Transformation of cells by the src oncogene results in elevated tyrosine phosphorylation of two related proteins, p80 and p85 (p80/85). Immunostaining with specific monoclonal antibodies reveals a striking change of subcellular localization of p80/85 in src-transformed cells. p80/85 colocalizes with F-actin in peripheral extensions of normal cells and rosettes (podosomes) of src-transformed cells. Sequence analysis of cDNA clones encoding p80/85 reveals an amino-terminal domain composed of six copies of a direct tandem repeat, each repeat containing 37 amino acids, a carboxyl-terminal SH3 domain, and an interdomain region composed of a highly charged acidic region and a region rich in proline, serine, and threonine. The multidomain structure of p80/85 and its colocalization with F-actin in normal and src-transformed cells suggest that these proteins may associate with components of the cytoskeleton and contribute to organization of cell structure (Wu, 1991).
Two related cellular proteins, p80 and p85 (cortactin), become phosphorylated on tyrosine in pp60src-transformed cells and in cells stimulated with certain growth factors. The amino-terminal half of cortactin is comprised of multiple copies of an internal, tandem 37-amino acid repeat. The carboxyl-terminal half contains a distal SH3 domain. Cortactin is an F-actin-binding protein. The binding to F-actin is specific and saturable. The amino-terminal repeat region appears to be both necessary and sufficient to mediate actin binding, whereas the SH3 domain has no apparent effect on the actin-binding activity. Cortactin, present in several different cell types, is enriched in cortical structures such as membrane ruffles and lamellipodia. The properties of cortactin indicate that it may be important for microfilament-membrane interactions as well as transducing signals from the cell surface to the cytoskeleton. The name cortactin is suggested, reflecting the cortical subcellular localization and its actin-binding activity (Wu, 1993).
Cortactin, a prominent substrate for pp60(c-src), is a filamentous actin (F-actin) binding protein. 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. 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 for cortactin as an F-actin modulator in tyrosine kinase-regulated cytoskeleton reorganization (Huang, 1997).
Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Cortactin is localized with the actin-related protein (Arp) 2/3 complex at sites of actin polymerization within the lamellipodia. Two distinct sequence motifs of cortactin contribute to its interaction with the cortical actin network: the fourth of six tandem repeats and the amino-terminal acidic region (NTA). Cortactin variants lacking either the fourth tandem repeat or the NTA fail to localize at the cell periphery. Tandem repeat four was necessary for cortactin to stably bind F-actin in vitro. The NTA region interacts directly with the Arp2/3 complex based on affinity chromatography, immunoprecipitation assays, and binding assays using purified components. Cortactin variants containing the NTA region are inefficient at promoting Arp2/3 actin nucleation activity. These data provide strong evidence that cortactin is specifically localized to sites of dynamic cortical actin assembly via simultaneous interaction with F-actin and the Arp2/3 complex. Cortactin interacts via its Src homology 3 (SH3) domain with ZO-1 and the SHANK family of postsynaptic density 95/dlg/ZO-1 homology (PDZ) domain-containing proteins, suggesting that cortactin contributes to the spatial organization of sites of actin polymerization coupled to selected cell surface transmembrane receptor complexes (Weed, 2000).
Cortactin is a c-src substrate associated with sites of dynamic actin assembly at the leading edge of migrating cells. Cortactin binds to Arp2/3 complex, the essential molecular machine for nucleating actin filament assembly. Cortactin is shown to activate Arp2/3 complex, based on direct visualization of filament networks and pyrene actin assays. Strikingly, cortactin potently inhibits the debranching of filament networks. When cortactin is added in combination with the active VCA fragment of N-WASp, they synergistically enhance Arp2/3-induced actin filament branching. The N-terminal acidic and F-actin binding domains of cortactin are 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 (Weaver, 2001).
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. How cortactin and the constitutively active VCA domain of N-WASp interact with Arp2/3 complex has been investigated. Structural studies show that cortactin is a thin, elongated monomer. Chemical crosslinking studies demonstrate 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 are mutually exclusive; however, cortactin NTA does not inhibit VCA crosslinking to Arp2 or ARPC1/p40, nor does it inhibit activation of Arp2/3 complex by VCA. A saturating concentration of cortactin NTA modestly lowers the binding affinity of VCA for Arp2/3; the results of this experiment provide further evidence for ternary complex formation. Consistent with a common binding site on Arp3, a saturating concentration of VCA abolishes binding of cortactin to Arp2/3 complex. It is concluded that 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 (Weaver, 2002).
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. 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; see Drosophila Verprolin 1). GST-cortactin interacts with WIP in an SH3-dependent manner. The subcellular localization of cortactin and WIP coincides at the cell periphery. WIP increases 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. It is concluded that 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 (Kinley, 2003).
