[Show abstract][Hide abstract] ABSTRACT: The biogenesis of exosomes, small secreted vesicles involved in signalling processes, remains incompletely understood. Here, we report evidence that the syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin control the formation of exosomes. Syntenin interacts directly with ALIX through LYPX(n)L motifs, similarly to retroviral proteins, and supports the intraluminal budding of endosomal membranes. Syntenin exosomes depend on the availability of heparan sulphate, syndecans, ALIX and ESCRTs, and impact on the trafficking and confinement of FGF signals. This study identifies a key role for syndecan-syntenin-ALIX in membrane transport and signalling processes.
[Show abstract][Hide abstract] ABSTRACT: Epiboly, the spreading and the thinning of the blastoderm to cover the yolk cell and close the blastopore in fish embryos, is central to the process of gastrulation. Despite its fundamental importance, little is known about the molecular mechanisms that control this coordinated cell movement. By a combination of knockdown studies and rescue experiments in zebrafish (Danio rerio), we show that epiboly relies on the molecular networking of syntenin with syndecan heparan sulphate proteoglycans, which act as co-receptors for adhesion molecules and growth factors. Furthermore, we show that the interaction of syntenin with phosphatidylinositol 4,5-bisphosphate (PIP2) and with the small GTPase ADP-ribosylation factor 6 (Arf6), which regulate the endocytic recycling of syndecan, is necessary for epiboly progression. Analysis of the earliest cellular defects suggests a role for syntenin in the autonomous vegetal expansion of the yolk syncytial layer and the rearrangement of the actin cytoskeleton in extra-embryonic tissues, but not in embryonic cell fate determination. This study identifies the importance of the syntenin-syndecan-PIP2-Arf6 complex for the progression of fish epiboly and establishes its key role in directional cell movements during early development.
[Show abstract][Hide abstract] ABSTRACT: Phosphatidylinositol 5-phosphate (PtdIns5P) is emerging as a potential lipid messenger involved in several cell types, from plants to mammals. Expression of IpgD, a PtdIns(4,5)P(2) 4-phosphatase induces Src kinase and Akt, but not ERK activation and enhances interleukin II promoter activity in T-cells. Expression of a new PtdIns5P interacting domain blocks IpgD-induced T-cell activation and selective signaling molecules downstream of TCR triggering. Altogether, these data suggest that PtdIns5P may play a sensor function in setting the threshold of T-cell activation and contributing to maintain T-cell homeostasis.
[Show abstract][Hide abstract] ABSTRACT: Integrin-dependent adhesion sites consist of clustered integrins that transmit mechanical forces and provide signaling required
for cell survival and morphogenesis. Despite their importance, the regulation of integrin clustering by the cytoplasmic adapter
protein talin (Tal) and phosphatidylinositol (PI)-4,5-biphosphate (PI(4,5)P2) lipids nor their dynamic coupling to the actin cytoskeleton is fully understood. By using a Tal-dependent integrin clustering
assay in intact cells, we identified a PI(4,5)P2-binding basic ridge spanning across the F2 and F3 domains of the Tal head that regulates integrin clustering. Clustering
requires a new PI(4,5)P2-binding site in F2 and is negatively regulated by autoinhibitory interactions between F3 and the Tal rod (Tal-R). The release
of the Tal-R exposes a new β3-integrin–binding site in F3, enabling interaction with a membrane proximal acidic motif, which
involves the formation of salt bridges between K316 and K324 with E726 and D723, respectively. This interaction shields the β-integrin tail from reassociation with its α subunit, thereby maintaining the
integrin in a substrate-binding and clustering-competent form.
