Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis
JOURNAL OF CLINICAL INVESTIGATION
2014; 124 (12): 5275-5290
Establishment of epithelial polarity - GEF who's minding the GAP?
Journal of cell science
2014; 127 (Pt 15): 3205-15
Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing sarcoma-associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes.
View details for DOI 10.1172/JC172124
View details for Web of Science ID 000345677200020
View details for PubMedID 25401475
The Rho Guanine Nucleotide Exchange Factor Syx Regulates the Balance of Dia and ROCK Activities To Promote Polarized-Cancer-Cell Migration.
Molecular and cellular biology
2013; 33 (24): 4909-4918
Cell polarization is a fundamental process that underlies epithelial morphogenesis, cell motility, cell division and organogenesis. Loss of polarity predisposes tissues to developmental disorders and contributes to cancer progression. The formation and establishment of epithelial cell polarity is mediated by the cooperation of polarity protein complexes, namely the Crumbs, partitioning defective (Par) and Scribble complexes, with Rho family GTPases, including RhoA, Rac1 and Cdc42. The activation of different GTPases triggers distinct downstream signaling pathways to modulate protein-protein interactions and cytoskeletal remodeling. The spatio-temporal activation and inactivation of these small GTPases is tightly controlled by a complex interconnected network of different regulatory proteins, including guanine-nucleotide-exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine-nucleotide-dissociation inhibitors (GDIs). In this Commentary, we focus on current understanding on how polarity complexes interact with GEFs and GAPs to control the precise location and activation of Rho GTPases (Crumbs for RhoA, Par for Rac1, and Scribble for Cdc42) to promote apical-basal polarization in mammalian epithelial cells. The mutual exclusion of GTPase activities, especially that of RhoA and Rac1, which is well established, provides a mechanism through which polarity complexes that act through distinct Rho GTPases function as cellular rheostats to fine-tune specific downstream pathways to differentiate and preserve the apical and basolateral domains. This article is part of a Minifocus on Establishing polarity. For further reading, please see related articles: 'ERM proteins at a glance' by Andrea McClatchey (J. Cell Sci. 127, 3199-3204). 'Integrins and epithelial cell polarity' by Jessica Lee and Charles Streuli (J. Cell Sci. 127, 3217-3215).
View details for DOI 10.1242/jcs.153197
View details for PubMedID 24994932
TEM4 is a junctional Rho GEF required for cell-cell adhesion, monolayer integrity and barrier function
JOURNAL OF CELL SCIENCE
2013; 126 (15): 3271-3277
The role of RhoA in promoting directed cell migration has been complicated by studies showing that it is activated both in the front and the rear of migrating cells. We report here that the RhoA-specific guanine nucleotide exchange factor Syx is required for the polarity of actively migrating brain and breast tumor cells. This function of Syx is mediated by the selective activation of the RhoA downstream effector Dia1, the subsequent reorganization of microtubules, and the downregulation of focal adhesions and actin stress fibers. The data argue that directed cell migration requires the precise spatiotemporal regulation of Dia1 and ROCK activities in the cell. The recruitment of Syx to the cell membrane and the subsequent selective activation of Dia1 signaling, coupled with the suppression of ROCK and activation of cofilin-mediated actin reorganization, plays a key role in establishing cell polarity during directed cell migration.
View details for DOI 10.1128/MCB.00565-13
View details for PubMedID 24126053
Phosphorylation-mediated 14-3-3 Protein Binding Regulates the Function of the Rho-specific Guanine Nucleotide Exchange Factor (RhoGEF) Syx
JOURNAL OF BIOLOGICAL CHEMISTRY
2013; 288 (9): 6640-6650
Signaling events mediated by Rho family GTPases orchestrate cytoskeletal dynamics and cell junction formation. The activation of Rho GTPases is tightly regulated by guanine-nucleotide-exchange factors (GEFs). In this study, we identified a novel Rho-specific GEF called TEM4 (tumor endothelial marker 4) that associates with multiple members of the cadherin-catenin complex and with several cytoskeleton-associated proteins. Depending on confluence, TEM4 localized to either actin stress fibers or areas of cell-cell contact. The junctional localization of TEM4 was independent of actin binding. Depletion of endogenous TEM4 by shRNAs impaired Madin-Darby canine kidney (MDCK) and human umbilical vein endothelial cell (HUVEC) cell junctions, disrupted MDCK acini formation in 3D culture and negatively affected endothelial barrier function. Taken together, our findings implicate TEM4 as a novel and crucial junctional Rho GEF that regulates cell junction integrity and epithelial and endothelial cell function.