The mechanisms by which mammalian cells remodel the actin cytoskeleton in response to motogenic stimuli are complex and a topic of intense study. Dynamin 2 (Dyn2) is a large GTPase that interacts directly with several actin binding proteins, including cortactin. Dyn2 and cortactin function to mediate dynamic remodeling of the actin cytoskeleton in response to stimulation with the motogenic growth factor platelet-derived growth factor. On stimulation, Dyn2 and cortactin coassemble into large, circular structures on the dorsal cell surface. These 'waves' promote an active reorganization of actin filaments in the anterior cytoplasm and function to disassemble actin stress fibers. Importantly, inhibition of Dyn2 and cortactin function potently blocks the formation of waves and subsequent actin reorganization. These findings demonstrate that cortactin and Dyn2 function together in a supramolecular complex that assembles in response to growth factor stimulation and mediates the remodeling of actin to facilitate lamellipodial protrusion at the leading edge of migrating cells (Krueger, 2003).
The WASP and cortactin families constitute two distinct classes of Arp2/3 modulators in mammalian cells. Physical and functional interactions among the Arp2/3 complex, VCA (a functional domain of N-WASP), and cortactin were examined under conditions that were with or without actin polymerization. In the absence of actin, cortactin binds significantly weaker to the Arp2/3 complex than VCA. At concentrations of VCA 20-fold lower than cortactin, the association of cortactin with the Arp2/3 complex was nearly abolished. Analysis of the cells infected with Shigella demonstrate that N-WASP is located at the tip of the bacterium, whereas cortactin accumulates in the comet tail. Interestingly, cortactin promotes Arp2/3 complex-mediated actin polymerization and actin branching in the presence of VCA at a saturating concentration, and cortactin acquires 20 nm affinity for the Arp2/3 complex during actin polymerization. The interaction of VCA with the Arp2/3 complex is reduced in the presence of both cortactin and actin. Moreover, VCA reduces its affinity for Arp2/3 complex at branching sites that are stabilized by phalloidin. These data imply a novel mechanism for the de novo assembly of a branched actin network that involves a coordinated sequential interaction of N-WASP and cortactin with the Arp2/3 complex (Uruno, 2003).
Small GTPases of the Rho family regulate signaling pathways that control actin cytoskeletal structures. In Swiss 3T3 cells, RhoA activation leads to stress fiber and focal adhesion formation, Rac1 to lamellipoda and membrane ruffles, and Cdc42 to microspikes and filopodia. Several downstream molecules mediating these effects have been recently identified. Evidence is provided that the intracellular localization of the actin binding protein cortactin, a Src kinase substrate, is regulated by the activation of Rac1. Cortactin redistributes from the cytoplasm into membrane ruffles as a result of growth factor-induced Rac1 activation, and this translocation is blocked by expression of dominant negative Rac1N17. Expression of constitutively active Rac1L61 evokes the translocation of cortactin from cytoplasmic pools into peripheral membrane ruffles. Expression of mutant forms of the serine/threonine kinase PAK1, a downstream effector of Rac1 and Cdc42, recently demonstrated to trigger cortical actin polymerization and membrane ruffling, also leads to the translocation of cortactin to the cell cortex, although this is effectively blocked by coexpression of Rac1N17. Collectively these data provide evidence for cortactin as a putative target of Rac1-induced signal transduction events involved in membrane ruffling and lamellipodia formation (Weed, 1998).
Rho family GTPases control cell migration and participate in the regulation of cancer metastasis. Invadopodia, associated with invasive tumour cells, are crucial for cellular invasion and metastasis. To study Rac1 GTPase in invadopodia dynamics, a genetically encoded, single-chain Rac1 fluorescence resonance energy (FRET) transfer biosensor was developed. The biosensor shows Rac1 activity exclusion from the core of invadopodia, and higher activity when invadopodia disappear, suggesting that reduced Rac1 activity is necessary for their stability, and Rac1 activation is involved in disassembly. Photoactivating Rac1 at invadopodia confirmed this previously unknown Rac1 function. This study describes an invadopodia disassembly model, where a signalling axis involving TrioGEF, Rac1, Pak1, and phosphorylation of cortactin, causes invadopodia dissolution. This mechanism is critical for the proper turnover of invasive structures during tumour cell invasion, where a balance of proteolytic activity and locomotory protrusions must be carefully coordinated to achieve a maximally invasive phenotype (Moshfegh, 2014).
The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. The cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods it has been demonstrated that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD [Dyn 2(aa)DeltaPRD] sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape (McNiven, 2000).
