The Syk Kinase Promotes Mammary Epithelial Integrity and Inhibits Breast Cancer Invasion by Stabilizing the E-Cadherin/Catenin Complex.

While first discovered in immunoreceptor signaling, the Syk protein kinase behaves as a tumor and metastasis suppressor in epithelial cells. Its reduced expression in breast and other carcinomas is correlated with decreased survival and increased metastasis risk, but its action mechanism remains largely unknown. Using phosphoproteomics we found that Syk phosphorylated E-cadherin and α-, ß-, and p120-catenins on multiple tyrosine residues that concentrate at intercellular junctions. Increased Syk expression and activation enhanced E-cadherin/catenin phosphorylation, promoting their association and complex stability. In human breast cancer cells, Syk stimulated intercellular aggregation, E-cadherin recruitment and retention at adherens junctions, and promoted epithelial integrity, whereas it inhibited cell migration and invasion. Opposite effects were obtained with Syk knockdown or non-phosphorylatable mutant E-cadherin expression. Mechanistically, Syk stimulated the interaction of the E-cadherin/catenin complex with zonula occludens proteins and the actin cytoskeleton. Conditional Syk knockout in the lactating mouse mammary gland perturbed alveologenesis and disrupted E-cadherin localization at adherens junctions, corroborating the observations in cells. Hence, Syk is involved in the maintenance of the epithelial integrity of the mammary gland via the phosphorylation and stabilization of the E-cadherin/catenin adherens junction complex, thereby inhibiting cell migration and malignant tumor invasion.

Reconstruction and signal propagation analysis of the Syk signaling network in breast cancer cells.

The ability to build in-depth cell signaling networks from vast experimental data is a key objective of computational biology. The spleen tyrosine kinase (Syk) protein, a well-characterized key player in immune cell signaling, was surprisingly first shown by our group to exhibit an onco-suppressive function in mammary epithelial cells and corroborated by many other studies, but the molecular mechanisms of this function remain largely unsolved. Based on existing proteomic data, we report here the generation of an interaction-based network of signaling pathways controlled by Syk in breast cancer cells. Pathway enrichment of the Syk targets previously identified by quantitative phospho-proteomics indicated that Syk is engaged in cell adhesion, motility, growth and death. Using the components and interactions of these pathways, we bootstrapped the reconstruction of a comprehensive network covering Syk signaling in breast cancer cells. To generate in silico hypotheses on Syk signaling propagation, we developed a method allowing to rank paths between Syk and its targets. We first annotated the network according to experimental datasets. We then combined shortest path computation with random walk processes to estimate the importance of individual interactions and selected biologically relevant pathways in the network. Molecular and cell biology experiments allowed to distinguish candidate mechanisms that underlie the impact of Syk on the regulation of cortactin and ezrin, both involved in actin-mediated cell adhesion and motility. The Syk network was further completed with the results of our biological validation experiments. The resulting Syk signaling sub-networks can be explored via an online visualization platform.

Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells.

The lysosomal protease cathepsin D (Cath-D) is overproduced in breast cancer cells (BCC) and supports tumor growth and metastasis formation. Here, we describe the mechanism whereby Cath-D is accumulated in the nucleus of ERα-positive (ER+) BCC. We identified TRPS1 (tricho-rhino-phalangeal-syndrome 1), a repressor of GATA-mediated transcription, and BAT3 (Scythe/BAG6), a nucleo-cytoplasmic shuttling chaperone protein, as new Cath-D-interacting nuclear proteins. Cath-D binds to BAT3 in ER+ BCC and they partially co-localize at the surface of lysosomes and in the nucleus. BAT3 silencing inhibits Cath-D accumulation in the nucleus, indicating that Cath-D nuclear targeting is controlled by BAT3. Fully mature Cath-D also binds to full-length TRPS1 and they co-localize in the nucleus of ER+ BCC where they are associated with chromatin. Using the LexA-VP16 fusion co-activator reporter assay, we then show that Cath-D acts as a transcriptional repressor, independently of its catalytic activity. Moreover, microarray analysis of BCC in which Cath-D and/or TRPS1 expression were silenced indicated that Cath-D enhances TRPS1-mediated repression of several TRPS1-regulated genes implicated in carcinogenesis, including PTHrP, a canonical TRPS1 gene target. In addition, co-silencing of TRPS1 and Cath-D in BCC affects the transcription of cell cycle, proliferation and transformation genes, and impairs cell cycle progression and soft agar colony formation. These findings indicate that Cath-D acts as a nuclear transcriptional cofactor of TRPS1 to regulate ER+ BCC proliferation and transformation in a non-proteolytic manner.

