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PubMed2004

Erk/Src phosphorylation of cortactin acts as a switch on-switch off mechanism that controls its ability to activate N-WASP.

The Arp2/3 complex can be independently activated to initiate actin polymerization by the VCA domain of WASP family members and by the acidic N-terminal and F-actin-binding repeat region of cortactin, which possesses a C-terminal SH3 domain. Cortactin is a target for phosphorylation by Src tyrosine kinases and by serine/threonine kinases that include Erk. Here we demonstrate that cortactin binds N-WASP and WASP via its SH3 domain, induces in vitro N-WASP-mediated actin polymerization, and colocalizes with N-WASP and WASP at sites of active actin polymerization. Erk phosphorylation and a mimicking S405,418D double mutation enhanced cortactin binding and activation of N-WASP. In contrast, Src phosphorylation inhibited the ability of cortactin previously phosphorylated by Erk, and that of S405,418D double mutant cortactin, to bind and activate N-WASP. Furthermore, Y-->D mutation of three tyrosine residues targeted by Src (Y421, Y466, and Y482) inhibited the ability of S405,418D cortactin to activate N-WASP. We propose that Erk phosphorylation liberates the SH3 domain of cortactin from intramolecular interactions with proline-rich regions, causing it to synergize with WASP and N-WASP in activating the Arp2/3 complex, and that Src phosphorylation terminates cortactin activation of N-WASP and WASP.

Actin-Related Protein 2↗

PubMed2006

Cortactin overexpression regulates actin-related protein 2/3 complex activity, motility, and invasion in carcinomas with chromosome 11q13 amplification.

Carcinoma cell motility and invasion are prerequisites for tumor cell metastasis, which requires regulation of the actin cytoskeleton. Cortactin is an actin-related protein 2/3 (Arp2/3) complex-activating and filamentous (F)-actin-binding protein that is implicated in tumor cell motility and metastasis, partially by its ability to become tyrosine phosphorylated. Cortactin is encoded by the CTTN gene and maps to chromosome 11q13, a region amplified in many carcinomas, including head and neck squamous cell carcinoma (HNSCC). CTTN gene amplification is associated with lymph node metastasis and poor patient outcome, and cortactin overexpression enhances motility in tumor cells lacking 11q13 amplification. However, a direct link between increased motility and invasion has not been reported in tumor cells with chromosome 11q13 amplification and cortactin overexpression. In this study, we have examined the relationship between CTTN amplification and tumor cell motility in HNSCC. In 11 of 39 (28%) HNSCC cases, cortactin overexpression determined by immunohistochemistry correlates with lymph node metastasis and CTTN gene amplification. HNSCC cells containing cortactin gene amplification and protein overexpression display increased binding and activation of Arp2/3 complex, and were more motile and invasive than HNSCC cells lacking CTTN amplification. Down-regulation of cortactin expression in CTTN-amplified HNSCC cells by small interfering RNA impairs HNSCC motility and invasion. Treatment of HNSCC cells with the epidermal growth factor receptor inhibitor gefitinib inhibits HNSCC motility and down-regulates cortactin tyrosine phosphorylation. These data suggest that cortactin may be a valid prognostic and therapeutic marker for invasive and metastatic HNSCC and other carcinomas with 11q13 amplification.

Actin-Related Protein 2-3 Complex↗

PubMed2004

Cortactin regulates cell migration through activation of N-WASP.

Cortactin is an actin-associated scaffolding protein that regulates cell migration. Amplification of the human gene, EMS1, has been detected in breast, head and neck tumors, where it correlates with increased invasiveness. Cortactin can regulate actin dynamics directly via its N-terminal half, which can bind and activate the Arp2/3 complex. The C-terminal portion of cortactin, however, is thought to have limited function in its regulation of the actin polymerization machinery. In this report, we identify a role for the cortactin C-terminus in regulating cell migration and, more specifically, actin dynamics. Overexpression of either full-length cortactin or cortactin C-terminus is sufficient to enhance migration of mammary epithelial cells. In vitro, cortactin binds to and activates, via its SH3 domain, a regulator of the Arp2/3 complex, neural Wiskott Aldrich Syndrome protein (N-WASP). This in vitro activation of N-WASP is likely to be important in vivo, as cortactin-enhanced migration is dependent upon N-WASP. Thus, our results suggest that cortactin has multiple mechanisms by which it can recruit and modulate the actin machinery and ultimately regulate cell migration.

Amino Acid Sequence↗

PubMed2001

Cortactin potentiates bone metastasis of breast cancer cells.