The GTPase dynamin is required for endocytic vesicle formation. Dynamin has also been implicated in regulating the actin cytoskeleton, but the mechanism by which it does so is unclear. Through interactions via its proline-rich domain (PRD), dynamin binds several proteins, including cortactin, profilin, syndapin, and murine Abp1, that regulate the actin cytoskeleton. The interaction of dynamin2 and cortactin was investigated in regulating actin assembly in vivo and in vitro. When expressed in cultured cells, a dynamin2 mutant with decreased affinity for GTP decreases actin dynamics within the cortical actin network. Expressed mutants of cortactin that have decreased binding of Arp2/3 complex or dynamin2 also decrease actin dynamics. Dynamin2 influences actin nucleation by purified Arp2/3 complex and cortactin in vitro in a biphasic manner. Low concentrations of dynamin2 enhance actin nucleation by Arp2/3 complex and cortactin, and high concentrations are inhibitory. Dynamin2 promotes the association of actin filaments nucleated by Arp2/3 complex and cortactin with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing lipid vesicles. GTP hydrolysis alters the organization of the filaments and the lipid vesicles. It is concluded that dynamin2, through an interaction with cortactin, regulates actin assembly and actin filament organization at membranes (Schafer, 2002).
The actin cytoskeleton is believed to contribute to the formation of clathrin-coated pits, although the specific components that connect actin filaments with the endocytic machinery are unclear. Cortactin is an F-actin-associated protein, localizes within membrane ruffles in cultured cells, and is a direct binding partner of the large GTPase dynamin. This direct interaction with a component of the endocytic machinery suggests that cortactin may participate in one or several endocytic processes. Therefore, the goal of this study was to test whether cortactin associates with clathrin-coated pits and participates in receptor-mediated endocytosis. Morphological experiments with either anti-cortactin antibodies or expressed red fluorescence protein-tagged cortactin reveal a striking colocalization of cortactin and clathrin puncta at the ventral plasma membrane. Consistent with these observations, cells microinjected with these antibodies exhibit a marked decrease in the uptake of labeled transferrin and low-density lipoprotein while internalization of the fluid marker dextran is unchanged. Cells expressing the cortactin Src homology three domain also exhibited markedly reduced endocytosis. These findings suggest that cortactin is an important component of the receptor-mediated endocytic machinery, where, together with actin and dynamin, it regulates the scission of clathrin pits from the plasma membrane. Thus, cortactin provides a direct link between the dynamic actin cytoskeleton and the membrane pinchase dynamin that supports vesicle formation during receptor-mediated endocytosis (Cao, 2003).
BALB/c 3T3 cells require a prolonged exposure to fibroblast growth factor (FGF)-1 for the stimulation of maximal DNA synthesis, and this event correlates with the tyrosine phosphorylation of novel proteins late in G1, including a protein termed p80/p85. The cDNA encoding p80/p85 has been purified, sequenced, and cloned; it is the murine homolog of the chicken cortactin gene and a member of the human hematopoietic specific-1 gene family. Immunochemical analysis of m-cortactin-tyrosine phosphorylation in response to FGF-1 demonstrates a biphasic phosphorylation pattern both as a weak immediate-early and strong mid to late G1 response protein. Because the chicken cortactin gene was originally isolated as a substrate for v-Src, FGF-1 may influence the enzymatic activity of other cell-associated tyrosine kinases that utilize p80/p85 (cortactin) as a polypeptide substrate (Zhan, 1993).
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. Phosphorylation of cortactin tyrosine residues in the carboxy terminus is shown to require 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 is elevated in response to Src, epidermal growth factor receptor and Rac1 activation, and tyrosine 421 phosphorylated cortactin localizes 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 a hierarchical manner that is closely coordinated with its ability to regulate actin dynamics (Head, 2003).
The F-actin binding protein cortactin is an important regulator of cytoskeletal dynamics, and a prominent target of various tyrosine kinases. Tyrosine phosphorylation of cortactin has been suggested to reduce its F-actin crosslinking capability. Whether a reciprocal relationship exists was investigated, i.e. whether the polymerization state of actin impacts on cortactin tyrosine phosphorylation. Actin depolymerization by Latrunculin B (LB) induces robust phosphorylation of C-terminal tyrosine residues of cortactin. Conversely, F-actin stabilization by jasplakinolide, which redistributes cortactin to F-actin containing patches, preventes cortactin phosphorylation triggered by hypertonic stress or LB. Using cell lines deficient in candidate tyrosine kinases, it was found that the F-actin depolymerization-induced cortactin phosphorylation is mediated by the Fyn/Fer kinase pathway, independent of Src and c-Abl. LB causes modest Fer activation and strongly facilitates the association between Fer and cortactin. Interestingly, the F-actin binding region within the cortactin N-terminus is essential for the efficient phosphorylation of C-terminal tyrosine residues. Investigating the structural requirements for the Fer-cortactin association, it was found that phosphorylation-incompetent cortactin still binds to Fer: the isolated N-terminus associates with Fer, and the C-terminus alone is insufficient for binding. Thus, the cortactin N-terminus participates in the Fer-cortactin interaction, which cannot be fully due to the binding of the Fer SH2 domain to C-terminal tyrosines of cortactin. Taken together, F-actin stabilization prevents whereas depolymerization promotes cortactin tyrosine phosphorylation. The depolymerization-induced phosphorylation is mediated by Fer, and requires the actin-binding domain of cortactin. These results define a novel F-actin-dependent pathway that may serve as a feedback mechanism during cytoskeleton remodeling (Fan, 2004).