Centrosomal targeting of Syk kinase is controlled by its catalytic activity and depends on microtubules and the dynein motor.

The nonreceptor Syk kinase is detected in epithelial cells, where it acts as a tumor suppressor, in addition to its well-established role in immunoreceptor-based signal transduction in hematopoietic cells. Thus, several carcinomas and melanomas have subnormal concentrations of Syk. Although Syk is mainly localized at the plasma membrane, it is also present in centrosomes, where it is involved in the control of cell division. The mechanisms responsible for its centrosomal localization and action are unknown. We used wild-type and mutant fluorescent Syk fusion proteins in live-cell imaging (fluorescence recovery after photobleaching, total internal reflection fluorescence, and photoactivation) combined with mathematical modeling to demonstrate that Syk is actively transported to the centrosomes via the microtubules and that this transport depends on the dynein/dynactin molecular motor. Syk can only target the centrosomes if its kinase activity is intact and it is catalytically active at the centrosomes. We showed that the autophosphorylated Y130 Syk residue helps to uncouple Syk from the plasma membrane and to promote its translocation to the centrosome, suggesting that the subcellular location of Syk depends on its autophosphorylation on specific tyrosine residues. We have thus established the details of how Syk is trafficked intracellularly and found evidence that its targeting to the centrosomes is controlled by autophosphorylation.

Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis.

Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs.

The Syk tyrosine kinase localizes to the centrosomes and negatively affects mitotic progression.

We showed previously that the spleen tyrosine kinase Syk is expressed by mammary epithelial cells and that it suppresses malignant growth of breast cancer cells. The exact molecular mechanism of its tumor-suppressive activity remains, however, to be identified. Here, we show that Syk colocalizes and copurifies with the centrosomal component gamma-tubulin and exhibits a catalytic activity within the centrosomes. Moreover, its centrosomal localization depends on its intact kinase activity. Centrosomal Syk expression is persistent in interphase but promptly drops during mitosis, obviously resulting from its ubiquitinylation and proteasomal degradation. Conversely, unrestrained exogenous expression of a fluorescently tagged Discosoma sp. red fluorescent protein (DsRed)-Syk chimera engenders abnormal cell division and cell death. Transient DsRed-Syk overexpression triggers an abrupt cell death lacking hallmarks of classic apoptosis but reminiscent of mitotic catastrophe. Surviving stable DsRed-Syk-transfected cells exhibit multipolar mitotic spindles and contain multiple abnormally sized nuclei and supernumerary centrosomes, revealing anomalous cell division. Taken together, these results show that Syk is a novel centrosomal kinase that negatively affects cell division. Its expression is strictly controlled in a spatiotemporal manner, and centrosomal Syk levels need to decline to allow customary progression of mitosis.

HIV-1 Tat enters T cells using coated pits before translocating from acidified endosomes and eliciting biological responses.