The Journal of Cell Biology 11/2009; 187(5):715-731. DOI:10.1083/jcb.200908134 · 9.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Downstream of tyrosine kinase (Dok) proteins Dok-1 and Dok-2 are involved in T cell homeostasis maintenance. Dok protein tyrosine phosphorylation plays a key role in establishing negative feedback loops of T cell signaling. These structurally related adapter molecules contain a pleckstrin homology (PH) domain generally acting as a lipid/protein-interacting module. We show that the presence of this PH domain is necessary for the tyrosine phosphorylation of Dok proteins and their negative functions in T cells. We find that Dok-1/Dok-2 PH domains bind in vitro to the rare phosphoinositide species, phosphatidylinositol 5-phosphate (PtdIns5P). Dok tyrosine phosphorylation correlates with PtdIns5P production in T cells upon TCR triggering. Furthermore, we demonstrate that PtdIns5P increase regulates Dok tyrosine phosphorylation in vivo. Together, our data identify a novel lipid mediator in T cell signaling and suggest that PH-PtdIns5P interactions regulate T cell responses.
The Journal of Immunology 05/2009; 182(7):3974-8. DOI:10.4049/jimmunol.0804172 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Arf6-specific exchange factor EFA6 coordinates membrane trafficking with actin cytoskeleton remodeling. It localizes to the plasma membrane where it catalyzes Arf6 activation and induces the formation of actin-based membrane ruffles. We have shown previously that the pleckstrin homology (PH) domain of EFA6 was responsible for its membrane localization. In this study we looked for the partners of the PH domain at the plasma membrane. Mutations of the conserved basic residues suspected to be involved in the binding to phosphoinositides redistribute EFA6-PH to the cytosol. In addition, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) breakdown also leads to the solubilization of EFA6-PH. Direct binding measured by surface plasmon resonance gives an apparent affinity of approximately 0.5 microm EFA6-PH for PI(4,5)P2. Moreover, we observed in vitro that the catalytic activity of EFA6 is strongly increased by PI(4,5)P2. These results indicate that the plasma membrane localization of EFA6-PH is based on its interaction with PI(4,5)P2, and this interaction is necessary for an optimal catalytic activity of EFA6. Furthermore, we demonstrated by fluorescence recovery after photobleaching and Triton X-100 detergent solubility experiments that in addition to the phophoinositides, EFA6-PH is linked to the actin cytoskeleton. We observed both in vivo and in vitro that EFA6-PH interacts directly with F-actin. Finally, we demonstrated that EFA6 could bind simultaneously filamentous actin and phospholipids vesicles. Our results explain how the exchange factor EFA6 via its PH domain could coordinate at the plasma membrane actin cytoskeleton organization with membrane trafficking.
[Show abstract][Hide abstract] ABSTRACT: Wnt signaling pathways are essential for embryonic patterning, and they are disturbed in a wide spectrum of diseases, including cancer. An unresolved question is how the different Wnt pathways are supported and regulated. We previously established that the postsynaptic density 95/disc-large/zona occludens (PDZ) protein syntenin binds to syndecans, Wnt coreceptors, and known stimulators of protein kinase C (PKC)alpha and CDC42 activity. Here, we show that syntenin also interacts with the C-terminal PDZ binding motif of several Frizzled Wnt receptors, without compromising the recruitment of Dishevelled, a key downstream Wnt-signaling component. Syntenin is coexpressed with cognate Frizzled during early development in Xenopus. Overexpression and down-regulation of syntenin disrupt convergent extension movements, supporting a role for syntenin in noncanonical Wnt signaling. Syntenin stimulates c-jun phosphorylation and modulates Frizzled 7 signaling, in particular the PKCalpha/CDC42 noncanonical Wnt signaling cascade. The syntenin-Frizzled 7 binding mode indicates syntenin can accommodate Frizzled 7-syndecan complexes. We propose that syntenin is a novel component of the Wnt signal transduction cascade and that it might function as a direct intracellular link between Frizzled and syndecans.
