outline a novel mechanism of signal chain abrogation through sequestration of a limiting component by specific protein-protein interactions.     

Rapostlin is a novel effector of Rnd2 GTPase inducing neurite branching

Rho family GTPases are central regulators of neuronal morphology. Recently, Rnd proteins, Rnd1, Rnd2, and Rnd3/RhoE, have been identified as new members of Rho family GTPases. Of these, Rnd2 is specifically expressed in neurons in brain; however, the signaling pathways of Rnd2 are not known. Here we have performed a yeast two-hybrid screen using Rnd2 as a bait and identified a novel Rnd2-effector protein, expressed predominantly in brain. We named it Rapostlin (apostle of Rnd2). Rapostlin has two functional domains, Fer-CIP4 homology (FCH) domain at the amino terminus and SH3 (Src homology 3) domain at the carboxyl terminus. In in vitro binding assays, Rapostlin specifically binds to Rnd2 among the Rho family GTPases in a GTP-dependent manner, and the Rnd2-binding domain of Rapostlin is localized between FCH and SH3 domains. Rapostlin directly binds to microtubules, and the amino-terminal region containing the FCH domain of Rapostlin is essential for this interaction. In PC12 cells, Rapostlin induces neurite branching in response to Rnd2, and at least the amino-terminal region of Rapostlin is necessary for this activity. Therefore, Rapostlin is the first effector of Rnd2, regulating neurite branch formation.     

Yeast dynamin Vps1 and amphiphysin Rvs167 function together during endocytosis

Dynamins are a conserved family of proteins involved in many membrane fusion and fission events. Previously, the dynamin-related protein Vps1 was shown to localize to endocytic sites, and yeast carrying deletions for genes encoding both the BAR domain protein Rvs167 and Vps1 had a more severe endocytic scission defect than either deletion alone. Vps1 and Rvs167 localize to endocytic sites at the onset of invagination and disassemble concomitant with inward vesicle movement. Rvs167-GFP localization is reduced in cells lacking vps1 suggesting that Vps1 influences Rvs167 association with the endocytic complex. Unlike classical dynamins, Vps1 does not have a proline-arginine domain that could interact with SH3 domain-containing proteins. Thus, while Rvs167 has an SH3 domain, it is not clear how an interaction would be mediated. Here, we demonstrate an interaction between Rvs167 SH3 domain and the single type I SH3-binding motif in Vps1. Mutant Vps1 that cannot bind Rvs167 rescues all membrane fusion/fission functions associated with Vps1 except for endocytic function, demonstrating the specificity and mechanistic importance of the interaction. In vitro, an Rvs161/Rvs167 heterodimer can disassemble Vps1 oligomers. Overall, the data support the idea that Vps1 and the amphiphysins function together to mediate scission during endocytosis in yeast.     

P-selectin targeting to secretory lysosomes of Rbl-2H3 cells.

The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes.     

Camptothecin induces the transit of FasL trimers to the cell surface in apoptotic HEp-2 cells

Fas ligand (L) is a membrane protein from the tumor necrosis factor (TNF) family. It induces apoptosis upon contact with its Fas/CD95/APO1 receptor. Trimerization of FasL on the surface of effector cells is essential in the binding of the Fas trimer of the target cells. The receptor then recruits an adaptor and caspase-like proteins which lead apoptosis. This paper reports on the fate of FasL in HEp-2 cells committed to apoptosis by induction with campthotecin. Our main results demonstrated that in non-apoptotic cells, FasL aggregates in the cytoplasm forming trimers of 120 kDa. Apoptosis increases the trimeric FasL species, but also induces its dissociation into monomers of 35 kDa. In conclusion, camptothecin appears to perturb the Fas and FasL segregation in the cytoplasm by promoting the transit of FasL to the cell surface, thus fostering a process of autocrine or paracrine apoptosis. FasL is trimerized prior to Fas/FasL complex formation, and after apoptosis, FasL undergoes an intense turnover.     

Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis

The conserved FER-CIP4 homology (FCH) domain is found in the pombe Cdc15 homology (PCH) protein family members, including formin-binding protein 17 (FBP17). However, the amino acid sequence homology extends beyond the FCH domain. We have termed this region the extended FC (EFC) domain. We found that FBP17 coordinated membrane deformation with actin cytoskeleton reorganization during endocytosis. The EFC domains of FBP17, CIP4, and other PCH protein family members show weak homology to the Bin-amphiphysin-Rvs (BAR) domain. The EFC domains bound strongly to phosphatidylserine and phosphatidylinositol 4,5-bisphosphate and deformed the plasma membrane and liposomes into narrow tubules. Most PCH proteins possess an SH3 domain that is known to bind to dynamin and that recruited and activated neural Wiskott-Aldrich syndrome protein (N-WASP) at the plasma membrane. FBP17 and/or CIP4 contributed to the formation of the protein complex, including N-WASP and dynamin-2, in the early stage of endocytosis. Furthermore, knockdown of endogenous FBP17 and CIP4 impaired endocytosis. Our data indicate that PCH protein family members couple membrane deformation to actin cytoskeleton reorganization in various cellular processes.     