The HIV-1 Tat protein is secreted by infected cells. Extracellular Tat can affect bystander uninfected T cells and induce numerous biological responses such as apoptosis and cytokine secretion. Tat is likely involved in several immune disorders during AIDS. Nevertheless, it is not known whether Tat triggers cell responses directly upon binding to signaling receptors at the plasma membrane or after delivery to the cytosol. The pathway that enables Tat to reach the cytosol is also unclear. Here we visualized Tat within T-cell-coated pits and endosomes. Moreover, inhibitors of clathrin/AP-2-mediated uptake such as chlorpromazine, activated RhoA, or dominant-negative mutants of Eps15, intersectin, dynamin, or rab5 impaired Tat delivery to the cytosol by preventing its endocytosis. Molecules neutralizing low endosomal pH or Hsp90 inhibitors abolished Tat entry at a later stage by blocking its endosomal translocation, as directly shown using a cell-free translocation assay. Finally, endosomal pH neutralization prevented Tat from inducing T-cell responses such as NF-kappaB activation, apoptosis, and interleukin secretion, indicating that cytosolic delivery is required for Tat signaling. Hence, Tat enters T cells essentially like diphtheria toxin, using clathrin-mediated endocytosis before low-pH-induced and Hsp90-assisted endosomal translocation. Cell responses are then induced from the cytosol.

Delta-promoted filopodia mediate long-range lateral inhibition in Drosophila.

Drosophila thoracic mechanosensory bristles originate from cells that are singled out from 'proneural' groups of competent epithelial cells. Neural competence is restricted to individual sensory organ precursors (SOPs) by Delta/Notch-mediated 'lateral inhibition', whereas other cells in the proneural field adopt an epidermal fate. The precursors of the large macrochaetes differentiate separately from individual proneural clusters that comprise about 20-30 cells or as heterochronic pairs from groups of more than 100 cells, whereas the precursors of the small regularly spaced microchaetes emerge from even larger proneural fields. This indicates that lateral inhibition might act over several cell diameters; it was difficult to reconcile with the fact that the inhibitory ligand Delta is membrane-bound until the observation that SOPs frequently extend thin processes offered an attractive hypothesis. Here we show that the extension of these planar filopodia--a common attribute of wing imaginal disc cells--is promoted by Delta and that their experimental suppression reduces Notch signalling in distant cells and increases bristle density in large proneural groups, showing that these membrane specializations mediate long-range lateral inhibition.

A conserved C-terminal domain of EFA6-family ARF6-guanine nucleotide exchange factors induces lengthening of microvilli-like membrane protrusions.

We recently reported the identification of EFA6 (exchange factor for ARF6), a brain-specific Sec7-domain-containing guanine nucleotide exchange factor that works specifically on ARF6. Here, we have characterized the product of a broadly expressed gene encoding a novel 1056 amino-acid protein that we have named EFA6B. We show that EFA6B, which contains a Sec7 domain that is highly homologous to EFA6, works as an ARF6-specific guanine exchange factor in vitro. Like EFA6, which will be referred to as EFA6A from now on, EFA6B is involved in membrane recycling and colocalizes with ARF6 in actin-rich membrane ruffles and microvilli-like protrusions on the dorsal cell surface in transfected baby hamster kidney cells. Strikingly, homology between EFA6A and EFA6B is not limited to the Sec7 domain but extends to an adjacent pleckstrin homology (PH) domain and a approximately 150 amino-acid C-terminal region containing a predicted coiled coil motif. Association of EFA6A with membrane ruffles and microvilli-like structures depends on the PH domain, which probably interacts with phosphatidylinositol 4,5-biphosphate. Moreover, we show that overexpression of the PH domain/C-terminal region of EFA6A or EFA6B in the absence of the Sec7 domain promotes lengthening of dorsal microvillar protrusions. This morphological change requires the integrity of the coiled-coil motif. Lastly, database analysis reveals that the EFA6-family comprises at least four members in humans and is conserved in multicellular organisms throughout evolution. Our results suggest that EFA6 family guanine exchange factors are modular proteins that work through the coordinated action of the catalytic Sec7 domain to promote ARF6 activation, through the PH domain to regulate association with specific subdomains of the plasma membrane and through the C-terminal region to control actin cytoskeletal reorganization.