Gene amplification of the chromosome 11q13 in breast cancer and squamous carcinomas in the head and neck results in frequent overexpression of cortactin, a prominent substrate of Src-related tyrosine kinases in the cell cortical areas. To investigate the role of cortactin in tumor progression, we analyzed MDA-MB-231 breast cancer cells overexpressing green fluorescent protein-tagged murine cortactin (GFP-cortactin) and a cortactin mutant deficient in tyrosine phosphorylation under the control of a retroviral vector. Injection of MDA-MB-231 cells overexpressing GFP-cortactin into nude mice through cardiac ventricles caused bone osteolysis at a frequency approximately 85% higher than that of cells expressing the vector alone, whereas injection of cells overexpressing the mutant deficient in tyrosine phosphorylation induced 74% fewer osteolytic metastases as compared with the control group. Interestingly, the cells expressing either GFP-cortactin or the mutant did not show significant differences in growth in vitro or when injected m.f.p. in vivo. On the other hand, the cells overexpressing GFP-cortactin but not the mutant acquired a >60% enhanced capability for transendothelial invasion and endothelial cell adhesion. These data suggest that cortactin contributes to tumor metastasis by enhancing the interaction of tumor cells with endothelial cells and the invasion of tumor cells into bone tissues.

Animals↗

PubMed1997

Upregulation of cortactin expression during the maturation of megakaryocytes.

Cortactin is a potent filamentous actin-binding protein acting as a prominent substrate of Src tyrosine kinases. We have evaluated cortactin expression in a series of murine tissues and shown an abundant expression of cortactin in megakaryocytes and platelets. Cortactin, but not its related protein HS1, is upregulated during the phorbol 12-myristate 13-acetate (PMA)-mediated maturation of a human megakaryoblastic cell line CMK. Although the expression of Src-related kinases is also upregulated more rapidly than cortactin in PMA-treated CMK cells, tyrosine phosphorylation of cortactin appears to be only transiently elevated 4 days after PMA stimulation. In addition, cortactin expression is induced by thrombopoietin and interleukin-3 in megakaryocytes derived from murine bone marrow cells. Thus, cortactin represents a megakaryocyte-specific gene in bone marrow and the interaction of Src kinases with cortactin may be involved in the maturation of megakaryocytes.

Animals↗

PubMed2005

Cortactin promotes cell motility by enhancing lamellipodial persistence.

BACKGROUND: Lamellipodial protrusion, which is the first step in cell movement, is driven by actin assembly and requires activity of the Arp2/3 actin-nucleating complex. However, it is unclear how actin assembly is dynamically regulated to support effective cell migration. RESULTS: Cells deficient in cortactin have impaired cell migration and invasion. Kymography analyses of live-cell imaging studies demonstrate that cortactin-knockdown cells have a selective defect in the persistence of lamellipodial protrusions. The motility and protrusion defects are fully rescued by cortactin molecules, provided both the Arp2/3 complex and F-actin binding sites are intact. Consistent with this requirement for simultaneous contacts with Arp2/3 and F-actin, cortactin is recruited by Arp2/3 complex to lamellipodia and binds with a higher affinity to ATP/ADP-Pi-F-actin than to ADP-F-actin. In situ labeling of lamellipodia revealed that the relative levels of free barbed ends of actin filaments are reduced by over 30% in the cortactin-knockdown cells; however, there is no change in Arp2/3-complex localization to lamellipodia. Cortactin-knockdown cells also have a selective defect in the assembly of new adhesions in protrusions, as assessed by analysis of GFP-paxillin dynamics in living cells. CONCLUSIONS: Cortactin enhances lamellipodial persistence, at least in part through regulation of Arp2/3 complex. The presence of cortactin also enhances the rate of new adhesion formation in lamellipodia. In vivo, these functions may be important during directed cell motility.

Actin-Related Protein 2↗

PubMed2004

Interaction of cortactin and Arp2/3 complex is required for sphingosine-1-phosphate-induced endothelial cell remodeling.

Sphingosine-1-phosphate (S1P) induces capillary formation of endothelial cells on Matrigel in accompany with actin assembly and accumulation of cortactin and Arp2/3 complex at the cell-leading edge. Suppression of cortactin expression with a cortactin antisense oligo significantly impaired S1P-induced capillary formation, migration of endothelial cells, and actin assembly at the cell periphery. Overexpression of wild-type cortactin tagged by green fluorescent protein (GFP) increased the S1P-induced tube formation and cell motility, whereas the cells overexpressing the mutant formed poorly capillary network and became less motile in response to S1P. Analysis of distribution in Triton X-100 insoluble fractions demonstrated that the cortactin mutant inhibited the association of wild-type cortactin and Arp2/3 complex with the actin-enriched complex. Furthermore, actin polymerization at and distribution of Arp2/3 complex as well as endogenous cortactin into the cell-leading edge mediated by S1P was disturbed. These data suggest that the interaction between cortactin and Arp2/3 complex plays an important role in S1P-mediated remodeling of endothelial cells.