Proper regulation of cell morphogenesis and migration by adhesion and growth-factor receptors requires Abl-family tyrosine kinases. Several substrates of Abl-family kinase have been identified, but they are unlikely to mediate all of the downstream actions of these kinases on cytoskeletal structure. A human protein microarray was used to identify the actin-regulatory protein cortactin as a novel substrate of the Abl and Abl-related gene (Arg) nonreceptor tyrosine kinases. Cortactin stimulates cell motility, and its upregulation in several cancers correlates with poor prognosis. Even though cortactin can be tyrosine phosphorylated by Src-family kinases in vitro, Abl and Arg are more adept at binding and phosphorylating cortactin. Importantly, platelet-derived growth-factor (PDGF)-induced cortactin phosphorylation on three tyrosine residues requires Abl or Arg. Cortactin triggers F-actin-dependent dorsal waves in fibroblasts after PDGF treatment and thus results in actin reorganization and lamellipodial protrusion. Evidence is provided that Abl/Arg-mediated phosphorylation of cortactin is required for this PDGF-induced dorsal-wave response. The results reveal that Abl-family kinases target cortactin as an effector of cytoskeletal rearrangements in response to PDGF (Boyle, 2007).
Two genes (EMS1 and PRAD1/cyclin D1) in the chromosome 11q13 region have been identified that are frequently coamplified and overexpressed in human breast cancer and in squamous cell carcinomas of the head and neck. The 80/85-kDa protein that is encoded by the EMS1 gene shows a high homology (85%) to a chicken protein that was recently identified as a substrate for the src oncogene. Immunocytochemistry reveals that in epithelial cells, the human EMS1 protein is localized mainly in the cytoplasm and, to a very low extent, in protruding leading lamellae of the cell. However, in carcinoma cells that constitutively overexpress the protein as a result of amplification of the EMS1 gene, the protein, except in cytoplasm, accumulates in the podosome-like adherens junctions associated with the cell-substratum contact sites. The protein was not found in intercellular adherens junctions. These findings, and the previously reported observations in src-transformed chicken embryo fibroblasts, suggest that the EMS1 protein is involved in regulating the interactions between components of adherens-type junctions. Since amplification of the 11q13 region has been associated with an enhanced invasive potential of these tumors, overexpression and concomitant accumulation of the EMS1 protein in the cell-substratum contact sites might, therefore, contribute to the invasive potential of these tumor cells (Schuuring, 1993).
Classical cadherin adhesion molecules are key determinants of cell-cell recognition during development and in post-embryonic life. A decisive step in productive cadherin-based recognition is the conversion of nascent adhesions into stable zones of contact. It is increasingly clear that such contact zone extension entails active cooperation between cadherin adhesion and the force-generating capacity of the actin cytoskeleton. Cortactin has recently emerged as an important regulator of actin dynamics in several forms of cell motility. Cortactin is recruited to cell-cell adhesive contacts in response to homophilic cadherin ligation. Notably, cortactin accumulates preferentially, with Arp2/3, at cell margins where adhesive contacts are being extended. Recruitment of cortactin is accompanied by a ligation-dependent biochemical interaction between cortactin and the cadherin adhesive complex. Inhibition of cortactin activity in cells blocks Arp2/3-dependent actin assembly at cadherin adhesive contacts, significantly reduces cadherin adhesive contact zone extension, and perturbs both cell morphology and junctional accumulation of cadherins in polarized epithelia. Together, these findings identify a necessary role for cortactin in the cadherin-actin cooperation that supports productive contact formation (Helwani, 2004).
The number and shape of dendritic spines are influenced by activity and regulated by molecules that organize the actin cytoskeleton of spines. Cortactin is an F-actin binding protein and activator of the Arp2/3 actin nucleation machinery that also interacts with the postsynaptic density (PSD) protein Shank. Cortactin is concentrated in dendritic spines of cultured hippocampal neurons, and the N-terminal half of the protein containing the Arp2/3 and F-actin binding domains is necessary and sufficient for spine targeting. Knockdown of cortactin protein by short-interfering RNA (siRNA) results in depletion of dendritic spines in hippocampal neurons, whereas overexpression of cortactin causes elongation of spines. In response to synaptic stimulation and NMDA receptor activation, cortactin redistributes rapidly from spines to dendritic shaft, correlating with remodeling of the actin cytoskeleton, implicating cortactin in the activity-dependent regulation of spine morphogenesis (Hering, 2003).
Delta-catenin is a neuronal protein that contains 10 Armadillo motifs and binds to the juxtamembrane segment of classical cadherins. Delta-catenin interacts with cortactin in a tyrosine phosphorylation-dependent manner. This interaction occurs within a region of the delta-catenin sequence that is also essential for the neurite elongation effects. Src family kinases can phosphorylate delta-catenin and bind to delta-catenin through its polyproline tract. Under conditions when tyrosine phosphorylation is reduced, delta-catenin binds to cortactin and cells extend unbranched primary processes. Conversely, increasing tyrosine phosphorylation disrupts the delta-catenin-cortactin complex. When RhoA is inhibited, delta-catenin enhances the effects of Rho inhibition on branching. It is concluded that delta-catenin contributes to setting a balance between neurite elongation and branching in the elaboration of a complex dendritic tree (Martinez, 2003).
