The Experts below are selected from a list of 5577 Experts worldwide ranked by ideXlab platform
Scott A Weed - One of the best experts on this subject based on the ideXlab platform.
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Cortactin phosphorylation by casein kinase 2 regulates actin related protein 2 3 complex activity invadopodia function and tumor cell invasion
Molecular Cancer Research, 2019Co-Authors: Steven M Markwell, Dorothy A Schafer, Amanda Gatesman Ammer, Erik T Interval, Jessica L Allen, Brenen W Papenberg, River A Hames, Johnathan E Castano, Scott A WeedAbstract:Malregulation of the actin cytoskeleton enhances tumor cell motility and invasion. The actin-binding protein Cortactin facilitates branched actin network formation through activation of the actin-related protein (Arp) 2/3 complex. Increased Cortactin expression due to gene amplification is observed in head and neck squamous cell carcinoma (HNSCC) and other cancers, corresponding with elevated tumor progression and poor patient outcome. Arp2/3 complex activation is responsible for driving increased migration and extracellular matrix (ECM) degradation by governing invadopodia formation and activity. Although Cortactin-mediated activation of Arp2/3 complex and invadopodia regulation has been well established, signaling pathways responsible for governing Cortactin binding to Arp2/3 are unknown and potentially present a new avenue for anti-invasive therapeutic targeting. Here we identify casein kinase (CK) 2α phosphorylation of Cortactin as a negative regulator of Arp2/3 binding. CK2α directly phosphorylates Cortactin at a conserved threonine (T24) adjacent to the canonical Arp2/3 binding motif. Phosphorylation of Cortactin T24 by CK2α impairs the ability of Cortactin to bind Arp2/3 and activate actin nucleation. Decreased invadopodia activity is observed in HNSCC cells with expression of CK2α phosphorylation-null Cortactin mutants, shRNA-mediated CK2α knockdown, and with the CK2α inhibitor Silmitasertib. Silmitasertib inhibits HNSCC collective invasion in tumor spheroids and orthotopic tongue tumors in mice. Collectively these data suggest that CK2α-mediated Cortactin phosphorylation at T24 is critical in regulating Cortactin binding to Arp2/3 complex and pro-invasive activity, identifying a potential targetable mechanism for impairing HNSCC invasion. Implications: This study identifies a new signaling pathway that contributes to enhancing cancer cell invasion. Visual Overview:http://mcr.aacrjournals.org/content/molcanres/17/4/987/F1.large.jpg.
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Src binds Cortactin through an SH2 domain cystine-mediated linkage.
Journal of cell science, 2012Co-Authors: Jason V Evans, Amanda G Ammer, John E Jett, Chris A Bolcato, Jason C Breaux, Karen H Martin, Mark V Culp, Peter M Gannett, Scott A WeedAbstract:Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates Cortactin binding and tyrosine phosphorylation, but how Src interacts with Cortactin is unknown. Here we demonstrate that Src binds Cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and Cortactin C112/246 in the Cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-Cortactin binding, eliminates Cortactin phosphorylation by Src, and prevents Src SH2 domain binding to Cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and Cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated Cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and Cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.
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Revisiting the ERK/Src Cortactin switch.
Communicative & integrative biology, 2011Co-Authors: Laura C. Kelley, Amanda Gatesman Ammer, Karen H Martin, Karen E. Hayes, Scott A WeedAbstract:The filamentous (F)-actin regulatory protein Cortactin plays an important role in tumor cell movement and invasion by promoting and stabilizing actin related protein (Arp)2/3-mediated actin networks necessary for plasma membrane protrusion. Cortactin is a substrate for ERK1/2 and Src family kinases, with previous in vitro findings demonstrating ERK1/2 phosphorylation of Cortactin as a positive and Src phosphorylation as a negative regulatory event in promoting Arp2/3 activation through neuronal Wiskott Aldrich Syndrome protein (N-WASp). Evidence for this regulatory Cortactin “switch” in cells has been hampered due to the lack of phosphorylation-specific antibodies that recognize ERK1/2-phosphorylated Cortactin. Our findings with phosphorylation-specific antibodies against these ERK1/2 sites (pS405 and pS418) indicate that Cortactin can be co-phosphorylated at 405/418 and tyrosine residues targeted by Src family tyrosine kinases. These results indicate that the ERK/Src Cortactin switch is not the sole mech...