Actin-Related Protein 2↗

PubMed2001

Cortactin promotes and stabilizes Arp2/3-induced actin filament network formation.

Cortactin is a c-src substrate associated with sites of dynamic actin assembly at the leading edge of migrating cells. We previously showed that cortactin binds to Arp2/3 complex, the essential molecular machine for nucleating actin filament assembly. In this study, we demonstrate that cortactin activates Arp2/3 complex based on direct visualization of filament networks and pyrene actin assays. Strikingly, cortactin potently inhibited the debranching of filament networks. When cortactin was added in combination with the active VCA fragment of N-WASp, they synergistically enhanced Arp2/3-induced actin filament branching. The N-terminal acidic and F-actin binding domains of cortactin were both necessary to activate Arp2/3 complex. These results support a model in which cortactin modulates actin filament dendritic nucleation by two mechanisms, (1) direct activation of Arp2/3 complex and (2) stabilization of newly generated filament branch points. By these mechanisms, cortactin may promote the formation and stabilization of the actin network that drives protrusion at the leading edge of migrating cells.

Actin Cytoskeleton↗

PubMed2001

Hepatocyte Growth Factor/scatter factor-induces phosphorylation of cortactin in A431 cells in a Src kinase-independent manner.

The Hepatocyte Growth Factor receptor transduces proliferating and scattering signals in epithelial and endothelial cells. We have explored potential interactions of the HGF/SF receptor beta-subunit (p145(beta MET)) with F-actin binding partners aiming to identify novel downstream effectors implicated in HGF/SF pluripotent signalling. Cortactin, a p80/85 F-actin binding protein, was found phosphorylated on tyrosine in response to HGF-SF in A431 human epidermoid carcinoma cells, expressing the HGF/SF receptor (c-MET). The HGF/SF receptor was enriched in the detergent-insoluble fraction and was found to co-precipitate with cortactin and to associate in vitro with cortactin. The Grb2 small adapter protein known to associate via its Src homology 2 domain (SH2) with the MET C-terminus, was also associated with cortactin. Transient transfection of A431 cells with dominant-negative Grb2 constructs has revealed that the Grb2-C-SH3 domain possesses a central role in cortactin phosphorylation in response to HGF/SF. Finally, tyrosine phosphorylation of cortactin was found uncoupled of endogenous c-Src kinase activity, thus further supporting the hypothesis that cortactin is a direct target of the MET kinase. We propose that cortactin may constitute a docking site for MET-derived signals within the cytoskeleton.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Down-regulation of the filamentous actin cross-linking activity of cortactin by Src-mediated tyrosine phosphorylation.

Cortactin, a prominent substrate for pp60(c-src), is a filamentous actin (F-actin) binding protein. We show here that cortactin can promote sedimentation of F-actin at centrifugation forces under which F-actin is otherwise not able to be precipitated. Electron microscopic analysis after negative staining further revealed that actin filaments in the presence of cortactin are cross-linked into bundles of various degrees of thickness. Hence, cortactin is also an F-actin cross-linking protein. We also demonstrate that the optimal F-actin cross-linking activity of cortactin requires a physiological pH in a range of 7.3-7.5. Furthermore, pp60(c-src) phosphorylates cortactin in vitro, resulting in a dramatic reduction of its F-actin cross-linking activity in a manner depending on levels of tyrosine phosphorylation. In addition, pp60(c-src) moderately inhibits the F-actin binding activity of cortactin. This study presents the first evidence that pp60(c-src) can directly regulate the activity of its substrate toward the cytoskeleton and implies a role of cortactin as an F-actin modulator in tyrosine kinase-regulated cytoskeleton reorganization.

Actins↗

PubMed2004

Pulmonary endothelial cell barrier enhancement by sphingosine 1-phosphate: roles for cortactin and myosin light chain kinase.

