A dynamin-cortactin-Arp2/3 complex mediates actin reorganization in growth factor-stimulated cells

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. In this study, we demonstrate that 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 blocked 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.     

The Helicobacter pylori CagA protein induces cortactin dephosphorylation and actin rearrangement by c-Src inactivation

The gastric pathogen Helicobacter pylori translocates the CagA protein into epithelial cells by a type IV secretion process. Translocated CagA is tyrosine phosphorylated (CagA(P-Tyr)) on specific EPIYA sequence repeats by Src family tyrosine kinases. Phos phorylation of CagA induces the dephosphorylation of as yet unidentified cellular proteins, rearrangements of the host cell actin cytoskeleton and cell scattering. We show here that CagA(P-Tyr) inhibits the catalytic activity of c-Src in vivo and in vitro. c-Src inactivation leads to tyrosine dephosphorylation of the actin binding protein cortactin. Concomitantly, cortactin is specifically redistributed to actin-rich cellular protrusions. c-Src inactivation and cortactin dephosphorylation are required for rearrangements of the actin cytoskeleton. Moreover, CagA(P-Tyr)-mediated c-Src inhibition downregulates further CagA phosphorylation through a negative feedback loop. This is the first report of a bacterial virulence factor that inhibits signalling of a eukaryotic tyrosine kinase and on a role of c-Src inactivation in host cell cytoskeletal rearrangements.     

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.     

Cortactin: an Achilles' heel of the actin cytoskeleton targeted by pathogens

Cortactin is an actin-binding protein and a central regulator of the actin cytoskeleton. Importantly, cortactin is also a common target exploited by microbes during infection. Its involvement in disease development is exemplified by a variety of pathogenic processes, such as pedestal formation [enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC)], invasion (Shigella, Neisseria, Rickettsia, Chlamydia, Staphylococcus and Cryptosporidium), actin-based motility (Listeria, Shigella and vaccinia virus) and cell scattering (Helicobacter). Recent progress turns our attention to how cortactin function can be regulated by serine and tyrosine phosphorylation. This has an important impact on how pathogens abuse cortactin to modulate the architecture of the host actin cytoskeleton.     

Tyrosine phosphorylation of Kv1.2 modulates its interaction with the actin-binding protein cortactin

Tyrosine phosphorylation evokes functional changes in a variety of ion channels. Modulation of the actin cytoskeleton also affects the function of some channels. Little is known about how these avenues of ion channel regulation may interact. We report that the potassium channel Kv1.2 associates with the actin-binding protein cortactin and that the binding is modulated by tyrosine phosphorylation. Immunocytochemical and biochemical analyses show that Kv1.2 and cortactin co-localize to the cortical actin cytoskeleton at the leading edges of the cell. Binding assays using purified recombinant proteins reveal a 19-amino acid span within the carboxyl terminus of Kv1.2 that is necessary for direct cortactin binding. Phosphorylation of specific tyrosines within the C terminus of Kv1.2 attenuates that binding. In HEK293 cells, activation of the M1 muscarinic acetylcholine receptor evokes tyrosine phosphorylation-dependent suppression of Kv1.2 ionic current. We show that M1 receptor activation also reduces the interaction of cortactin with Kv1.2 and that mutant Kv1.2 channels deficient for cortactin binding exhibit strongly attenuated ionic current. These results demonstrate a dynamic, phosphorylation-dependent interaction between Kv1.2 and the actin cytoskeleton-binding protein cortactin and suggest a role for that interaction in the regulation of Kv1.2 ionic current.     

Cortactin phosphorylated by ERK1/2 localizes to sites of dynamic actin regulation and is required for carcinoma lamellipodia persistence

Background

Tumor cell motility and invasion is governed by dynamic regulation of the cortical actin cytoskeleton. The actin-binding protein cortactin is commonly upregulated in multiple cancer types and is associated with increased cell migration. Cortactin regulates actin nucleation through the actin related protein (Arp)2/3 complex and stabilizes the cortical actin cytoskeleton. Cortactin is regulated by multiple phosphorylation events, including phosphorylation of S405 and S418 by extracellular regulated kinases (ERK)1/2. ERK1/2 phosphorylation of cortactin has emerged as an important positive regulatory modification, enabling cortactin to bind and activate the Arp2/3 regulator neuronal Wiskott-Aldrich syndrome protein (N-WASp), promoting actin polymerization and enhancing tumor cell movement.

