Alternative splicing of the actin binding domain of human cortactin affects cell migration

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. Here we report the identification of two alternative splice variants (SV1 and SV2) 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.     

Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/3 complex

Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Here, we report that 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 failed 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 were 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.     

Cortactin affects cell migration by regulating intercellular adhesion and cell spreading

Cortactin is an F-actin binding protein that stabilizes F-actin networks and promotes actin polymerization by activating the Arp2/3 complex. Overexpression of cortactin, as observed in several human cancers, stimulates cell migration, invasion, and experimental metastasis; however, the underlying mechanism is not understood. To investigate the importance of cortactin in cell migration, we downregulated its expression using RNA interference (RNAi). Stable downregulation of cortactin in HBL100 breast epithelial cells resulted in (i) decreased cell migration and invasion, (ii) enhanced cell-cell adhesion, and (iii) accelerated cell spreading. These phenotypic changes were reversed by expression of RNAi-resistant mouse cortactin. Cortactin colocalized with cadherin and beta-catenin in adherens junctions, consistent with its role in intercellular adhesion. Remarkably, cortactin deficiency did not affect lamellipodia formation. Instead, downregulation of cortactin in human squamous carcinoma cells that overexpress cortactin changed the cytoskeletal organization. We conclude that increased levels of cortactin, as found in human carcinomas, promote cell migration and invasion by reducing cell spreading and intercellular adhesive strength.     

Differential localization and sequence analysis of capping protein beta- subunit isoforms of vertebrates

Capping protein nucleates the assembly of actin filaments and stabilizes actin filaments by binding to their barbed ends. We describe here a novel isoform of the beta subunit of chicken capping protein, the beta 2 isoform, which arises by alternative splicing. The chicken beta 1 isoform and the beta 2 isoform are identical in their amino acid sequence except for a short region at the COOH terminus; this region of the beta subunit has been implicated in binding actin. Human and mouse cDNAs of the beta 1 and beta 2 isoforms also were isolated and among these vertebrates, the COOH-terminal region of each isoform is highly conserved. In contrast, comparison of the sequences of the vertebrate beta subunit COOH-termini to those of lower eukaryotes shows no similarities. The beta 2 isoform is the predominant isoform of nonmuscle tissues and the beta 1 isoform, which was first characterized in studies of capping protein from chicken muscle, is the predominant isoform of muscle tissues, as shown by immunoblots probed with isoform- specific antibodies and by RNAse protection analysis of mRNAs. The beta 2 isoform also is a component of dynactin complex from brain, which contains the actin-related protein Arp1. Both beta-subunit isoforms are expressed in cardiac muscle but they have non-overlapping subcellular distributions. The beta 1 isoform is at Z-discs of myofibrils, and the beta 2 isoform is enriched at intercalated discs; in cardiac myocytes grown in culture, the beta 2 isoform also is a component of cell-cell junctions and at sites where myofibrils contact the sarcolemma. The biochemical basis for the differential distribution of capping protein isoforms is likely due to interaction with specific proteins at Z-discs and cell-cell junctions, or to preferential association with different actin isoforms. Thus, vertebrates have developed isoforms of capping protein that associate with distinct actin-filament arrays.     

Arg/Abl2 modulates the affinity and stoichiometry of binding of cortactin to f-actin

The Abl family nonreceptor tyrosine kinase Arg/Abl2 interacts with cortactin, an Arp2/3 complex activator, to promote actin-driven cell edge protrusion. Both Arg and cortactin bind directly to filamentous actin (F-actin). While protein-protein interactions between Arg and cortactin have well-characterized downstream effects on the actin cytoskeleton, it is unclear whether and how Arg and cortactin affect each other's actin binding properties. We employ actin cosedimentation assays to show that Arg increases the stoichiometry of binding of cortactin to F-actin at saturation. Using a series of Arg deletion mutants and fragments, we demonstrate that the Arg C-terminal calponin homology domain is necessary and sufficient to increase the stoichiometry of binding of cortactin to F-actin. We also show that interactions between Arg and cortactin are required for optimal affinity between cortactin and the actin filament. Our data suggest a mechanism for Arg-dependent stimulation of binding of cortactin to F-actin, which may facilitate the recruitment of cortactin to sites of local actin network assembly.     