We recently reported the critical importance of Rac GTPase-dependent cortical actin rearrangement in the augmentation of pulmonary endothelial cell (EC) barrier function by sphingosine 1-phosphate (S1P). We now describe functional roles for the actin-binding proteins cortactin and EC myosin light chain kinase (MLCK) in mediating this response. Antisense down-regulation of cortactin protein expression significantly inhibits S1P-induced barrier enhancement in cultured human pulmonary artery EC as measured by transendothelial electrical resistance (TER). Immunofluorescence studies reveal rapid, Rac-dependent translocation of cortactin to the expanded cortical actin band following S1P challenge, where colocalization with EC MLCK occurs within 5 min. Adenoviral overexpression of a Rac dominant negative mutant attenuates TER elevation by S1P. S1P also induces a rapid increase in cortactin tyrosine phosphorylation (within 30 s) critical to subsequent barrier enhancement, since EC transfected with a tyrosine-deficient mutant cortactin exhibit a blunted TER response. Direct binding of EC MLCK to the cortactin Src homology 3 domain appears essential to S1P barrier regulation, since cortactin blocking peptide inhibits both S1P-induced MLC phosphorylation and peak S1P-induced TER values. These data support novel roles for the cytoskeletal proteins cortactin and EC MLCK in mediating lung vascular barrier augmentation evoked by S1P.

Actins↗

PubMed2004

Cortactin and Crk cooperate to trigger actin polymerization during Shigella invasion of epithelial cells.

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.

Actins↗

PubMed2003

Fgd1, the Cdc42 GEF responsible for Faciogenital Dysplasia, directly interacts with cortactin and mAbp1 to modulate cell shape.

FGD1 mutations result in Faciogenital Dysplasia (FGDY), an X-linked human disease that affects skeletal formation and embryonic morphogenesis. FGD1 and Fgd1, the mouse FGD1 ortholog, encode guanine nucleotide exchange factors (GEF) that specifically activate Cdc42, a Rho GTPase that controls the organization of the actin cytoskeleton. To further understand FGD1/Fgd1 signaling and begin to elucidate the molecular pathophysiology of FGDY, we demonstrate that Fgd1 directly interacts with cortactin and mouse actin-binding protein 1 (mAbp1), actin-binding proteins that regulate actin polymerization through the Arp2/3 complex. In yeast two-hybrid studies, cortactin and mAbp1 Src homology 3 (SH3) domains interact with a single Fgd1 SH3-binding domain (SH3-BD), and biochemical studies show that the Fgd1 SH3-BD directly binds to cortactin and mAbp1 in vitro. Immunoprecipitation studies show that Fgd1 interacts with cortactin and mAbp1 in vivo and that Fgd1 SH3-BD mutations disrupt binding. Immunocytochemical studies show that Fgd1 colocalizes with cortactin and mAbp1 in lamellipodia and membrane ruffles, and that Fgd1 subcellular targeting is dynamic. By using truncated cortactin proteins, immunocytochemical studies show that the cortactin SH3 domain targets Fgd1 to the subcortical actin cytoskeleton, and that abnormal Fgd1 localization results in actin cytoskeletal abnormalities and significant changes in cell shape and viability. Thus, this study provides novel in vitro and in vivo evidence that Fgd1 specifically and directly interacts with cortactin and mAbp1, and that these interactions play an important role in regulating the actin cytoskeleton and, subsequently, cell shape.

Actin Cytoskeleton↗

PubMed2006

Roles of cortactin, an actin polymerization mediator, in cell endocytosis.

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.

Actin-Related Protein 2↗

PubMed2004

Cortactin associates with N-cadherin adhesions and mediates intercellular adhesion strengthening in fibroblasts.

The regulation of N-cadherin-mediated intercellular adhesion strength in fibroblasts is poorly characterized; this is due, in part, to a lack of available quantitative models. We used a recombinant N-cadherin chimeric protein and a Rat 2 fibroblast, donor-acceptor cell model, to study the importance of cortical actin filaments and cortactin in the strengthening of N-cadherin adhesions. In wash-off assays, cytochalasin D (1 microM) reduced intercellular adhesion by threefold, confirming the importance of cortical actin filaments in strengthening of N-cadherin-mediated adhesions. Cortactin, an actin filament binding protein, spatially colocalized to, and directly associated with, nascent N-cadherin adhesion complexes. Transfection of Rat-2 cells with cortactin-specific, RNAi oligonucleotides reduced cortactin protein by 85% and intercellular adhesion by twofold compared with controls (P<0.005) using the donor-acceptor model. Cells with reduced cortactin exhibited threefold less N-cadherin-mediated intercellular adhesion strength compared with controls in wash-off assays using N-cadherin-coated beads. Immunoprecipitation and immunoblotting showed that N-cadherin-associated cortactin was phosphorylated on tyrosine residue 421 after intercellular adhesion. While tyrosine phosphorylation of cortactin was not required for recruitment to N-cadherin adhesions it was necessary for cadherin-mediated intercellular adhesion strength. Thus cortactin, and phosphorylation of its tyrosine residues, are important for N-cadherin-mediated intercellular adhesion strength.