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Cortactin is a functional target of e cadherin activated src family kinases in mcf7 epithelial monolayers
Journal of Biological Chemistry, 2009Co-Authors: Gang Ren, Scott A Weed, Falak Helwani, Suzie Verma, Robert W Mclachlan, Alpha S YapAbstract:Src family kinases (SFKs) signal in response to E-cadherin to support cadherin adhesion and the integrity of cell-cell contacts (McLachlan, R. W., Kraemer, A., Helwani, F. M., Kovacs, E. M., and Yap, A. S. (2007) Mol. Biol. Cell 18, 3214-3223). We now identify the actin-regulatory protein, Cortactin, as a target of E-cadherin-activated SFK signaling. Tyr-phosphorylated Cortactin was found at cell-cell contacts in established epithelial monolayers, and Cortactin became acutely tyrosine-phosphorylated when E-cadherin adhesion was engaged. In all circumstances, Cortactin tyrosine phosphorylation was blocked by inhibiting SFK signaling. Importantly, Tyr-phosphorylated Cortactin was necessary to preserve the integrity of cadherin contacts and the perijunctional actin cytoskeleton. Moreover, expression of a phosphomimetic Cortactin mutant could prevent SFK blockade from disrupting cadherin organization, thereby placing Cortactin functionally downstream of SFK signaling at cadherin adhesions. We conclude that SFK and Cortactin constitute an important signaling pathway that functionally links E-cadherin adhesion and the actin cytoskeleton.
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Role of c-Abl in Cortactin phosphorylation
Cancer Research, 2007Co-Authors: Karen Hayes, Laura Gibson, Mark Auble, Ann Marie Pendergast, Scott A WeedAbstract:AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 3749 Cortactin is an actin-binding protein and Src kinase substrate that activates the actin-related protein (Arp 2/3) complex and has been shown to facilitate actin cytoskeleton organization. Tyrosine phosphorylation of Cortactin is associated with increased motility and invasiveness of tumor cells. c-Abl is a non-receptor tyrosine kinase that regulates cytoskeletal dynamics and tumor cell metastasis. Here we demonstrate that Cortactin and c-Abl interact, and that Cortactin is a potential c-Abl substrate. Screening of SH2 domain arrays with Cortactin peptides phosphorylated at tyrosine 421 or 466 demonstrate a direct interaction with the c-Abl SH2 domain. Cortactin and c-Abl colocalize at the cell periphery and in dorsal waves in Src, Yes, and Fyn deficient fibroblasts (SYF) as determined by confocal microscopy. Expression of a kinase active c-Abl variant in SYF cells leds to increased phosphorylation of Cortactin at tyrosine 421. This was not observed in cells expressing either wild type or kinase inactive c-Abl constructs. Treatment of SYF cells expressing activated c-Abl with the Abl inhibitor, imatinib mesylate, substantially diminished Cortactin phosphorylation. c-Abl phopshorylates Cortactin in vitro at tyrosines 421 and 466 in a concentration-dependent manner. Collectively, these results indicate that c-Abl activation leads to Cortactin tyrosine phosphorylation without the involvement of Src-family kinases, with Cortactin serving as a direct substrate. The role of c-Abl induced phosphorylation of Cortactin on cellular motility and invasion is currently under investigation.
Xi Zhan - One of the best experts on this subject based on the ideXlab platform.
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Cortactin is implicated in murine zygotic development
Experimental cell research, 2009Co-Authors: Helin Zhang, Elizabeth P. Smith, Thomas A. Blanpied, Xi ZhanAbstract:Cortactin is a cortex-enriched protein implicated in Arp2/3 complex-mediated actin polymerization. However, the physiological role of Cortactin remains unknown. We have generated a mouse strain in which the allele of murine Cortactin was disrupted by a gene trapping vector. The resulting heterozygous mice developed normally and were fertile, but embryonic fibroblasts derived from heterozygous animals displayed partial impairment in PDGF-induced membrane ruffling. No homozygous offspring or early embryos even at the two-cell stage were detected. Analysis of oocytes revealed a gradual decrease in the detection of homozygous zygotes after fertilization. In normal oocytes arrested at meiotic metaphase II (MII), Cortactin immunoreactivity was detected in an apical layer that overlies the maternal chromosome and overlaps with a polarized cortex enriched with actin. The formation of the polarized Cortactin layer was diminished upon treatment with latrunculin B, an actin polymerization inhibitor. After resumption of meiosis II, the majority of Cortactin protein was accumulated into the second polar body. Microinjection of MII-arrested eggs with either Cortactin antibody or RNA encoding a Cortactin mutant deficient in Arp2/3 complex binding disrupted the integrity of the actin cap and inhibited emission of the second polar body triggered by parthenogenesis. Our data suggest that Cortactin plays an important role in the mechanics of asymmetric division in oocytes.