Behavior of antithrombin III isoforms on immobilized heparins. Evidence that the isoforms bind to different numbers of low-affinity heparin sites

Antithrombin III exists in plasma as major and minor isoforms differing in affinity for heparin. The nature of the binding of each purified isoform to immobilized heparins was investigated. Unfractionated, mixed-affinity heparin bound each isoform with both high affinity and concentration-dependent low affinity. The isoforms were resolved when filtered through low-affinity heparin (heparin repeatedly passed over immobilized antithrombin III) columns. Following chemical modification of a specific tryptophan residue required for heparin binding, each isoform failed to bind to either low-affinity or mixed-affinity heparin-agarose, but elution of the modified higher-affinity isoform was retarded on both gels. Because the modified lower-affinity isoform eluted with the similarly sized bovine serum albumin in these experiments, the difference in isoform affinity for heparin appears to be the result of a unique, secondary heparin-binding site in the higher-affinity isoform that can bind a heparin site with low affinity for antithrombin III. This interpretation was supported by the chromatographic behavior of the isoforms on mixed-affinity agarose during reverse gradient elution. Two other populations of each of the tryptophan-modified isoforms were identified. Since these isoforms bound tightly to mixed-affinity heparin-agarose but eluted at lower salt concentrations than the corresponding unmodified isoforms, both isoforms may contain additional secondary sites that interact weakly with heparin. A general model of heparin-antithrombin III interaction is proposed in which a high-affinity heparin site initially interacts with a primary site on antithrombin III. The subsequent conformational change leads to a cooperative, entropy-driven association between secondary sites on the protein and low-affinity sites on heparin, stabilizing antithrombin III in its activated form.     

Association of cortactin with developing neuromuscular specializations

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 synaptic vesicles (SVs), and postsynaptic specialization, consisting of clusters of acetylcholine receptors (AChRs). Previous studies have shown that the 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, we examined the localization of cortactin 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 molecule to induce the formation of AChR clusters and SV clusters and the localization of cortactin was followed by immunofluorescence. In untreated muscle cells, cortactin is often co-localized with spontaneously formed AChR clusters. After cells were treated with beads, cortactin became localized at bead-induced AChR clusters at their earliest appearance (1 h after the addition of beads). This association was most reliably detected at the early stage of the clustering process. On the presynaptic side, cortactin localization could be detected as early as 10 min after the bead-neurite contact was established. Cortactin-enriched contacts later showed 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.     

Differential modulation of cadherin-mediated cell-cell adhesion by platelet endothelial cell adhesion molecule-1 isoforms through activation of extracellular regulated kinases

The role of platelet endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cell-cell interactions and its contribution to cadherin-mediated cell adhesion are poorly understood. Such studies have been difficult because all known endothelial cells express PECAM-1. We have used Madin-Darby canine kidney (MDCK) cells as a model system in which to evaluate the role of PECAM-1 isoforms that differ in their cytoplasmic domains in cell-cell interactions. MDCK cells lack endogenous PECAM-1 but form cell-cell junctions similar to those of endothelial cells, in which PECAM-1 is concentrated. MDCK cells were transfected with two isoforms of murine PECAM-1, Delta15 and Delta14&15, the predominant isoforms expressed in vivo. Expression of the Delta15 isoform resulted in apparent dedifferentiation of MDCK cells concomitant with the loss of adherens junctions, down-regulation of E-cadherin, alpha- and beta-catenin expression, and sustained activation of extracellular regulated kinases. The Delta15 isoform was not concentrated at cell-cell contacts. In contrast, the Delta14&15 isoform localized to sites of cell-cell contact and had no effect on MDCK cell morphology, cadherin/catenin expression, or extracellular regulated kinase activity. Thus, the presence of exon 14 in the cytoplasmic domain of PECAM-1 has dramatic effects on the ability of cells to maintain adherens junctions and an epithelial phenotype. Therefore, changes in the expression of exon 14 containing PECAM-1 isoforms, which we have observed during development, may have profound functional consequences.     

