Cortactin Modulates Lung Endothelial Apoptosis Induced by Cigarette Smoke.

Cigarette smoke (CS) is the primary cause of Chronic Obstructive Pulmonary Disease (COPD), and an important pathophysiologic event in COPD is CS-induced apoptosis in lung endothelial cells (EC). Cortactin (CTTN) is a cytoskeletal actin-binding regulatory protein with modulation by Src-mediated tyrosine phosphorylation. Based upon data demonstrating reduced CTTN mRNA levels in the lungs of smokers compared to non-smokers, we hypothesized a functional role for CTTN in CS-induced mitochondrial ROS generation and apoptosis in lung EC. Exposure of cultured human lung EC to CS condensate (CSC) led to the rearrangement of the actin cytoskeleton and increased CTTN tyrosine phosphorylation (within hours). Exposure to CS significantly increased EC mitochondrial ROS generation and EC apoptosis. The functional role of CTTN in these CSC-induced EC responses was explored using cortactin siRNA to reduce its expression, and by using a blocking peptide for the CTTN SH3 domain, which is critical to cytoskeletal interactions. CTTN siRNA or blockade of its SH3 domain resulted in significantly increased EC mitochondrial ROS and apoptosis and augmented CSC-induced effects. Exposure of lung EC to e-cigarette condensate demonstrated similar results, with CTTN siRNA or SH3 domain blocking peptide increasing lung EC apoptosis. These data demonstrate a novel role for CTTN in modulating lung EC apoptosis induced by CS or e-cigarettes potentially providing new insights into COPD pathogenesis.

Cortactin function in invadopodia.

Actin remodeling plays an essential role in diverse cellular processes such as cell motility, vesicle trafficking or cytokinesis. The scaffold protein and actin nucleation promoting factor Cortactin is present in virtually all actin-based structures, participating in the formation of branched actin networks. It has been involved in the control of endocytosis, and vesicle trafficking, axon guidance and organization, as well as adhesion, migration and invasion. To migrate and invade through three-dimensional environments, cells have developed specialized actin-based structures called invadosomes, a generic term to designate invadopodia and podosomes. Cortactin has emerged as a critical regulator of invadosome formation, function and disassembly. Underscoring this role, Cortactin is frequently overexpressed in several types of invasive cancers. Herein we will review the roles played by Cortactin in these specific invasive structures.

Cortactin and fascin-1 regulate extracellular vesicle release by controlling endosomal trafficking or invadopodia formation and function.

Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function.

Live-cell imaging of multiple endogenous mRNAs permits the direct observation of RNA granule dynamics.

Here, we developed two pairs of high-contrast chemical probes and their RNA aptamers with distinct readout channels that permitted simultaneous live-cell imaging of endogenous ß-actin and cortactin mRNAs. Application of this technology allowed the direct observation of the formation process of stress granules, protein-RNA assemblies essential for cellular response to the environment.

Cortactin stabilization of actin requires actin-binding repeats and linker, is disrupted by specific substitutions, and is independent of nucleotide state.

The actin-binding protein cortactin promotes the formation and maintenance of actin-rich structures, including lamellipodial protrusions in fibroblasts and neuronal dendritic spines. Cortactin cellular functions have been attributed to its activation of the Arp2/3 complex, which stimulates actin branch nucleation, and to its recruitment of Rho family GTPase regulators. Cortactin also binds actin filaments and significantly slows filament depolymerization, but the mechanism by which it does so and the relationship between actin binding and stabilization are unclear. Here we elucidated the cortactin regions that are necessary and sufficient for actin filament binding and stabilization. Using actin cosedimentation assays, we found that the cortactin repeat region binds actin but that the adjacent linker region is required for binding with the same affinity as full-length cortactin. Using total internal reflection fluorescence microscopy to measure the rates of single filament actin depolymerization, we observed that cortactin-actin interactions are sufficient to stabilize actin filaments. Moreover, conserved charged residues in repeat 4 were necessary for high-affinity actin binding, and substitution of these residues significantly impaired cortactin-mediated actin stabilization. Cortactin bound actin with higher affinity than did its paralog, hematopoietic cell-specific Lyn substrate 1 (HS1), and the effects on actin stability were specific to cortactin. Finally, cortactin stabilized ADP-actin filaments, indicating that the stabilization mechanism does not depend on the actin nucleotide state. Together, these results indicate that cortactin binding to actin is necessary and sufficient to stabilize filaments in a concentration-dependent manner, specific to conserved residues in the cortactin repeats, and independent of the actin nucleotide state.

MiR-448 downregulates CTTN to inhibit cell proliferation and promote apoptosis in glioma.