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Receptor-mediated endocytosis involves tyrosine phosphorylation of Cortactin.
The Journal of biological chemistry, 2007Co-Authors: Jianwei Zhu, Xian-chun Zeng, Kang Zhou, Xi ZhanAbstract:Efficient internalization of cell surface receptors requires actin polymerization mediated by Arp2/3 complex and Cortactin, a prominent substrate of the protein-tyrosine kinase Src. However, the significance of Cortactin tyrosine phosphorylation in endocytosis is unknown. We found that overexpression of a Cortactin mutant deficient in tyrosine phosphorylation decreased transferrin uptake. Suppression of Cortactin expression by RNA interference also reduced transferrin internalization. Such inhibition was effectively rescued by overexpressing wild-type Cortactin but not a Cortactin mutant deficient in tyrosine phosphorylation or a mutant with deletion of the Src homology 3 domain. Likewise, purified phosphorylation-null Cortactin failed to restore the formation of clathrin-coated vesicles in a Cortactin-depleted cell extract. In vitro analysis revealed that Src-mediated phosphorylation enhanced the association of Cortactin with dynamin-2 in a tyrosine phosphorylation-dependent manner. Quantitative analysis demonstrated that Src enhances the affinity of Cortactin for dynamin-2 by more than 3-fold. On the other hand, Src-treated dynamin-2 had no effect on its interaction with Cortactin. These data indicate that Src kinase is implicated in clathrin-mediated endocytosis by phosphorylation of Cortactin.
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endothelial cell Cortactin phosphorylation by src contributes to polymorphonuclear leukocyte transmigration in vitro
Circulation Research, 2006Co-Authors: Lin Yang, Xi Zhan, Jennifer R. Kowalski, Sheila M. Thomas, Francis W LuscinskasAbstract:The underlying mechanisms that regulate leukocyte transendothelial migration through the vascular endothelium remain unclear. Cortactin is a substrate of Src tyrosine kinases and a regulator of cytoskeletal dynamics. Previous studies demonstrated a role for Src phosphorylation of Cortactin in clustering of E-selectin and intercellular cell adhesion molecule-1 around adherent leukocytes. In the current study, we used an in vitro flow model to investigate the role of Src-induced Cortactin phosphorylation in endothelium during polymorphonuclear leukocyte (PMN) transmigration through human umbilical vein endothelium (HUVEC) monolayers preactivated with tumor necrosis factor-α. Inhibition of Src in HUVEC using Src kinase inhibitors PP2 and SU6656 reduced PMN transmigration by 45±8% and 36±6%, respectively. Live cell imaging of green fluorescent protein–tagged Cortactin in HUVEC revealed redistribution of Cortactin in the region surrounding transmigrating PMN. Knockdown of Cortactin in HUVEC by small interferin...
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Roles of Cortactin, an Actin Polymerization Mediator, in Cell Endocytosis
Acta biochimica et biophysica Sinica, 2006Co-Authors: Li Chen, Jianwei Zhu, Zhiwei Wang, Xi ZhanAbstract:Cortactin, an actin-binding protein and a substrate of Src, is encoded by the EMS1 oncogene. Cortactin is known to activate Arp2/3 complex-mediated actin polymerization and interact with dynamin, a large GTPase and proline rich domain-containing protein. Transferrin endocytosis was significantly reduced in cells by knock-down of Cortactin expression as well as in vivo introduction of Cortactin immunoreagents. Cortactin-dynamin interaction displayed morphologically dynamic co-distribution with a change in the endocytosis level in cells treated with an actin depolymerization reagent, cytochalasin D. In an in vitro beads assay, a branched actin network was recruited onto dynamin-coated beads in a Cortactin Src homology domain 3 (SH3)-dependent manner. In addition, Cortactin was found to function in the late stage of clathrin coated vesicle formation. Taken together, Cortactin is required for optimal clathrin mediated endocytosis in a dynamin directed manner.
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Cortactin mediated morphogenic cell movements during zebrafish (Danio rerio) gastrulation.
Science in China. Series C Life sciences, 2005Co-Authors: Peijun Zhang, Xi ZhanAbstract:Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether Cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of Cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a Cortactin specific monoclonal antibody demonstrated that Cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that Cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, Cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with Cortactin and Arp3 antibodies also revealed that Cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that Cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.
Alissa M Weaver - One of the best experts on this subject based on the ideXlab platform.
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Cortactin promotes exosome secretion by controlling branched actin dynamics.