Cortactin is an actin-binding protein that contains several potential signaling motifs including a Src homology 3 (SH3) domain at the distal C terminus. Translocation of cortactin to specific cortical actin structures and hyperphosphorylation of cortactin on tyrosine have been associated with the cortical cytoskeleton reorganization induced by a variety of cellular stimuli. The function of cortactin in these processes is largely unknown in part due to the lack of information about cellular binding partners for cortactin. A novel cortactin-binding protein of approximately 180 kDa has been identified by yeast two-hybrid interaction screening. The interaction of cortactin with this 180-kDa protein was confirmed by both in vitro and in vivo methods, and the SH3 domain of cortactin was found to direct this interaction. Since this protein represents the first reported natural ligand for the cortactin SH3 domain it has been designated CortBP1 for cortactin-binding protein 1. CortBP1 contains two recognizable sequence motifs within its C-terminal region, including a consensus sequence for cortactin SH3 domain-binding peptides and a sterile alpha motif. Northern and Western blot analyses indicate that CortBP1 is expressed predominately in brain tissue. Immunofluorescence studies revealed colocalization of CortBP1 with cortactin and cortical actin filaments in lamellipodia and membrane ruffles in fibroblasts expressing CortBP1. Colocalization of endogenous CortBP1 and cortactin is also observed in growth cones of developing hippocampal neurons, implicating CortBP1 and cortactin in cytoskeleton reorganization during neurite outgrowth (Du, 1998).
Growth factor regulation of the cortical actin cytoskeleton is fundamental to a wide variety of cellular processes. The cortical actin-associated protein, cortactin, regulates the formation of dynamic actin networks via the actin-related protein (Arp)2/3 complex and hence is a key mediator of such responses. In order to reveal novel roles for this versatile protein, a proteomics-based approach was used to isolate cortactin-interacting proteins. This identified several proteins, including CD2-associated protein (CD2AP), as targets for the cortactin Src homology 3 domain. Co-immunoprecipitation of CD2AP with cortactin occurs at endogenous expression levels, is transiently induced by epidermal growth factor (EGF) treatment, and requires the cortactin Src homology 3 domain. The CD2AP-binding site for cortactin maps to the second of three proline-rich regions. Because CD2AP is closely related to Cbl-interacting protein of 85 kDa (CIN85), which regulates growth factor receptor down-regulation via complex formation with Cbl and endophilin, whether the CD2AP-cortactin complex performs a similar function was investigated. EGF treatment of cells leads to transient association of Cbl and the epidermal growth factor receptor (EGFR) with a constitutive CD2AP-endophilin complex. Cortactin is recruited into this complex with slightly delayed kinetics compared with Cbl and the EGFR. Immunofluorescence analysis revealed that the EGFR, CD2AP, and cortactin co-localize in regions of EGF-induced membrane ruffles. Therefore, by binding both CD2AP and the Arp2/3 complex, cortactin links receptor endocytosis to actin polymerization, which may facilitate the trafficking of internalized growth factor receptors (Lynch, 2003).
The regulation of protein tyrosine phosphorylation is an important aspect during the cell cycle. From G2-M transition to mitotic anaphase, phosphorylation of Tyr421, Tyr466 and Tyr482 of cortactin, an actin-filament associated protein, is dramatically induced. The phosphorylated cortactin is almost exclusively associated with centrosomes or spindle poles during mitosis. At G2-M transition prior to the breakdown of the nuclear envelope, two duplicated centrosomes migrate towards opposite ends of the nucleus to form the spindle poles. This centrosome-separation process and also the start of mitosis are inhibited or delayed by the depolymerization of actin filaments. Also inhibited is the separation of centrosomes when a truncated form of cortactin is expressed, whose C-terminus contains the tyrosine phosphorylation region but lacks the actin-binding domains. Mutations were introduced at the tyrosine phosphorylation sites in the truncated C-terminus of cortactin, and it was found that the C-terminus could no longer interfere with centrosome separation process. This study shows that, cortactin phosphorylated at Tyr421, Tyr466 and Tyr482 mediates the actin-filament-driven centrosome separation at G2-M transition by providing a bridge between the centrosome and actin-filaments (Wang, 2008).