Molecular biology of the cell 05/2008; 19(4):1594-604. DOI:10.1091/mbc.E07-08-0832 · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The syndecan transmembrane proteoglycans are involved in the organization of the actin cytoskeleton and have important roles as cell surface receptors during cell-matrix interactions. We have shown that the syndecan-4 cytoplasmic domain (4L) forms oligomeric complexes that bind to and stimulate PKCalpha activity in the presence of PtdIns(4,5)P2, emphasizing the importance of multimerization in the regulation of PKCalpha activation. Oligomerization of the cytoplasmic domain of syndecan-4 is regulated either positively by PtdIns(4,5)P2 or negatively by phosphorylation of serine 183. Phosphorylation results in reduced PKCalpha activity by inhibiting PtdIns(4,5)P2-dependent oligomerization of the syndecan-4 cytoplasmic domain. Data from NMR and gel-filtration chromatography show that the phosphorylated cytoplasmic domain (p-4L) exists as a dimer, similar to 4L, but not as higher-order oligomers. NMR analysis showed that the overall conformation of p-4L is a compact intertwined dimer with an unusually symmetric clamp shape, and its molecular surface is mostly positively charged. The two parallel strands form a cavity in the center of the dimeric twist. An especially marked effect of phosphorylation of the syndecan-4 cytoplasmic domain is a dramatic conformational change near the C2 region that ablates an interaction site with the PDZ domain of syntenin. Wound healing studies further suggest that syndecan-4 phosphorylation might influence cell migration behavior. We conclude that the phosphorylation (Ser183) of syndecan-4 can play a critical role as a molecular switch to regulate its functions through conformational change.
[Show abstract][Hide abstract] ABSTRACT: Syndecans are heparan sulfate proteoglycans that modulate the activity of several growth factors and cell adhesion molecules. PDZ domains in the adaptor protein syntenin interact with syndecans and with the phosphoinositide PIP(2), which is involved in the regulation of the actin cytoskeleton and membrane trafficking. Here, we show that the syntenin PDZ domain-PIP(2) interaction controls Arf6-mediated syndecan recycling through endosomal compartments. FGF receptor accompanies syndecan along the syntenin-mediated recycling pathway, in a heparan sulfate- and FGF-dependent manner. Syndecans that cannot recycle via this pathway become trapped intracellularly and inhibit cell spreading. This syntenin-mediated syndecan recycling pathway may regulate the surface availability of a number of cell adhesion and signaling molecules.
[Show abstract][Hide abstract] ABSTRACT: PDZ (Postsynaptic density protein, Disc large, Zona occludens) domains are protein-protein interaction modules that predominate in submembranous scaffolding proteins. Recently, we showed that the PDZ domains of syntenin-1 also interact with phosphatidylinositol 4,5-bisphosphate (PIP2) and that this interaction controls the recruitment of the protein to the plasma membrane. Here we evaluate the general importance of PIP2-PDZ domain interactions. We report that most PDZ proteins bind weakly to PIP2, but that syntenin-2, the closest homolog of syntenin-1, binds with high affinity to PIP2 via its PDZ domains. Surprisingly, these domains target syntenin-2 to nuclear PIP2 pools, in nuclear speckles and nucleoli. Targeting to these sites is abolished by treatments known to affect these PIP2 pools. Mutational and domain-swapping experiments indicate that high-affinity binding to PIP2 requires both PDZ domains of syntenin-2, but that its first PDZ domain contains the nuclear PIP2 targeting determinants. Depletion of syntenin-2 disrupts the nuclear speckles-PIP2 pattern and affects cell survival and cell division. These findings show that PIP2-PDZ domain interactions can directly contribute to subnuclear assembly processes.
The EMBO Journal 08/2005; 24(14):2556-65. DOI:10.1038/sj.emboj.7600722 · 10.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Wnt signaling pathways,are essential for embryonic patterning and are disturbed in a wide spectrum of diseases, including cancer. An unresolved question is how the different Wnt pathways,are supported and regulated. We previously established that the PDZ protein syntenin binds to syndecans, Wnt co-receptors and known stimulators of PKCα and CDC42 activity. Here, we show that syntenin also interacts with the C-terminal PDZ binding motif ofseveral Frizzled Wnt receptors, without compromising the recruitment of Dishevelled, a key downstream ,Wnt-signaling component. Syntenin is co-expressed with cognate Frizzled during early development ,in Xenopus. Overexpression and down-regulation of syntenin disrupt convergent extension movements, supporting a role for syntenin in non-canonical