The Journal of cell biology, 2016Co-Authors: Seema Sinha, Nan Hyung Hong, Daisuke Hoshino, Kellye C. Kirkbride, Nathan E. Grega-larson, Motoharu Seiki, Matthew J. Tyska, Alissa M WeaverAbstract:Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein Cortactin promotes exosome secretion. Knockdown or overexpression of Cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that Cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by Cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, Cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to Cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which Cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites.
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pi 3 5 p2 controls endosomal branched actin dynamics by regulating Cortactin actin interactions
Journal of Cell Biology, 2015Co-Authors: Nan Hyung Hong, Alissa M WeaverAbstract:Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator Cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that Cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to Cortactin, thereby antagonizing Cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove Cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to Cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced Cortactin localization to late endosomes. Knockdown of Cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes Cortactin from late endosomal branched actin networks and thereby promotes net actin turnover.
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Cortactin controls cell motility and lamellipodial dynamics by regulating ecm secretion
Current Biology, 2011Co-Authors: Bong Hwan Sung, Xiaodong Zhu, Irina Kaverina, Alissa M WeaverAbstract:Summary Background Branched actin assembly is critical for both cell motility and membrane trafficking. The branched actin regulator Cortactin is generally considered to promote cell migration by controlling leading-edge lamellipodial dynamics. However, recent reports indicate that lamellipodia are not required for cell movement, suggesting an alternate mechanism. Results Because Cortactin also regulates membrane trafficking and adhesion dynamics, we hypothesized that altered secretion of extracellular matrix (ECM) and/or integrin trafficking might underlie motility defects of Cortactin-knockdown (KD) cells. Consistent with a primary defect in ECM secretion, both motility and lamellipodial defects of Cortactin-KD cells were fully rescued by plating on increasing concentrations of exogenous ECM. Furthermore, Cortactin-KD cell speed defects were rescued on cell-free autocrine ECM produced by control cells, but not on ECM produced by Cortactin-KD cells. Investigation of the mechanism revealed that whereas endocytosed fibronectin (FN) is redeposited at the basal cell surface by control cells, Cortactin-KD cells exhibit defective FN secretion and abnormal FN retention in a late endocytic/lysosomal compartment. Cortactin-KD motility and FN deposition defects were phenocopied by KD in control cells of the lysosomal fusion regulator synaptotagmin-7. Rescue of Cortactin-KD cells by expression of Cortactin-binding domain mutants revealed that interaction with the Arp2/3 complex and actin filaments is essential for rescue of both cell motility and autocrine ECM secretion phenotypes, whereas binding of SH3-domain partners is not required. Conclusions Efficient cell motility, promoted by Cortactin regulation of branched actin networks, involves processing and resecretion of internalized ECM from a late endosomal/lysosomal compartment.
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aggressiveness of hnscc tumors depends on expression levels of Cortactin a gene in the 11q13 amplicon
Oncogene, 2009Co-Authors: Emily S Clark, Brandee T Brown, Amy S Whigham, Avtandyl Kochaishvili, Wendell G Yarbrough, Alissa M WeaverAbstract:11q13 amplification is a late-stage event in several cancers that is often associated with poor prognosis. Among 11q13-amplified genes, the actin assembly protein Cortactin/CTTN is considered a likely candidate for direct involvement in tumor progression because of its cell motility-enhancing functions. We modulated Cortactin expression in head and neck squamous cell carcinoma (HNSCC) cell lines. Cortactin expression levels directly correlated with tumor size, vascularization and cell proliferation in an orthotopic HNSCC in vivo model. In contrast, under normal in vitro culture conditions, Cortactin expression levels had no effect on cell proliferation. However, cell lines in which Cortactin expression was reduced by knockdown (KD) grew poorly in vitro under harsh conditions of growth factor deprivation, anchorage independence and space constraint. In contrast, overexpression of Cortactin enhanced in vitro growth under the same harsh conditions. Surprisingly, defects in growth factor-independent proliferation of Cortactin-KD cells were rescued by coculture with Cortactin-expressing cells. As the cocultured cells are separated by permeable filters, Cortactin-expressing cells must secrete growth-supporting autocrine factors to rescue the Cortactin-KD cells. Overall, Cortactin expression modulates multiple cellular traits that may allow survival in a tumor environment, suggesting that the frequent overexpression of Cortactin in tumors is not an epiphenomenon but rather promotes tumor aggressiveness.
