Cortactin, a Z0-1 interacting protein

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 had 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).

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 activation to lamellipoda and membrane ruffles, and Cdc42 to microspikes and filopodia. Recently identified have been several downstream molecules mediating these effects. 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 that has recently been 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).

Rabbit anti-Drosophila Cortactin antibody can also react with a bacterially expressed protein containing the mouse cortactin 37-amino acid repeat domain fused to maltose binding protein. Using this antibody, Western blot analysis of tissue lysates from a 4-day postnatal mouse has detected 80- and 85-kDa proteins. These proteins are fairly abundant in brain and testis but not so in liver and kidney. Western blot analysis using the rat anti-Drosophila Cortactin antiserum also yields the same pattern as that using an anti-chicken p80/85 (cortactin) monoclonal antibody, which cross-reacts with mouse cortactin. These results indicate that both the rabbit and rat anti-Drosophila Cortactin antibodies can cross-react with mouse cortactin. Mouse cortactin is shown to associate with mouse ZO-1 in vivo (Katsube, 1998).

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 reduce 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 prominent substrate for pp60(c-src), is a filamentous actin (F-actin) binding protein. Cortactin can promote sedimentation of F-actin at centrifugation forces below which F-actin is otherwise not able to be precipitated. Electron microscopic analysis after negative staining further reveals 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. pp60(c-src) phosphorylates cortactin in vitro, resulting in a dramatic reduction of its F-actin cross-linking activity in a manner dependent upon 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 (Huang, 1997).

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 identifed 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 analysis indicate that CortBP1 is expressed predominately in brain tissue. Immunofluorescence studies reveal 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).

Catenin pp120 is the prototype of a subfamily of Armadillo proteins, comprising ARVCF, p0071, delta-catenin/NPRAP, and plakophilins 1 and 2. Characterization of the nonreceptor tyrosine kinase FER has identified a tight physical association with catenin pp120 and has led to the suggestion that FER may be involved in cell-cell signaling. The majority of FER is localized to the cytoplasmic fraction where it forms a complex with the actin-binding protein cortactin. The Src homology 2 sequence of FER is required for directly binding cortactin, and phosphorylation of the FER-cortactin complex is up-regulated in cells treated with peptide growth factors. Using a dominant-negative mutant of FER, evidence is provided that FER kinase activity is required for the growth factor-dependent phosphorylation of cortactin. These data suggest that cortactin is likely to be a direct substrate of FER. These observations provide additional support for a role for FER in mediating signaling from the cell surface, via growth factor receptors, to the cytoskeleton. The nature of the FER-cortactin interaction, and their putative enzyme-substrate relationship, support the previous proposal that one of the functions of the Src homology 2 sequences of nonreceptor tyrosine kinases is to provide a binding site for their preferred substrates (Kim, 1998).

The Fer protein belongs to the fes/fps family of nontransmembrane receptor tyrosine kinases. Fer has been shown to be a cortactin-interacting protein (Kim, 1998). Lack of success in attempts to establish a permanent cell line overexpressing Fer at significant levels suggests a strong negative selection against too much Fer protein and points to a critical cellular function for Fer. Using a tetracycline-regulatable expression system, overexpression of Fer in embryonic fibroblasts evokes a massive rounding up, and the subsequent detachment of the cells from the substratum, which eventually leads to cell death. Induction of Fer expression coincides with increased complex formation between Fer and the cadherin/src-associated substrate p120(cas) and elevated tyrosine phosphorylation of p120(cas). beta-Catenin also exhibits clearly increased phosphotyrosine levels, and Fer and beta-catenin are found to be in complex. Significantly, although the levels of alpha-catenin, beta-catenin, and E-cadherin are unaffected by Fer overexpression, decreased amounts of alpha-catenin and beta-catenin are coimmunoprecipitated with E-cadherin, demonstrating a dissolution of adherens junction complexes. A concomitant decrease in levels of phosphotyrosine in the focal adhesion-associated protein p130 is also observed. Together, these results provide a mechanism for explaining the phenotype of cells overexpressing Fer and indicate that the Fer tyrosine kinase has a function in the regulation of cell-cell adhesion (Rosato, 1998).

Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of fibroblasts and epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s), as well as the exact biochemical nature of the cytoskeletal change, remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac-phosphatidylinositol 4,5-biphosphate (PIP2)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP2 binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP2 inhibits the actin-EMS1 interaction, HS1 reverses the PIP2 effect. Thus, it is proposed that PIP2, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton (He, 1998).

N-syndecan (syndecan-3) is a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and is suggested to mediate the neurite growth-promoting signal from cell matrix-bound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. N18 neuroblastoma cells were transfected with a rat N-syndecan cDNA and it was shown that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinase-active fractions that bind to the cytosolic moiety of N-syndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. It is suggested that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway (Kinnunen, 1998).

During the development of the neuromuscular junction (NMJ), motoneurons grow to the muscle cell and the nerve-muscle contact triggers the development of both presynaptic specialization, consisting of clusters of synaptic vesicles (SVs), and postsynaptic specialization, consisting of clusters of acetylcholine receptors (AChRs). Activation of tyrosine kinases and the local assembly of an actin-based cytoskeletal specialization are involved in the development of both types of specializations. To understand the link between tyrosine phosphorylation and the assembly of the cytoskeleton, the localization of cortactin was examined in relationship to synaptic development. Cortactin is a 80/85 kD F-actin binding protein and is a substrate for tyrosine kinases. It contains a proline-rich motif and an SH3 domain and is localized at sites of active F-actin assembly. Using a monoclonal antibody against cortactin, its localization at developing NMJs in culture was observed. To understand the spatial and temporal relationship between cortactin and developing synaptic structures, cultured muscle cells and spinal neurons from Xenopus embryos were treated with beads coated with heparin-binding growth-associated molecules to induce the formation of AChR clusters and SV clusters. The localization of cortactin was followed by immunofluorescence. In untreated muscle cells, cortactin is often co-localized with spontaneously formed AChR clusters. After cells are treated with beads, cortactin becomes localized at bead-induced AChR clusters at their earliest appearance (1 h after the addition of beads). This association is most reliably detected at the early stage of the clustering process. On the presynaptic side, cortactin localization can be detected as early as 10 min after the bead-neurite contact is established. Cortactin-enriched contacts later show concentration of F-actin (at 1 h) and clusters of SVs (at 24 h). These data suggest that cortactin mediates the local assembly of the cytoskeletal specialization triggered by the synaptogenic signal on both nerve and muscle (Peng, 1997).

ZO-1 and development

The mouse preimplantation embryo has been used to investigate the de novo synthesis of tight junctions during trophectoderm epithelial differentiation. Individual components of the tight junction assemble in a temporal sequence: membrane assembly of the cytoplasmic plaque protein ZO-1 occurs 12 hours before that of cingulin, a 140 kDa cytoplasmic constituent of junctions. Subsequently, two alternatively spliced isoforms of ZO-1 (alpha+ and alpha-) associate with the junction; they differ in the presence or absence of an 80 residue alpha domain. The temporal and spatial expression of these ZO-1 isoforms has been investigated at different stages of preimplantation development. ZO-1alpha- mRNA is present in oocytes and all preimplantation stages, whilst ZO-1alpha+ transcripts are first detected in embryos at the morula stage, close to the time of blastocoele formation. mRNAs for both isoforms are detected in trophectoderm and ICM cells. Immunoprecipitation of 35S-labelled embryos also shows synthesis of ZO-1alpha- throughout cleavage, whereas synthesis of ZO-1alpha+ is only apparent from the blastocyst stage. In addition, both isoforms are phosphorylated at the early blastocyst stage (Sheth, 1997).