Methodology/Principal Findings

In this report we have developed phosphorylation-specific antibodies against phosphorylated cortactin S405 and S418 to analyze the subcellular localization of this cortactin form in tumor cells and patient samples by microscopy. We evaluated the interplay between cortactin S405 and S418 phosphorylation with cortactin tyrosine phosphorylation in regulating cortactin conformational forms by Western blotting. Cortactin is simultaneously phosphorylated at S405/418 and Y421 in tumor cells, and through the use of point mutant constructs we determined that serine and tyrosine phosphorylation events lack any co-dependency. Expression of S405/418 phosphorylation-null constructs impaired carcinoma motility and adhesion, and also inhibited lamellipodia persistence monitored by live cell imaging.

Conclusions/Significance

Cortactin phosphorylated at S405/418 is localized to sites of dynamic actin assembly in tumor cells. Concurrent phosphorylation of cortactin by ERK1/2 and tyrosine kinases enables cells with the ability to regulate actin dynamics through N-WASp and other effector proteins by synchronizing upstream regulatory pathways, confirming cortactin as an important integration point in actin-based signal transduction. Reduced lamellipodia persistence in cells with S405/418A expression identifies an essential motility-based process reliant on ERK1/2 signaling, providing additional understanding as to how this pathway impacts tumor cell migration.     

Characterization of the EMS1 Gene and its Product,Human Cortactin

We have identified a novel gene, EMSl, that is consistently amplified and overexpressed in human carcinomas with an amplification of the chromosome 11q13 region. Comparisons of the EMSl sequences with those present in the GenBank databases revealed a high identity with chicken cortactin. Southern and western blot analyses confirm the high sequence conservation during evolution. An antiserum specific for human cortactin, showed in gene transfer experiments that both human p80 and p85 isoforms are encoded by the EMSl cDNA. Further comparisons demonstrated an high sequence and structural homology with HSl that is implicated in signal transduction in lymphoid cells only. Expression of EMSl/cortactin mRNA was restricted to tumor cell lines derived from non-lymphoid origin. Cortactin contains (i) a filamentous actin binding tandem repeat domain, (ii) a proline-rich SH3-binding and (iii) a SH3 domain that is common in proteins involved in signal transduction. Our data suggest that human EMSl/cortactin has a function in signal transmission between cell-matrix contact sites and the cytoskeleton and, as such, its overexpression due to 11q13 amplification might effect adhesive properties of human carcinomas.     

Cortactin, an 80/85-kilodalton pp60src substrate, is a filamentous actin-binding protein enriched in the cell cortex

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. We report that 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. We suggest the name cortactin, reflecting the cortical subcellular localization and its actin-binding activity.     

Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape

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. We have observed that 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 we demonstrate 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.     

Src-mediated cortactin phosphorylation regulates actin localization and injurious blebbing in acinar cells

Suprastimulation of pancreatic acini is a well-known model for pancreatitis, and it is characterized by actin reorganization and cell blebbing. Currently, however, the mechanisms underlying regulation of these aberrant cytoskeletal and membrane dynamics and how they contribute to cell injury are unclear. We observed that suprastimulation results in a rapid activation of Src and relocalization of the actin-binding protein cortactin from the apical to the basolateral domain at the necks of membrane blebs. Furthermore, Src-mediated cortactin tyrosine phosphorylation was markedly increased after suprastimulation. Pretreatment of acini with Src inhibitors or expression of a cortactin tyrosine phospho-inhibitory mutant reduced actin redistribution and bleb formation induced by suprastimulation in vitro. Importantly, inhibition of Src activity in rat models of suprastimulation-induced pancreatitis substantially reduced disease severity, as indicated by a reduction in serum amylase and pancreatic edema and a striking improvement in tissue histology. These findings indicate a novel, disease-relevant role for Src-mediated cortactin phosphorylation in aberrant reorganization of the actin cytoskeleton, a mechanism that is likely to have implications in other types of cell injury. In addition, they suggest a potential use for Src inhibitors as an approach to reduce cell injury.     