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Cortactin is an essential regulator of matrix metalloproteinase secretion and extracellular matrix degradation in invadopodia
Cancer Research, 2007Co-Authors: Emily S Clark, Amy S Whigham, Wendell G Yarbrough, Alissa M WeaverAbstract:Invadopodia are branched actin-rich structures associated with extracellular matrix (ECM) degradation that collectively form the invasive machinery of aggressive cancer cells. Cortactin is a prominent component and a specific marker of invadopodia. Amplification of Cortactin is associated with poor prognosis in head and neck squamous cell carcinomas (HNSCC), possibly because of its activity in invadopodia. Although the role of Cortactin in invadopodia has been attributed to signaling and actin assembly, it is incompletely understood. We made HNSCC cells deficient in Cortactin by RNA interference knockdown methods. In these Cortactin knockdown cells, invadopodia were reduced in number and lost their ability to degrade ECM. In the reverse experiment, overexpression of Cortactin dramatically increased ECM degradation, far above and beyond the effect on formation of actin/Arp3-positive invadopodia puncta. Secretion of matrix metalloproteinases (MMP) MMP-2 and MMP-9, as well as plasma membrane delivery of MT1-MMP correlated closely with Cortactin expression levels. MMP inhibitor treatment of control cells mimicked the Cortactin knockdown phenotype, with abolished ECM degradation and fewer invadopodia, suggesting a positive feedback loop in which degradation products from MMP activity promote new invadopodia formation. Collectively, these data suggest that a major role of Cortactin in invadopodia is to regulate the secretion of MMPs and point to a novel mechanism coupling dynamic actin assembly to the secretory machinery, producing enhanced ECM degradation and invasiveness. Furthermore, these data provide a possible explanation for the observed association between Cortactin overexpression and enhanced invasiveness and poor prognosis in HNSCC patients.
Thomas J Parsons - One of the best experts on this subject based on the ideXlab platform.
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hdac6 modulates cell motility by altering the acetylation level of Cortactin
Molecular Cell, 2007Co-Authors: Xiaohong Zhang, Thomas J Parsons, Yingtao Zhang, Nancy Olashaw, Zhigang Yua, Sarah Yong, Alexis Salasburgos, Joh M Koome, Xiangjiao Yang, Tsopang YaoAbstract:Histone deacetylase 6 (HDAC6) is a tubulin-specific deacetylase that regulates microtubule-dependent cell movement. In this study, we identify the F-actin-binding protein Cortactin as a HDAC6 substrate. We demonstrate that HDAC6 binds Cortactin and that overexpression of HDAC6 leads to hypoacetylation of Cortactin, whereas inhibition of HDAC6 activity leads to Cortactin hyperacetylation. HDAC6 alters the ability of Cortactin to bind F-actin by modulating a "charge patch" in its repeat region. Introduction of charge-preserving or charge-neutralizing mutations in this Cortactin repeat region correlates with the gain or loss of F-actin binding ability, respectively. Cells expressing a charge-neutralizing Cortactin mutant were less motile than control cells or cells expressing a charge-preserving mutant. These findings suggest that, in addition to its role in microtubule-dependent cell motility, HDAC6 influences actin-dependent cell motility by altering the acetylation status of Cortactin, which, in turn, changes the F-actin binding activity of Cortactin.
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Cortactin and crk cooperate to trigger actin polymerization during shigella invasion of epithelial cells
Journal of Cell Biology, 2004Co-Authors: Laurence Bougneres, Scott A Weed, Thomas J Parsons, Andrei V. Karginov, Stephane Girardin, Jean Christophe Olivomarin, Philippe J Sansonetti, Guy Tran Van NhieuAbstract:Shigella, the causative agent of bacillary dysentery, invades epithelial cells in a process involving Src tyrosine kinase signaling. Cortactin, a ubiquitous actin-binding protein present in structures of dynamic actin assembly, is the major protein tyrosine phosphorylated during Shigella invasion. Here, we report that RNA interference silencing of Cortactin expression, as does Src inhibition in cells expressing kinase-inactive Src, interferes with actin polymerization required for the formation of cellular extensions engulfing the bacteria. Shigella invasion induced the recruitment of Cortactin at plasma membranes in a tyrosine phosphorylation–dependent manner. Overexpression of wild-type forms of Cortactin or the adaptor protein Crk favored Shigella uptake, and Arp2/3 binding–deficient Cortactin derivatives or an Src homology 2 domain Crk mutant interfered with bacterial-induced actin foci formation. Crk was shown to directly interact with tyrosine-phosphorylated Cortactin and to condition Cortactin-dependent actin polymerization required for Shigella uptake. These results point at a major role for a Crk–Cortactin complex in actin polymerization downstream of tyrosine kinase signaling.