Interaction of protein kinase C with filamentous actin: isozyme specificity resulting from divergent phorbol ester and calcium dependencies

The mechanism of activation of protein kinase C isoforms by filamentous actin (F-actin) was investigated with respect to isozyme specificity and phorbol ester and Ca(2+) dependencies. It was found that the "conventional" (cPKC), alpha, betaI, betaII, and gamma, "novel" (nPKC) delta and epsilon, and "atypical" (aPKC) zeta isoforms were each activated by F-actin with varying potencies. The level of activity along with the affinity for binding to F-actin was further potentiated by the phorbol ester 4beta-12-O-tetradecanoylphorbol 13-acetate (TPA), the potency of which again varied for each isoform. By contrast to the other cPKC isoforms, the level of cPKC-gamma activity was unaffected by TPA, as was also the case for aPKC-zeta. It was found that whereas in the absence of F-actin the soluble form of cPKC-betaI contained two phorbol ester binding sites of low and high affinity, respectively, as previously reported for cPKC-alpha [Slater et al. (1998) J. Biol. Chem. 273, 23160-23168], the F-actin-bound form of the isozyme contained only a single site of relatively low affinity. The level of TPA required to induce cPKC-alpha, -betaI, and -betaII activity and the binding of these isozymes to F-actin was reduced in the presence of Ca(2+). By contrast, the activity of cPKC-gamma was unaffected by Ca(2+), as were the activities of nPKC-delta and -epsilon and aPKC-zeta, as expected. Thus, the interaction with F-actin appears to be a general property of each of the seven PKC isozymes tested. However, isoform specificity may, in part, be directed by differences in the phorbol ester and Ca(2+) dependences, which, with the notable exception of cPKC-gamma, appear to resemble those observed for the activation of each isoform by membrane association. The observation that cPKC isoforms may translocate to F-actin as well as the membrane as a response to an elevation of Ca(2+) levels may allow for the functional coupling of fluctuations of intracellular Ca(2+) levels through cPKC to F-actin cytoskeleton-mediated processes.     

Structural and Functional Diversity of Novel Coronin 1C (CRN2) Isoforms in Muscle

Coronin 1C (synonyms: coronin-3, CRN2), a WD40 repeat-containing protein involved in cellular actin dynamics, is ubiquitously expressed in human tissues. Here, we report on the identification and functional characterization of two novel coronin 1C isoforms, referred to as CRN2i2 and CRN2i3, which also associate with F-actin. Analyses of the coronin 1C gene disclosed a single promoter containing binding sites for myogenic regulatory factors and an alternative first exon 1b present in intron 1, which give rise to the novel isoforms. Chromatin immunoprecipitation studies demonstrate MyoD binding to a region of the CRN2 gene, which contains a highly conserved E-box element in exon 1a. Gel-filtration assays suggest that the largest isoform 3 exists as a monomer, in contrast to isoform 1 and isoform 2 appearing as trimers. CRN2i3, which can be induced by MyoD, is exclusively expressed in well-differentiated myoblasts as well as in mature skeletal muscle tissue. In human skeletal muscle, CRN2i3 is a novel component of postsynaptic neuromuscular junctions and thin filaments of myofibrils. Together, our findings postulate a role for CRN2 isoforms in the structural and functional organization of F-actin in highly ordered protein complexes.     