Src kinase activity and SH2 domain regulate the dynamics of Src association with lipid and protein targets

Src functions depend on its association with the plasma membrane and with specific membrane-associated assemblies. Many aspects of these interactions are unclear. We investigated the functions of kinase, SH2, and SH3 domains in Src membrane interactions. We used FRAP beam-size analysis in live cells expressing a series of c-Src-GFP proteins with targeted mutations in specific domains together with biochemical experiments to determine whether the mutants can generate and bind to phosphotyrosyl proteins. Wild-type Src displays lipid-like membrane association, whereas constitutively active Src-Y527F interacts transiently with slower-diffusing membrane-associated proteins. These interactions require Src kinase activity and SH2 binding, but not SH3 binding. Furthermore, overexpression of paxillin, an Src substrate with a high cytoplasmic population, competes with membrane phosphotyrosyl protein targets for binding to activated Src. Our observations indicate that the interactions of Src with lipid and protein targets are dynamic and that the kinase and SH2 domain cooperate in the membrane targeting of Src.     

The Lck SH3 domain negatively regulates localization to lipid rafts through an interaction with c-Cbl.

Lck is a member of the Src family of protein-tyrosine kinases and is essential for T cell development and function. Lck is localized to the inner surface of the plasma membrane and partitions into lipid rafts via dual acylation on its N terminus. We have tested the role of Lck binding domains in regulating Lck localization to lipid rafts. A form of Lck containing a point mutation inactivating the SH3 domain (W97ALck) was preferentially localized to lipid rafts compared with wild type or SH2 domain-inactive (R154K) Lck when expressed in Lck-deficient J.CaM1 cells. W97ALck incorporated more of the radioiodinated version of palmitic acid, 16-[(125)I]iodohexadecanoic acid. Overexpression of c-Cbl, a ligand of the Lck SH3 domain, depleted Lck from lipid rafts in Jurkat cells. Additionally, Lck localization to lipid rafts was enhanced in c-Cbl-deficient T cells. The association of Lck with c-Cbl in vivo required a functional SH3 domain. These results suggest a model whereby the SH3 domain negatively regulates basal localization of Lck to lipid rafts via association with c-Cbl.     

SIMPLE/LITAF expression induces the translocation of the ubiquitin ligase itch towards the lysosomal compartments

LITAF is a small cellular protein with an unknown function. The C-terminus of LITAF contains a highly conserved domain termed the SIMPLE-like domain (SLD), while the N-terminus contains two PPXY motifs that mediate protein-protein interactions with WW-domain containing proteins. LITAF also harbors two endosome/lysosome targeting sequences at its C-terminus, but there has been conflicting reports regarding its intracellular localization. Here, we demonstrate that LITAF is localized to the late endosome/lysosomal compartment in a variety of cell lines. We also show that Itch, a WW-domain containing protein, and LITAF strongly interact and that this interaction depends on the two PPXY motifs in the N-terminus of LITAF. Interestingly, co-expression of LITAF with Itch induces major changes in Itch intracellular localization, bringing Itch from the trans-Golgi network to lysosomes. We show that this re-localization is dependent upon the interaction with the PPXY sequences of LITAF, since disruption of these binding motifs completely abrogates Itch re-localization.     

Characterization of interactions of Nck with Sos and dynamin

One of the adaptor proteins, Nck, comprises a single SH2 domain and three SH3 domains that are important in protein-protein interactions. The in vivo association of Nck with the guanine nucleotide exchange factor Sos has been well documented; however, the precise nature of the interaction is unclear. To determine which SH3 domains are involved in the Nck-Sos interaction, individual SH3 domains of Nck were generated as glutathione S-transferase fusion proteins. We found that exclusively the third (C-terminal) SH3 domain of Nck has the ability to bind to Sos. In addition, in [35S]methionine labelled K562 cells, a 100,000 Mr protein was found to be associated with the third SH3 domain of Nck. This protein was identified as dynamin, a GTP-binding protein that has been implicated in clathrin-coated vesicle formation. Dynamin and Nck co-precipitated when cell lysates were immunoprecipitated with anti-Nck antibody. These data suggest that Nck may contribute to Ras activation and the function of dynamin in membrane trafficking through its third SH3 domain.