Actins↗

PubMed2000

Transient suppression of cortactin ectopically induces large telencephalic neurons towards a GABAergic phenotype.

Excitatory and inhibitory neuronal cell fates require specific expression of both neurotransmitter and morphological phenotypes. The role of the F-actin cytoskeleton in morphological phenotypes has been well documented, but its role in neurotransmitter phenotype expression remains unknown. Here we present evidence that the F-actin binding protein cortactin participates in determining both aspects of cell fate in large telencephalic neurons. We show that the expression of cortactin was upregulated early in development just prior to appearance of GABAergic neurons in the chick telencephalon at embryonic day 6. This program was faithfully maintained in primary neuronal cultures derived from E6 telencephalon, where immature neurons differentiate either to large pyramidal and large stellate excitatory neurons or to small inhibitory GABAergic neurons. Immunostaining revealed that cortactin was enriched in areas of membrane budding, growth cones, and in the cell cortex of immature neurons. With differentiation, intense punctate staining was also observed in an extraction-resistant cytosolic compartment of the soma and processes. More importantly, suppression of cortactin by inhibition of cortactin mRNA translation with antisense oligonucleotides caused permanent phenotypic changes. Specifically, a transient suppression of cortactin was achieved in immature neurons with a single exposure to antisense oligonucleotides. This inhibition first induced both the expression of mRNA and the enzymatic activity of GAD significantly earlier than in control neurons. Second, cortactin-suppressed large projectional neurons exhibited significantly shorter processes and growth cones with protrusive filopodia and an enlarged lamellipodia veil. Most importantly, this remodeling of neuritic outgrowth in projectional somata was accompanied by the ectopic induction of GABA (*-aminobutyric acid) expression. Considering this data altogether, it appears that cortactin may function to suppress concurrently several parameters of the GABAergic program in large developing neurons.

Animals↗

PubMed1995

Differentiation dependent expression of tensin and cortactin in chicken osteoclasts.

The expression and localization of tensin and cortactin were examined in osteoclast precursors in comparison with isolated osteoclasts on various substrates. Initially, the ability of hen monocytes to differentiate into osteoclasts was evaluated on plastic or glass, and compared to differentiation on bone. Specifically, monocytes were isolated from the medullary bones of egg-laying hens maintained on a Ca-deficient diet. Differentiation was monitored morphologically and by quantitation of the ability to form Howship's lacunae in bone slices or resorb radiolabeled bone particles of 20-53 microns diameter. These cells differentiated into tartrate resistant acid phosphatase (TRAP)-positive, bone resorbing, multinucleated syncytia in the presence of cytosine-1-beta-D-arabinofuranoside in a time dependent manner (day 1-6). Differentiation into osteoclast-like cells was similar whether cultured on plastic, on glass, or on bone. When compared to GAP-DH control levels, tensin and cortactin mRNA levels increased by 7- and 10-fold, respectively, by day 6. Tensin and cortactin protein levels also increased by 6- and 15-fold, respectively, by day 6. Immunofluorescence of differentiating precursors showed that tensin localized between regions of cell to cell contact and colocalized with vinculin in podosomes of osteoclast-like cells and of real osteoclasts. Cortactin immunofluorescence was not detectable in monocytes but localized inside tensin/vinculin podosome structures after fusion into osteoclast-like cells and in freshly isolated osteoclasts. Both tensin and cortactin were associated with attachment complexes used by osteoclast-like cells and osteoclasts to resorb bone. Specifically, punctate cortactin staining was observed inside tensin staining which formed a double ring structure at the membrane/bone interface of resorbing osteoclasts. These data showed that tensin and cortactin can be used as osteoclast differentiation markers, that participate in attachment complexes used to resorb bone, and that tensin may participate in the fusion process of osteoclast precursors.

Animals↗

PubMed2006

Critical role of cortactin in actin ring formation and osteoclastic bone resorption.

Tyrosine kinase c-Src plays an essential role in ruffled border formation and bone resorption in osteoclasts; however, it is unclear how c-Src controls ruffled border formation during bone resorption. To address this question, we investigated the role of cortactin, a c-Src substrate, in osteoclasts. We found that cortactin showed colocalization with c-Src and actin rings in osteoclasts. Overexpression of cortactin stimulated actin ring formation in RAW 264.7 cells. In contrast, overexpression of Csk inhibited tyrosine phosphorylation of cortactin and binding of cortactin to c-Src. More importantly, overexertion of a mutant cortactin strongly suppressed actin ring formation and bone resorbing activity in osteoclasts. Collectively, our data indicate that cortactin controls osteoclastic bone resorption by regulating actin organization.

Actins↗