View details for DOI 10.1242/jcs.123869
View details for Web of Science ID 000322570200005
View details for PubMedID 23729734
p120 Catenin: An Essential Regulator of Cadherin Stability, Adhesion-Induced Signaling, and Cancer Progression
MOLECULAR BIOLOGY OF CADHERINS
2013; 116: 409-432
Syx is a Rho-specific guanine nucleotide exchange factor (GEF) that localizes at cell-cell junctions and promotes junction stability by activating RhoA and the downstream effector Diaphanous homolog 1 (Dia1). Previously, we identified several molecules, including 14-3-3 proteins, as Syx-interacting partners. In the present study, we show that 14-3-3 isoforms interact with Syx at both its N- and C-terminal regions in a phosphorylation-dependent manner. We identify the protein kinase D-mediated phosphorylation of serine 92 on Syx, and additional phosphorylation at serine 938, as critical sites for 14-3-3 association. Our data indicate that the binding of 14-3-3 proteins inhibits the GEF activity of Syx. Furthermore, we show that phosphorylation-deficient, 14-3-3-uncoupled Syx exhibits increased junctional targeting and increased GEF activity, resulting in the strengthening of the circumferential junctional actin ring in Madin-Darby canine kidney cells. These findings reveal a novel means of regulating junctional Syx localization and function by phosphorylation-induced 14-3-3 binding and further support the importance of Syx function in maintaining stable cell-cell contacts.
View details for DOI 10.1074/jbc.M112.432682
View details for Web of Science ID 000315820700063
View details for PubMedID 23335514
Rho GEFs in endothelial junctions: Effector selectivity and signaling integration determine junctional response.
2013; 1 (5): e27132
p120 catenin is the best studied member of a subfamily of proteins that associate with the cadherin juxtamembrane domain to suppress cadherin endocytosis. p120 also recruits the minus ends of microtubules to the cadherin complex, leading to junction maturation. In addition, p120 regulates the activity of Rho family GTPases through multiple interactions with Rho GEFs, GAPs, Rho GTPases, and their effectors. Nuclear signaling is affected by the interaction of p120 with Kaiso, a transcription factor regulating Wnt-responsive genes as well as transcriptionally repressing methylated promoters. Multiple alternatively spliced p120 isoforms and complex phosphorylation events affect these p120 functions. In cancer, reduced p120 expression correlates with reduced E-cadherin function and with tumor progression. In contrast, in tumor cells that have lost E-cadherin expression, p120 promotes cell invasion and anchorage-independent growth. Furthermore, p120 is required for Src-induced oncogenic transformation and provides a potential target for future therapeutic interventions.
View details for DOI 10.1016/B978-0-12-394311-8.00018-2
View details for Web of Science ID 000318528500019
View details for PubMedID 23481205
VEGF and Angiopoietin-1 exert opposing effects on cell junctions by regulating the Rho GEF Syx
JOURNAL OF CELL BIOLOGY
2012; 199 (7): 1103-1115
Rho GTPases are cytoskeleton-regulating proteins that mediate the formation of intercellular junctions. Their localized activation by Rho GEFs (guanine-nucleotide exchange factors) and the selective activation of downstream effectors have emerged as areas of active research in the cell adhesion field. We reported recently that the Rho-specific GEFs Syx (Synectin-binding RhoA exchange factor) and TEM4 (Tumor Endothelial Marker 4) are both essential for endothelial junction maturation and barrier function. Syx is recruited to cell contacts via its C-terminal PDZ binding motif and it's interaction with Mupp1 and the Crumbs polarity complex, while the junctional localization of TEM4 requires it's N-terminal domain and interaction with the cadherin-catenin complex. Our findings support multiple roles for RhoA in junction formation and maintenance. They also suggest that selective coupling of RhoA activation to Dia1 and/or ROCK signaling is critical for determining endothelial junction integrity.
View details for DOI 10.4161/tisb.27132
View details for PubMedID 24790803
Vascular endothelial growth factor (VEGF) and Ang1 (Angiopoietin-1) have opposing effects on vascular permeability, but the molecular basis of these effects is not fully known. We report in this paper that VEGF and Ang1 regulate endothelial cell (EC) junctions by determining the localization of the RhoA-specific guanine nucleotide exchange factor Syx. Syx was recruited to junctions by members of the Crumbs polarity complex and promoted junction integrity by activating Diaphanous. VEGF caused translocation of Syx from cell junctions, promoting junction disassembly, whereas Ang1 maintained Syx at the junctions, inducing junction stabilization. The VEGF-induced translocation of Syx from EC junctions was caused by PKD1 (protein kinase D1)-mediated phosphorylation of Syx at Ser(806), which reduced Syx association to its junctional anchors. In support of the pivotal role of Syx in regulating EC junctions, syx(-/-) mice had defective junctions, resulting in vascular leakiness, edema, and impaired heart function.
View details for DOI 10.1083/jcb.201207009
View details for Web of Science ID 000313154500010
View details for PubMedID 23253477