Cortactin, a filamentous actin cross-linking protein and a substrate of Src protein tyrosine kinase, is phosphorylated at tyrosine residues upon stimulation by extracellular signals. The filamentous actin cross-linking activity of cortactin is attenuated by Src. In vitro, tyrosine phosphorylation of cortactin occurs specifically within the region between the proline-rich sequence and the Src homology 3 domain. Among the nine tyrosine residues in this region, mutations at Tyr421, Tyr466, and Tyr482 significantly reduced Src-meditated tyrosine phosphorylation both in vitro and in vivo. Ectopic expression of wild-type cortactin in ECV304, a spontaneously transformed human umbilical endothelial cell line, results in an enhanced cell migration. In contrast, overexpression of a cortactin mutant deficient in tyrosine phosphorylation impairs the migration of endothelial cells. These findings reveal an intracellular signaling mechanism whereby the motility of endothelial cells is regulated by a Src-mediated tyrosine phosphorylation of cortactin (Huang, 1998).
Cortactin, a p80/85 protein first identified as a src kinase substrate, is thought to be involved in the signaling pathway of mitogenic receptors and adhesion molecules mediating cytoskeletal reorganization. The cortactin gene, EMS1, maps to chromosome 11q13, a region amplified in head and neck squamous cell carcinomas (HNSCC) and breast cancer, which display lymph node metastasis and an unfavorable clinical outcome. To further address the role of cortactin in the malignant phenotype of cells, cortactin was stably overexpressed in NIH3T3 fibroblasts and the effects of elevated cortactin on cellular proliferation, motility and invasiveness were evaluated. Cortactin overexpressing cells do not display any striking morphological changes, nor any significant differences in cell proliferation or saturation density as compared to control NIH3T3 cells. Furthermore, the cortactin overexpressing cells are anchorage dependent for growth. Interestingly, cortactin overexpressing cells are more motile and invasive in modified Boyden chamber assays. These results suggest that overexpression of cortactin may play a role in tumor progression by influencing tumor cell migration and invasion (Patel, 1998).
Invasive breast cancer cells have the ability to extend membrane protrusions, invadopodia, into the extracellular matrix (ECM). These structures are associated with sites of active matrix degradation. The amount of matrix degradation associated with the activity of these membrane protrusions has been shown to directly correlate with invasive potential. Microinjection of polyclonal anti-cortactin antibodies blocks matrix degradation at invadopodia, supporting the hypothesis that cortactin has a direct role in invasive behavior. MDA-MB-231, invasive breast cancer cells, were sheared from the surface of a gelatin matrix to isolate invadopodia. Cortactin, paxillin and protein kinase C (PKC) mu, a serine kinase, were co-immunoprecipitated as a complex from invadopodia-enriched membranes. the subcellular distribution of these proteins was confirmed by immunolocalization and Western blotting. In contrast to its presence in invasive cells, this complex of proteins was not detected in lysates from non-invasive cells that do not form invadopodia. Taken together, these data suggest that the formation of this cortactin-containing complex correlates with cellular invasiveness. It is hypothesized that this complex of molecules has a role in the formation and function of invadopodia during cellular invasion (Bowden, 1999).
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, 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 were examined. Injection of MDA-MB-231 cells overexpressing GFP-cortactin into nude mice through cardiac ventricles causes 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. In contrast, 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 (Li, 2001).
Cortactin is a filamentous actin (F-actin)-binding protein that regulates cytoskeletal dynamics by activating the Arp2/3 complex; it binds to F-actin by means of six N-terminal 'cortactin repeats'. Gene amplification of 11q13 and consequent overexpression of cortactin in several human cancers is associated with lymph node metastasis. Overexpression as well as tyrosine phosphorylation of cortactin has been reported to enhance cell migration, invasion, and metastasis. Two alternative splice variants (SV1 and SV2) have been identified that affect the cortactin repeats: SV1-cortactin lacks the 6th repeat (exon 11), whereas SV2-cortactin lacks the 5th and 6th repeats (exons 10 and 11). SV-1 cortactin is found co-expressed with wild type (wt)-cortactin in all tissues and cell lines examined, whereas the SV2 isoform is much less abundant. SV1-cortactin binds F-actin and promotes Arp2/3-mediated actin polymerization equally well as wt-cortactin, whereas SV2-cortactin shows reduced F-actin binding and polymerization. Alternative splicing of cortactin does not affect its subcellular localization or growth factor-induced tyrosine phosphorylation. However, cells that overexpress SV1- or SV2-cortactin show significantly reduced cell migration when compared with wt-cortactin-overexpressing cells. Thus, in addition to overexpression and tyrosine phosphorylation, alternative splicing of the F-actin binding domain of cortactin is a new mechanism by which cortactin influences cell migration (van Rossum, 2003).