Two-way interactions between the blastocyst trophectoderm and the uterine luminal epithelium are essential for implantation. The key events of this process are cell-cell contact of trophectoderm cells with uterine luminal epithelial cells; controlled invasion of trophoblast cells through the luminal epithelium and the basement membrane; transformation of uterine stromal cells surrounding the blastocyst into decidual cells, and protection of the 'semiallogenic' embryo from the mother's immunological responses. Because cell-cell contact between the trophectoderm epithelium and the luminal epithelium is essential for implantation in the mouse uterus, the expression of zonula occludens-1 (ZO-1) and E-cadherin, two molecules associated with epithelial cell junctions, was examined during the periimplantation period. Preimplantation uterine epithelial cells express both ZO-1 and E-cadherin. With the initiation and progression of implantation, ZO-1 and E-cadherin are expressed in stromal cells of the primary decidual zone (PDZ). As trophoblast invasion progresses, these two molecules are expressed in stroma in advance of the invading trophoblast cells. These results suggest that expression of these adherence and tight junction molecules in the PDZ functions as a permeability barrier to regulate access of immunologically competent maternal cells and/or molecules to the embryo and provides homotypic guidance of trophoblast cells in the endometrium (Paria, 1999).

The pattern and timing of membrane assembly of the two isoforms is also distinct. ZO-1alpha- is initially seen in punctate sites at the cell-cell contacts of compact 8-cell embryos. These sites then coalesce laterally along the membrane until by the late morula stage they completely surround each cell with a zonular belt. ZO-1alpha+ however, is first seen as perinuclear foci in late morulae, before assembling at the tight junction. Membrane assembly of ZO-1alpha+ first occurs during the 32-cell stage and is zonular just prior to the early blastocyst stage. Both isoforms are restricted to the trophectoderm lineage. Membrane assembly of ZO-1alpha+ and blastocoele formation are sensitive to brefeldin A, an inhibitor of intracellular trafficking beyond the Golgi complex. The tight junction transmembrane protein occludin co-localizes with ZO-1alpha+ at the perinuclear sites in late morulae and at the newly assembled cell junctions. These results provide direct evidence from a native epithelium that ZO-1 isoforms perform distinct roles in tight junction assembly. The late expression of ZO-1alpha+ and its apparent intracellular interaction with occludin may act as a final rate-limiting step in the synthesis of the tight junction, thereby regulating the time of junction sealing and blastocoele formation in the early embryo (Sheth, 1997).

The epithelial character of neuroepithelial cells was investigated in the context of neurogenesis by examining the presence of molecular components of tight junctions during the transition from the neural plate to the neural tube. Immunoreactivity for occludin, a transmembrane protein specific to tight junctions, is detected at the apical end of the lateral membrane of neuroepithelial cells throughout the chick neural plate. During neural tube closure, occludin disappears from all neuroepithelial cells. Correspondingly, functional tight junctions are present in the neural plate (embryonic day 8), but not in the neural tube (embryonic day 9). In contrast to occludin, expression of ZO-1, a peripheral membrane protein of tight junctions, increases from the neural plate to the neural tube stage, also being confined to the apical end of the lateral neuroepithelial cell membrane. This localization coincides with that of N-cadherin (see Drosophila Cadherin-N), whose expression increases concomitantly with the disappearance of occludin. It is proposed that the loss of tight junctions from neuroepithelial cells reflects an overall decrease in their epithelial nature, which precedes the generation of neurons (Aaku-Saraste, 1996).

Cadherin-based adhesions in the apical endfoot are required for active Notch signaling to control neurogenesis in vertebrates

The development of the vertebrate brain requires an exquisite balance between proliferation and differentiation of neural progenitors. Notch signaling plays a pivotal role in regulating this balance, yet the interaction between signaling and receiving cells remains poorly understood. This study found that numerous nascent neurons and/or intermediate neurogenic progenitors expressing the ligand of Notch retain apical endfeet transiently at the ventricular lumen that form adherens junctions (AJs) with the endfeet of progenitors. Forced detachment of the apical endfeet of those differentiating cells by disrupting AJs resulted in precocious neurogenesis that was preceded by the downregulation of Notch signaling. Both Notch1 and its ligand Dll1 are distributed around AJs in the apical endfeet, and these proteins physically interact with ZO-1, a constituent of the AJ. Furthermore, live imaging of a fluorescently tagged Notch1 demonstrated its trafficking from the apical endfoot to the nucleus upon cleavage. These results identified the apical endfoot as the central site of active Notch signaling to securely prohibit inappropriate differentiation of neural progenitors (Hatakeyama, 2014).

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