Novel kelch-like protein, KLEIP, is involved in actin assembly at cell-cell contact sites of Madin-Darby canine kidney cells

Dynamic rearrangements of cell-cell adhesion underlie a diverse range of physiological processes, but their precise molecular mechanisms are still obscure. Thus, identification of novel players that are involved in cell-cell adhesion would be important. We isolated a human kelch-related protein, Kelch-like ECT2 interacting protein (KLEIP), which contains the broad-complex, tramtrack, bric-a-brac (BTB)/poxvirus, zinc finger (POZ) motif and six-tandem kelch repeats. KLEIP interacted with F-actin and was concentrated at cell-cell contact sites of Madin-Darby canine kidney cells, where it colocalized with F-actin. Interestingly, this localization took place transiently during the induction of cell-cell contact and was not seen at mature junctions. KLEIP recruitment and actin assembly were induced around E-cadherin-coated beads placed on cell surfaces. The actin depolymerizing agent cytochalasin B inhibited this KLEIP recruitment around E-cadherin-coated beads. Moreover, constitutively active Rac1 enhanced the recruitment of KLEIP as well as F-actin to the adhesion sites. These observations strongly suggest that KLEIP is localized on actin filaments at the contact sites. We also found that N-terminal half of KLEIP, which lacks the actin-binding site and contains the sufficient sequence for the localization at the cell-cell contact sites, inhibited constitutively active Rac1-induced actin assembly at the contact sites. We propose that KLEIP is involved in Rac1-induced actin organization during cell-cell contact in Madin-Darby canine kidney cells.     

HDAC6 modulates cell motility by altering the acetylation level of cortactin

Histone deacetylase 6 (HDAC6) is a tubulin-specific deacetylase that regulates microtubule-dependent cell movement. In this study, we identify the F-actin-binding protein cortactin as a HDAC6 substrate. We demonstrate that HDAC6 binds cortactin and that overexpression of HDAC6 leads to hypoacetylation of cortactin, whereas inhibition of HDAC6 activity leads to cortactin hyperacetylation. HDAC6 alters the ability of cortactin to bind F-actin by modulating a "charge patch" in its repeat region. Introduction of charge-preserving or charge-neutralizing mutations in this cortactin repeat region correlates with the gain or loss of F-actin binding ability, respectively. Cells expressing a charge-neutralizing cortactin mutant were less motile than control cells or cells expressing a charge-preserving mutant. These findings suggest that, in addition to its role in microtubule-dependent cell motility, HDAC6 influences actin-dependent cell motility by altering the acetylation status of cortactin, which, in turn, changes the F-actin binding activity of cortactin.     

Cortactin modulates cell migration and ring canal morphogenesis during Drosophila oogenesis

Cortactin is a Src substrate that interacts with F-actin and can stimulate actin polymerization by direct interaction with the Arp2/3 complex. We have isolated complete loss-of-function mutants of the single Drosophila cortactin gene. Mutants are viable and fertile, showing that cortactin is not an essential gene. However, cortactin mutants show distinct defects during oogenesis. During oogenesis, Cortactin protein is enriched at the F-actin rich ring canals in the germ line, and in migrating border cells. In cortactin mutants, the ring canals are smaller than normal. A similar phenotype has been observed in Src64 mutants and in mutants for genes encoding Arp2/3 complex components, supporting that these protein products act together to control specific processes in vivo. Cortactin mutants also show impaired border cell migration. This invasive cell migration is guided by Drosophila EGFR and PDGF/VEGF receptor (PVR). We find that accumulation of Cortactin protein is positively regulated by PVR. Also, overexpression of Cortactin can by itself induce F-actin accumulation and ectopic filopodia formation in epithelial cells. We present evidence that Cortactin is one of the factors acting downstream of PVR and Src to stimulate F-actin accumulation. Cortactin is a minor contributor in this regulation, consistent with the cortactin gene not being essential for development.     