Cortactin is necessary for E-cadherin-mediated contact formation and actin reorganization

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. We now report that 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 blocked Arp2/3-dependent actin assembly at cadherin adhesive contacts, significantly reduced cadherin adhesive contact zone extension, and perturbed both cell morphology and junctional accumulation of cadherins in polarized epithelia. Together, our findings identify a necessary role for cortactin in the cadherin-actin cooperation that supports productive contact formation.     

Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.     

Cortactin interacts with WIP in regulating Arp2/3 activation and membrane protrusion

BACKGROUND: Modulation of actin cytoskeleton assembly is an integral step in many cellular events. A key regulator of actin polymerization is Arp2/3 complex. Cortactin, an F-actin binding protein that localizes to membrane ruffles, is an activator of Arp2/3 complex. RESULTS: A yeast two-hybrid screen revealed the interaction of the cortactin Src homology 3 (SH3) domain with a peptide fragment derived from a cDNA encoding a region of WASp-Interacting Protein (WIP). GST-cortactin interacted with WIP in an SH3-dependent manner. The subcellular localization of cortactin and WIP coincided at the cell periphery. WIP increased the efficiency of cortactin-mediated Arp2/3 complex activation of actin polymerization in a concentration-dependent manner. Lastly, coexpression of cortactin and WIP stimulated membrane protrusions. CONCLUSIONS: WIP, a protein involved in filopodia formation, binds to both actin monomers and cortactin. Thus, recruitment of actin monomers to a cortactin-activated Arp2/3 complex likely leads to the observed increase in cortactin activation of Arp2/3 complex by WIP. These data suggest that a cortactin-WIP complex functions in regulating actin-based structures at the cell periphery.     

Cortactin branches out: roles in regulating protrusive actin dynamics

Since its discovery in the early 1990's, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cytoskeletal organization, impacting the regulation and formation of actin-rich motility structures.     

Dynamin2 and cortactin regulate actin assembly and filament organization

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. We investigated the interaction of dynamin2 and cortactin in regulating actin assembly in vivo and in vitro. When expressed in cultured cells, a dynamin2 mutant with decreased affinity for GTP decreased actin dynamics within the cortical actin network. Expressed mutants of cortactin that have decreased binding of Arp2/3 complex or dynamin2 also decreased actin dynamics. Dynamin2 influenced actin nucleation by purified Arp2/3 complex and cortactin in vitro in a biphasic manner. Low concentrations of dynamin2 enhanced actin nucleation by Arp2/3 complex and cortactin, and high concentrations were inhibitory. Dynamin2 promoted the association of actin filaments nucleated by Arp2/3 complex and cortactin with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing lipid vesicles. GTP hydrolysis altered the organization of the filaments and the lipid vesicles. We conclude that dynamin2, through an interaction with cortactin, regulates actin assembly and actin filament organization at membranes.     

Interactions with Actin Monomers,Actin Filaments,and Arp2/3 Complex Define the Roles of WASP Family Proteins and Cortactin in Coordinately Regulating Branched Actin Networks

Arp2/3 complex is an important actin filament nucleator that creates branched actin filament networks required for formation of lamellipodia and endocytic actin structures. Cellular assembly of branched actin networks frequently requires multiple Arp2/3 complex activators, called nucleation promoting factors (NPFs). We recently presented a mechanism by which cortactin, a weak NPF, can displace a more potent NPF, N-WASP, from nascent branch junctions to synergistically accelerate nucleation. The distinct roles of these NPFs in branching nucleation are surprising given their similarities. We biochemically dissected these two classes of NPFs to determine how their Arp2/3 complex and actin interacting segments modulate their influences on branched actin networks. We find that the Arp2/3 complex-interacting N-terminal acidic sequence (NtA) of cortactin has structural features distinct from WASP acidic regions (A) that are required for synergy between the two NPFs. Our mutational analysis shows that differences between NtA and A do not explain the weak intrinsic NPF activity of cortactin, but instead that cortactin is a weak NPF because it cannot recruit actin monomers to Arp2/3 complex. We use TIRF microscopy to show that cortactin bundles branched actin filaments using actin filament binding repeats within a single cortactin molecule, but that N-WASP antagonizes cortactin-mediated bundling. Finally, we demonstrate that multiple WASP family proteins synergistically activate Arp2/3 complex and determine the biochemical requirements in WASP proteins for synergy. Our data indicate that synergy between WASP proteins and cortactin may play a general role in assembling diverse actin-based structures, including lamellipodia, podosomes, and endocytic actin networks.     