Missing in metastasis (MIM) gene encodes an actin binding protein that is expressed at low levels in a subset of malignant cell lines. MIM protein tagged by green fluorescent protein (GFP) colocalizes with cortactin, an Arp2/3 complex activator, and interacts directly with the SH3 domain of cortactin. Recombinant full-length MIM promotes markedly cortactin and Arp2/3 complex-mediated actin polymerization in an SH3 dependent manner. In contrast, MIM-CT, a short splicing variant of MIM, binds poorly to cortactin in vitro and is unable to enhance actin polymerization. Full-length MIM binds to G-actin with a similar affinity as N-WASP-VCA, a constitutively active form of N-WASP, and inhibits N-WASP-VCA-mediated actin polymerization as analysed in vitro. The significance of the association of MIM with cortactin and G-actin was evaluated in NIH3T3 cells expressing several MIM constructs. Overexpression of full-length wild-type MIM-GFP inhibited markedly the motility of NIH3T3 cells induced by PDGF and that of human vein umbilical endothelial cells induced by sphingosine 1 phosphate. However, an MIM mutant with deletion of the WH2 domain, which is responsible for G-actin binding, enhanced cell motility. The motility inhibition imposed by MIM was compromised in the cells overexpressing N-WASP. In contrast, deletion of an MIM proline-rich domain, which is required for an optimal binding to cortactin, substantiated the MIM-mediated inhibition of cell motility. These data imply that MIM regulates cell motility by modulating different Arp2/3 activators in a distinguished manner (Lin, 2005).
The regulation of protein tyrosine phosphorylation is an important aspect during the cell cycle. From G2-M transition to mitotic anaphase, phosphorylation of Tyr421, Tyr466 and Tyr482 of cortactin, an actin-filament associated protein, is dramatically induced. The phosphorylated cortactin is almost exclusively associated with centrosomes or spindle poles during mitosis. At G2-M transition prior to the breakdown of the nuclear envelope, two duplicated centrosomes migrate towards opposite ends of the nucleus to form the spindle poles. This centrosome-separation process and also the start of mitosis are inhibited or delayed by the depolymerization of actin filaments. Also inhibited is the separation of centrosomes when a truncated form of cortactin is expressed, whose C-terminus contains the tyrosine phosphorylation region but lacks the actin-binding domains. Mutations were introduced at the tyrosine phosphorylation sites in the truncated C-terminus of cortactin and it was found that the C-terminus could no longer interfere with centrosome separation process. This study shows that, cortactin phosphorylated at Tyr421, Tyr466 and Tyr482 mediates the actin-filament-driven centrosome separation at G2-M transition by providing a bridge between the centrosome and actin-filaments (Bershteyn, 2010).
The primary cilium is critical for transducing Sonic hedgehog (Shh) signaling, but the mechanisms of its transient assembly are poorly understood. Previously it has been shown that the actin regulatory protein Missing-in-Metastasis (MIM) regulates Shh signaling, but the nature of MIM's role was unknown. This study shows that MIM is required at the basal body of mesenchymal cells for cilia maintenance, Shh responsiveness, and de novo hair follicle formation. MIM knockdown results in increased Src kinase activity and subsequent hyperphosphorylation of the actin regulator Cortactin. Importantly, inhibition of Src or depletion of Cortactin compensates for the cilia defect in MIM knockdown cells, whereas overexpression of Src or phospho-mimetic Cortactin is sufficient to inhibit ciliogenesis. These results suggest that MIM promotes ciliogenesis by antagonizing Src-dependent phosphorylation of Cortactin and describe a mechanism linking regulation of the actin cytoskeleton with ciliogenesis and Shh signaling during tissue regeneration (Bershteyn, 2010).
This study shows that MIM promotes ciliogenesis by inhibiting Src kinase activation during G1. Decreased levels of MIM lead to upregulation of activated Src and subsequent hyperphosphorylation of multiple actin-associated Src substrates including Cortactin (CTTN), which promotes increased F-actin branching and polymerization. Importantly, either inhibition of the Src catalytic domain or removal of CTTN is sufficient to restore ciliogenesis in the absence of MIM, suggesting that MIM regulates an intricate balance of actin regulatory factors that affect cilia dynamics, but is not uniquely required for ciliogenesis. Among the critical functional consequences of this deregulation in mesenchymal cells are failure to respond to Shh signaling and inability to induce hair growth. Collectively, these data reveal a mechanism that coordinates ciliogenesis with cell cycle progression and provide a strong connection between actin cytoskeletal regulators, ciliogenesis, and Shh signaling during tissue regeneration (Bershteyn, 2010).
Much like the developing node and the limbs, the hair follicles rely on primary cilia to transduce signals from Shh and other morphogens. Reduction in MIM levels in the dermal compartment leads to severe lack of hair and immature follicles that appear arrested at stage 2-4 of anagen, similar to the phenotypes observed upon dermal cilia ablation through conditional deletion of ciliary structural components Ift88 or Kif3A or in Shh mutant skin. The fact that MIM KD disrupts primary cilia in the regenerated dermal papillae provides additional evidence that the cilia defect forms the basis of the hair follicle phenotype and underscores the importance for dermal primary cilia in hair follicle development. Thus, the results support a key role for MIM in dermal cilia regulation and Shh signaling during de novo hair formation and demonstrate the utility of the hair regeneration system for rapid screening, identification, and analysis of in vivo, cell type-specific regulators of hair formation (Bershteyn, 2010).