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Cortactin tyrosine phosphorylation requires rac1 activity and association with the cortical actin cytoskeleton
Molecular Biology of the Cell, 2003Co-Authors: Julie A Head, Thomas J Parsons, Dongyan Jiang, Lynda Zorn, Erik Schaefer, Scott A WeedAbstract:Cortactin is an F-actin binding protein that activates actin-related protein 2/3 complex and is localized within lamellipodia. Cortactin is a substrate for Src and other protein tyrosine kinases involved in cell motility, where its phosphorylation on tyrosines 421, 466, and 482 in the carboxy terminus is required for cell movement and metastasis. In spite of the importance of Cortactin tyrosine phosphorylation in cell motility, little is known regarding the structural, spatial, or signaling requirements regulating Cortactin tyrosine phosphorylation. Herein, we report that phosphorylation of Cortactin tyrosine residues in the carboxy terminus requires the aminoterminal domain and Rac1-mediated localization to the cell periphery. Phosphorylation-specific antibodies directed against tyrosine 421 and 466 were produced to study the regulation and localization of tyrosine phosphorylated Cortactin. Phosphorylation of Cortactin tyrosine 421 and 466 was elevated in response to Src, epidermal growth factor receptor and Rac1 activation, and tyrosine 421 phosphorylated Cortactin localized with F-actin in lamellipodia and podosomes. Cortactin tyrosine phosphorylation is progressive, with tyrosine 421 phosphorylation required for phosphorylation of tyrosine 466. These results indicate that Cortactin tyrosine phosphorylation requires Rac1-induced Cortactin targeting to cortical actin networks, where it is tyrosine phosphorylated in hierarchical manner that is closely coordinated with its ability to regulate actin dynamics.
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Cortactin interacts with wip in regulating arp2 3 activation and membrane protrusion
Current Biology, 2003Co-Authors: Andrew W. Kinley, Scott A Weed, Alissa M Weaver, John A. Cooper, Andrei V. Karginov, Eric A. Bissonette, Thomas J ParsonsAbstract:Abstract Background: Modulation of actin cytoskeleton assembly is an integral step in many cellular events. A key regulator of actin polymerization is Arp2/3 complex. Cortactin, an F-actin binding protein that localizes to membrane ruffles, is an activator of Arp2/3 complex. Results: A yeast two-hybrid screen revealed the interaction of the Cortactin Src homology 3 (SH3) domain with a peptide fragment derived from a cDNA encoding a region of WASp-Interacting Protein (WIP). GST-Cortactin interacted with WIP in an SH3-dependent manner. The subcellular localization of Cortactin and WIP coincided at the cell periphery. WIP increased the efficiency of Cortactin-mediated Arp2/3 complex activation of actin polymerization in a concentration-dependent manner. Lastly, coexpression of Cortactin and WIP stimulated membrane protrusions. Conclusions: WIP, a protein involved in filopodia formation, binds to both actin monomers and Cortactin. Thus, recruitment of actin monomers to a Cortactin-activated Arp2/3 complex likely leads to the observed increase in Cortactin activation of Arp2/3 complex by WIP. These data suggest that a Cortactin-WIP complex functions in regulating actin-based structures at the cell periphery.
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interaction of Cortactin and n wasp with arp2 3 complex
Current Biology, 2002Co-Authors: Alissa M Weaver, Thomas J Parsons, Andrei V. Karginov, John E. Heuser, Wei Lih Lee, John A. CooperAbstract:Abstract Background: Dynamic actin assembly is required for diverse cellular processes and often involves activation of Arp2/3 complex. Cortactin and N-WASp activate Arp2/3 complex, alone or in concert. Both Cortactin and N-WASp contain an acidic (A) domain that is required for Arp2/3 complex binding. Results: We investigated how Cortactin and the constitutively active VCA domain of N-WASp interact with Arp2/3 complex. Structural studies showed that Cortactin is a thin, elongated monomer. Chemical crosslinking studies demonstrated selective interaction of the Arp2/3 binding NTA domain of Cortactin (Cortactin NTA) with the Arp3 subunit and VCA with Arp3, Arp2, and ARPC1/p40. Cortactin NTA and VCA crosslinking to the Arp3 subunit were mutually exclusive; however, Cortactin NTA did not inhibit VCA crosslinking to Arp2 or ARPC1/p40, nor did it inhibit activation of Arp2/3 complex by VCA. We conducted an experiment in which a saturating concentration of Cortactin NTA modestly lowered the binding affinity of VCA for Arp2/3; the results of this experiment provided further evidence for ternary complex formation. Consistent with a common binding site on Arp3, a saturating concentration of VCA abolished binding of Cortactin to Arp2/3 complex. Conclusions: Under certain circumstances, Cortactin and N-WASp can bind simultaneously to Arp2/3 complex, accounting for their synergy in activation of actin assembly. The interaction of Cortactin NTA with Arp2/3 complex does not inhibit Arp2/3 activation by N-WASp, despite competition for a common binding site located on the Arp3 subunit. These results suggest a model in which Cortactin may bridge Arp2/3 complex to actin filaments via Arp3 and N-WASp activates Arp2/3 complex by binding Arp2 and/or ARPC1/p40.