Cortactin tyrosine phosphorylation requires Rac1 activity and association with the cortical actin cytoskeleton

Cortactin is an F-actin binding protein that activates actin-related protein 2/3 complex and is localized within lamellipodia. Cortactin is a substrate for Src and other protein tyrosine kinases involved in cell motility, where its phosphorylation on tyrosines 421, 466, and 482 in the carboxy terminus is required for cell movement and metastasis. In spite of the importance of cortactin tyrosine phosphorylation in cell motility, little is known regarding the structural, spatial, or signaling requirements regulating cortactin tyrosine phosphorylation. Herein, we report that phosphorylation of cortactin tyrosine residues in the carboxy terminus requires the aminoterminal domain and Rac1-mediated localization to the cell periphery. Phosphorylation-specific antibodies directed against tyrosine 421 and 466 were produced to study the regulation and localization of tyrosine phosphorylated cortactin. Phosphorylation of cortactin tyrosine 421 and 466 was elevated in response to Src, epidermal growth factor receptor and Rac1 activation, and tyrosine 421 phosphorylated cortactin localized with F-actin in lamellipodia and podosomes. Cortactin tyrosine phosphorylation is progressive, with tyrosine 421 phosphorylation required for phosphorylation of tyrosine 466. These results indicate that cortactin tyrosine phosphorylation requires Rac1-induced cortactin targeting to cortical actin networks, where it is tyrosine phosphorylated in hierarchical manner that is closely coordinated with its ability to regulate actin dynamics.     

Molecular cloning and functional characterization of chicken brain tau: isoforms with up to five tandem repeats

Tau is a major microtubule-associated protein in mammalian brain, where it exists as multiple isoforms that are produced from a single gene by alternative mRNA splicing. Here we present the first report on the structure and function of tau protein from a nonmammalian vertebrate. In the adult chicken brain, five main tau isoforms are expressed. One isoform has three tandem repeats, two isoforms have four repeats each, and two isoforms have five repeats each. Similar to mammalian tau, some chicken tau isoforms contain an amino-terminal insert of 53 amino acids. Unlike mammalian tau, a 34 amino acid insert in the proline-rich region upstream of the repeats is alternatively spliced in chicken tau. It is preceded by a constitutively expressed sequence of 17 amino acids that is absent in tau from human and rodent brains. The expression of chicken tau isoforms and their phosphorylation are developmentally regulated, similar to what has been described in mammalian brain. Functionally, chicken tau isoforms with five repeats have the greatest ability to promote microtubule assembly, followed by isoforms with four and three repeats, respectively. The 34 amino acid insert positively influences both the rate and the extent of microtubule assembly, whereas the 53 amino acid insert only influences the extent of assembly.     

The coiled-coil domain is required for HS1 to bind to F-actin and activate Arp2/3 complex

HS1 (hematopoietic lineage cell-specific protein 1), a substrate of protein tyrosine kinases in lymphocytes, binds to F-actin, and promotes Arp2/3 complex-mediated actin polymerization. However, the mechanism for the interaction between HS1 and F-actin has not yet been fully characterized. HS1 contains 3.5 tandem repeats, a coiled-coil region, and an SH3 domain at the C terminus. Unlike cortactin, which is closely related to HS1 and requires absolutely the repeat domain for F-actin binding, an HS1 mutant with deletion of the repeat domain maintains a significant F-actin binding activity. On the other hand, deletion of the coiled-coil region abolished the ability of HS1 to bind to actin filaments and to activate the Arp2/3 complex for actin nucleation and actin branching. Furthermore, a peptide containing the coiled-coil sequence only was sufficient for F-actin binding. Within cells overexpressing green fluorescent protein-tagged HS1 proteins, wild type HS1 co-localizes with cortical F-actin at the cell leading edge, whereas mutants with deletion of either the coiled-coil region or the repeat domain diffuse in the cytoplasm. Immunoprecipitation analysis reveals that the coiled-coil deletion mutant binds poorly to F-actin, whereas the mutant without the repeat domain fails to bind to both Arp2/3 complex and F-actin. These data suggest that the HS1 coiled-coil region acts synergistically with the repeat domain in the modulation of the Arp2/3 complex-mediated actin polymerization.