The data point to a model whereby MIM promotes ciliogenesis by antagonizing Src activity during G1. MIM's inhibitory effect on Src is highlighted by the fact that multiple Src substrates, including other kinases, scaffolding proteins, and actin cytoskeleton regulators known to affect cell proliferation, adhesion and migration, become hyperphosphorylated upon MIM KD. This finding provides a potential basis for the frequent alteration of MIM levels in metastatic cancers, as Src is a well-known oncogene and the hyperphosphorylated substrates that were detected are all associated with decreased cell adhesion and increased cell motility. However, with respect to Src-dependent cilia disassembly, the Src substrate CTTN appears to be a key downstream effector, since CTTN KD impairs cilia resorption and removal of CTTN is sufficient to restore cilia in MIM KD cells (Bershteyn, 2010).
Several independent lines of evidence support the model that ectopic Src kinase activity during G1 inhibits ciliogenesis. First, the cilia defect in MIM KD cells can be rescued by several small molecule inhibitors of the SFK family. Second, overexpression of just p60 Src in Src/Yes/Fyn triple knockout SYF-/- MEFs potently inhibits ciliogenesis and completely abolishes any Shh responsiveness, despite the fact that most of the cells are in G1/G0. Third, transient expression of a constitutively active Src Y527F mutant reveals basal body localization and inhibits ciliogenesis in SYF-/-MEFs and primary dermal cells in a cell-autonomous manner. Finally, fourth: transiently expressed kinase dead Src K295R mutant functions as a dominant-negative with respect to p-CTTN and induces more and longer cilia in dermal cells. These data point to the powerful inhibitory actions of Src kinase and the critical need to restrain its activity during G1 (Bershteyn, 2010).
Emerging data support the idea that the basal body coordinates ciliogenesis with the cell cycle. This regulation is likely based on a dynamic cell-cycle-dependent equilibrium between factors that promote cilia formation after cytokinesis and factors that promote cilia disassembly prior to mitosis. MIM and CTTN appear to be two such factors, with MIM promoting cilia formation and Src-activated p-CTTN promoting cilia disassembly (Bershteyn, 2010).
Based on these results, a model is proposed whereby MIM antagonism of p-CTTN serves as a switch to regulate the timing of ciliogenesis and coordinate it with the cell cycle. Thus during G1/S, when the relative ratio of MIM to p-CTTN is high, cilia are maintained. As the cell cycle progresses toward G2/M, activation of Src leads to increased p-CTTN levels, shifting the ratio of MIM to p-CTTN to low and inducing cilia disassembly. When MIM is depleted, activation of Src leads to upregulation of p-CTTN during G1, artificially shifting the ratio of MIM to p-CTTN to resemble what it normally is during G2/M. Interestingly MIM-depleted cells display ectopic p-Src and p-CTTN in the cytosol and at the basal body, suggesting that Src and CTTN functions outside the basal body may also contribute to cilia maintenance. This is supported by the finding that inhibition of Src or depletion of CTTN restores cilia in MIM KD cells and underscores the critical relationship between MIM and Src activity (Bershteyn, 2010).
These findings combined with multiple lines of published data suggest that MIM and CTTN antagonism affects ciliogenesis by regulating the actin cytoskeleton. First of all, the antagonism between MIM and CTTN appears to be conserved in evolution and 'recycled' for multiple actin-dependent cellular processes including Drosophila border cells migration and clathrin-mediated endocytosis (Quinones, 2010). Moreover, high levels of MIM relative to CTTN were shown in vitro to inhibit CTTN mediated actin polymerization (Lin, 2005). In contrast, phosphorylation by Src is known to increase CTTN affinity and nucleating activity for F-actin (Ammer, 2008; Lua, 2005; Kruchten, 2008). Moreover, recent studies in NIH 3T3 and HeLa cells found that p-CTTN Y466/Y421 is enriched at the centrosomes during G2/M and mediates actin-dependent centrosome separation (Wang, 2008). Consistent with all of these data, increased levels and disorganized F-actin staining is seen in MIM KD cells that lack cilia and vice versa, reduced F-actin staining in CTTN KD cells that fail to disassemble their cilia. In addition, disruption of F-actin with polymerization inhibitors causes cilia elongation and prevents cilia disassembly. Thus, while F-actin may be required for initial basal body motility and membrane docking, the data suggest that F-actin needs to be cleared locally during G1 to allow cilia elongation and maintenance and then reformed later in the cell cycle to promote cilia disassembly. Interestingly, a recent screen for genes involved in ciliogenesis identified several regulators of actin dynamics and vesicle trafficking, and actin clearing by the centrosome has been shown to be required in other directed membrane events such as exocytosis of lytic granules. Moreover, Septin2, known to play a role in actin dynamics, appears to play a role in maintaining the ciliary protein diffusion barrier and proper signaling. Therefore, actin regulatory proteins localized at the basal body such as MIM and CTTN could provide a general mechanism for regulating directional plasma membrane remodeling and proper cilium-dependent signaling (Bershteyn, 2010).
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