John A. Cooper - One of the best experts on this subject based on the ideXlab platform.
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Cortactin has an essential and specific role in osteoclast actin assembly
Molecular biology of the cell, 2006Co-Authors: Shandiz Tehrani, Roberta Faccio, Indra Chandrasekar, F. Patrick Ross, John A. CooperAbstract:Osteoclasts are essential for bone dynamics and calcium homeostasis. The cells form a tight seal on the bone surface, onto which they secrete acid and proteases to resorb bone. The seal is associated with a ring of actin filaments. Cortactin, a c-Src substrate known to promote Arp2/3-mediated actin assembly in vitro, is expressed in osteoclasts and localizes to the sealing ring. To address the role of Cortactin and actin assembly in osteoclasts, we depleted Cortactin by RNA interference. Cortactin-depleted osteoclasts displayed a complete loss of bone resorption with no formation of sealing zones. On nonosteoid surfaces, osteoclasts flatten with a dynamic, actin-rich peripheral edge that contains podosomes, filopodia, and lamellipodia. Cortactin depletion led to a specific loss of podosomes, revealing a tight spatial compartmentalization of actin assembly. Podosome formation was restored in Cortactin-depleted cells by expression of wild-type Cortactin or a Src homology 3 point mutant of Cortactin. In contrast, expression of a Cortactin mutant lacking tyrosine residues phosphorylated by Src did not restore podosome formation. Cortactin was found to be an early component of the nascent podosome belt, along with dynamin, supporting a role for Cortactin in actin assembly.
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Cortactin Interacts with WIP in Regulating Arp2/3 Activation and Membrane Protrusion
Current biology : CB, 2003Co-Authors: Andrew W. Kinley, Scott A Weed, Alissa M Weaver, John A. Cooper, Andrei V. Karginov, Eric A. Bissonette, J. Thomas ParsonsAbstract:Abstract Background: Modulation of actin cytoskeleton assembly is an integral step in many cellular events. A key regulator of actin polymerization is Arp2/3 complex. Cortactin, an F-actin binding protein that localizes to membrane ruffles, is an activator of Arp2/3 complex. Results: A yeast two-hybrid screen revealed the interaction of the Cortactin Src homology 3 (SH3) domain with a peptide fragment derived from a cDNA encoding a region of WASp-Interacting Protein (WIP). GST-Cortactin interacted with WIP in an SH3-dependent manner. The subcellular localization of Cortactin and WIP coincided at the cell periphery. WIP increased the efficiency of Cortactin-mediated Arp2/3 complex activation of actin polymerization in a concentration-dependent manner. Lastly, coexpression of Cortactin and WIP stimulated membrane protrusions. Conclusions: WIP, a protein involved in filopodia formation, binds to both actin monomers and Cortactin. Thus, recruitment of actin monomers to a Cortactin-activated Arp2/3 complex likely leads to the observed increase in Cortactin activation of Arp2/3 complex by WIP. These data suggest that a Cortactin-WIP complex functions in regulating actin-based structures at the cell periphery.
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Cortactin interacts with wip in regulating arp2 3 activation and membrane protrusion
Current Biology, 2003Co-Authors: Andrew W. Kinley, Scott A Weed, Alissa M Weaver, John A. Cooper, Andrei V. Karginov, Eric A. Bissonette, Thomas J ParsonsAbstract:Abstract Background: Modulation of actin cytoskeleton assembly is an integral step in many cellular events. A key regulator of actin polymerization is Arp2/3 complex. Cortactin, an F-actin binding protein that localizes to membrane ruffles, is an activator of Arp2/3 complex. Results: A yeast two-hybrid screen revealed the interaction of the Cortactin Src homology 3 (SH3) domain with a peptide fragment derived from a cDNA encoding a region of WASp-Interacting Protein (WIP). GST-Cortactin interacted with WIP in an SH3-dependent manner. The subcellular localization of Cortactin and WIP coincided at the cell periphery. WIP increased the efficiency of Cortactin-mediated Arp2/3 complex activation of actin polymerization in a concentration-dependent manner. Lastly, coexpression of Cortactin and WIP stimulated membrane protrusions. Conclusions: WIP, a protein involved in filopodia formation, binds to both actin monomers and Cortactin. Thus, recruitment of actin monomers to a Cortactin-activated Arp2/3 complex likely leads to the observed increase in Cortactin activation of Arp2/3 complex by WIP. These data suggest that a Cortactin-WIP complex functions in regulating actin-based structures at the cell periphery.