Alix protein is substrate of Ozz-E3 ligase and modulates actin remodeling in skeletal muscle

Alix/AIP1 is a multifunctional adaptor protein that participates in basic cellular processes, including membrane trafficking and actin cytoskeleton assembly, by binding selectively to a variety of partner proteins. However, the mechanisms regulating Alix turnover, subcellular distribution, and function in muscle cells are unknown. We now report that Alix is expressed in skeletal muscle throughout myogenic differentiation. In myotubes, a specific pool of Alix colocalizes with Ozz, the substrate-binding component of the muscle-specific ubiquitin ligase complex Ozz-E3. We found that interaction of the two endogenous proteins in the differentiated muscle fibers changes Alix conformation and promotes its ubiquitination. This in turn regulates the levels of the protein in specific subcompartments, in particular the one containing the actin polymerization factor cortactin. In Ozz(-/-) myotubes, the levels of filamentous (F)-actin is perturbed, and Alix accumulates in large puncta positive for cortactin. In line with this observation, we show that the knockdown of Alix expression in C2C12 muscle cells affects the amount and distribution of F-actin, which consequently leads to changes in cell morphology, impaired formation of sarcolemmal protrusions, and defective cell motility. These findings suggest that the Ozz-E3 ligase regulates Alix at sites where the actin cytoskeleton undergoes remodeling.     

Cortactin mediated morphogenic cell movements during zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) gastrulation

Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a cortactin specific monoclonal antibody demonstrated that cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with cortactin and Arp3 antibodies also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.     

Cortactin mediated morphogenic cell movements during zebrafish (Danio rerio) gastrulation

Cortactin, a filamentous actin (F-actin) associated protein and a prominent substrate of protein tyrosine kinase Src[1,2], is composed of several functional do-mains, including an amino terminal domain (NTA) that is rich in acidic residues, six and one half 37-amino-acid tandem repeating segments, an al-pha-helical motif, a less conserved region but rich in tyrosine, proline, serine and threonine residues, and a Src homology 3 (SH3) domain at the distal carboxyl terminus. In mammalian cells …     

Lymphocyte migration through brain endothelial cell monolayers involves signaling through endothelial ICAM-1 via a rho-dependent pathway

Lymphocyte extravasation into the brain is mediated largely by the Ig superfamily molecule ICAM-1. Several lines of evidence indicate that at the tight vascular barriers of the central nervous system (CNS), endothelial cell (EC) ICAM-1 not only acts as a docking molecule for circulating lymphocytes, but is also involved in transducing signals to the EC. In this paper, we examine the signaling pathways in brain EC following Ab ligation of endothelial ICAM-1, which mimics adhesion of lymphocytes to CNS endothelia. ICAM-1 cross-linking results in a reorganization of the endothelial actin cytoskeleton to form stress fibers and activation of the small guanosine triphosphate (GTP)-binding protein Rho. ICAM-1-stimulated tyrosine phosphorylation of the actin-associated molecule cortactin and ICAM-1-mediated, Ag/IL-2-stimulated T lymphocyte migration through EC monolayers were inhibited following pretreatment of EC with cytochalasin D. Pretreatment of EC with C3 transferase, a specific inhibitor of Rho proteins, significantly inhibited the transmonolayer migration of T lymphocytes, endothelial Rho-GTP loading, and endothelial actin reorganization, without affecting either lymphocyte adhesion to EC or cortactin phosphorylation. These data show that brain vascular EC are actively involved in facilitating T lymphocyte migration through the tight blood-brain barrier of the CNS and that this process involves ICAM-1-stimulated rearrangement of the endothelial actin cytoskeleton and functional EC Rho proteins.