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Interaction of Cortactin and N-WASp with Arp2/3 complex.
Current biology : CB, 2002Co-Authors: Alissa M Weaver, Andrei V. Karginov, J. Thomas Parsons, John E. Heuser, Wei Lih Lee, John A. CooperAbstract:Abstract Background: Dynamic actin assembly is required for diverse cellular processes and often involves activation of Arp2/3 complex. Cortactin and N-WASp activate Arp2/3 complex, alone or in concert. Both Cortactin and N-WASp contain an acidic (A) domain that is required for Arp2/3 complex binding. Results: We investigated how Cortactin and the constitutively active VCA domain of N-WASp interact with Arp2/3 complex. Structural studies showed that Cortactin is a thin, elongated monomer. Chemical crosslinking studies demonstrated selective interaction of the Arp2/3 binding NTA domain of Cortactin (Cortactin NTA) with the Arp3 subunit and VCA with Arp3, Arp2, and ARPC1/p40. Cortactin NTA and VCA crosslinking to the Arp3 subunit were mutually exclusive; however, Cortactin NTA did not inhibit VCA crosslinking to Arp2 or ARPC1/p40, nor did it inhibit activation of Arp2/3 complex by VCA. We conducted an experiment in which a saturating concentration of Cortactin NTA modestly lowered the binding affinity of VCA for Arp2/3; the results of this experiment provided further evidence for ternary complex formation. Consistent with a common binding site on Arp3, a saturating concentration of VCA abolished binding of Cortactin to Arp2/3 complex. Conclusions: Under certain circumstances, Cortactin and N-WASp can bind simultaneously to Arp2/3 complex, accounting for their synergy in activation of actin assembly. The interaction of Cortactin NTA with Arp2/3 complex does not inhibit Arp2/3 activation by N-WASp, despite competition for a common binding site located on the Arp3 subunit. These results suggest a model in which Cortactin may bridge Arp2/3 complex to actin filaments via Arp3 and N-WASp activates Arp2/3 complex by binding Arp2 and/or ARPC1/p40.
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interaction of Cortactin and n wasp with arp2 3 complex
Current Biology, 2002Co-Authors: Alissa M Weaver, Thomas J Parsons, Andrei V. Karginov, John E. Heuser, Wei Lih Lee, John A. CooperAbstract:Abstract Background: Dynamic actin assembly is required for diverse cellular processes and often involves activation of Arp2/3 complex. Cortactin and N-WASp activate Arp2/3 complex, alone or in concert. Both Cortactin and N-WASp contain an acidic (A) domain that is required for Arp2/3 complex binding. Results: We investigated how Cortactin and the constitutively active VCA domain of N-WASp interact with Arp2/3 complex. Structural studies showed that Cortactin is a thin, elongated monomer. Chemical crosslinking studies demonstrated selective interaction of the Arp2/3 binding NTA domain of Cortactin (Cortactin NTA) with the Arp3 subunit and VCA with Arp3, Arp2, and ARPC1/p40. Cortactin NTA and VCA crosslinking to the Arp3 subunit were mutually exclusive; however, Cortactin NTA did not inhibit VCA crosslinking to Arp2 or ARPC1/p40, nor did it inhibit activation of Arp2/3 complex by VCA. We conducted an experiment in which a saturating concentration of Cortactin NTA modestly lowered the binding affinity of VCA for Arp2/3; the results of this experiment provided further evidence for ternary complex formation. Consistent with a common binding site on Arp3, a saturating concentration of VCA abolished binding of Cortactin to Arp2/3 complex. Conclusions: Under certain circumstances, Cortactin and N-WASp can bind simultaneously to Arp2/3 complex, accounting for their synergy in activation of actin assembly. The interaction of Cortactin NTA with Arp2/3 complex does not inhibit Arp2/3 activation by N-WASp, despite competition for a common binding site located on the Arp3 subunit. These results suggest a model in which Cortactin may bridge Arp2/3 complex to actin filaments via Arp3 and N-WASp activates Arp2/3 complex by binding Arp2 and/or ARPC1/p40.