OBJECTIVE: miRNAs have been confirmed to be related to cell proliferation and apoptosis. In this study, we detected the potential effect of miR-448 on glioma cell proliferation and apoptosis. MATERIALS AND METHODS: miR-448 and CTTN expression levels were detected in glioma cell lines with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Cells were transfected with miR-448 mimics and inhibitor by using lipofectamine 2000 respectively. The proliferative ability of transfected cells was detected via methyl thiazolyl tetrazolium (MTT) and cell counting kit-8 (CCK8) assays. Cell apoptosis and cell-cycle were tested using flow cytometry. The regulatory correlation between miR-448 and CTTN was explored by bioinformatics analysis and luciferase reporter assay. RESULTS: Lower expression of miR-448 and higher level of CTTN were detected in glioma cells. MiR-448 could regulate cell proliferation, cell apoptosis, and cell cycle. CTTN was negatively regulated by miR-448. CONCLUSIONS: miR-448 downregulates CTTN to inhibit cell proliferation and promote apoptosis in glioma, which indicates a potential therapeutic target of glioma.

The repeat region of cortactin is intrinsically disordered in solution.

The multi-domain protein, cortactin, contains a 37-residue repeating motif that binds to actin filaments. This cortactin repeat region comprises 6½ similar copies of the motif and binds actin filaments. To better understand this region of cortactin, and its fold, we conducted extensive biophysical analysis. Size exclusion chromatography with multi-angle light scattering (SEC-MALS) reveals that neither constructs of the cortactin repeats alone or together with the adjacent helical region homo-oligomerize. Using circular dichroism (CD) we find that in solution the cortactin repeats resemble a coil-like intrinsically disordered protein. Small-angle X-ray scattering (SAXS) also indicates that the cortactin repeats are intrinsically unfolded, and the experimentally observed radius of gyration (R g) is coincidental to that calculated by the program Flexible-Meccano for an unfolded peptide of this length. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the domain contains limited hydrophobic core regions. These experiments therefore provide evidence that in solution the cortactin repeat region of cortactin is intrinsically disordered.

Cellular distribution of invadopodia is regulated by nanometer scale surface protein patterns.

Invadopodia are proteolytic structures formed by cancer cells. It is not known whether their cellular distribution can be regulated by the organization of the extracellular matrix or the organization of the golgi complex or whether they have an adhesion requirement. Here, we used electron beam lithography to fabricate fibronectin (FN) nanodots with isotropic and gradient micrometer scale spacings on K-casein and laminin backgrounds. Investigating cancer cells cultured on protein nanopatterns, we showed that (i) presence of FN nanodots on a K-casein background decreased percent of cells with neutral invadopodia polarization compared to FN control surfaces; (ii) presence of a gradient of FN nanodots on a K-casein background increased percent of cells with negative invadopodia polarization compared to FN control surfaces; (iii) polarization of the golgi complex was similar to that of invadopodia in agreement with a spatial link; (iv) local adhesion did not necessarily appear to be a prerequisite for invadopodia formation.

Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and cancer cell invasion.

Cancer cells exploit different strategies to escape from the primary tumor, gain access to the circulation, disseminate throughout the body, and form metastases, the leading cause of death by cancer. Invadopodia, proteolytically active plasma membrane extensions, are essential in this escape mechanism. Cortactin is involved in every phase of invadopodia formation, and its overexpression is associated with increased invadopodia formation, extracellular matrix degradation, and cancer cell invasion. To analyze endogenous cortactin domain function in these processes, we characterized the effects of nanobodies that are specific for the N-terminal acidic domain of cortactin and expected to target small epitopes within this domain. These nanobodies inhibit cortactin-mediated actin-related protein (Arp)2/3 activation, and, after their intracellular expression in cancer cells, decrease invadopodia formation, extracellular matrix degradation, and cancer cell invasion. In addition, one of the nanobodies affects Arp2/3 interaction and invadopodium stability, and a nanobody targeting the Src homology 3 domain of cortactin enabled comparison of 2 functional regions in invadopodium formation or stability. Given their common and distinct effects, we validate cortactin nanobodies as an instrument to selectively block and study distinct domains within a protein with unprecedented precision, aiding rational future generation of protein domain-selective therapeutic compounds.-Bertier, L., Boucherie, C., Zwaenepoel, O., Vanloo, B., Van Troys, M., Van Audenhove, I., Gettemans, J. Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and cancer cell invasion.

Fascin actin bundling controls podosome turnover and disassembly while cortactin is involved in podosome assembly by its SH3 domain in THP-1 macrophages and dendritic cells.

Podosomes are dynamic degrading devices present in myeloid cells among other cell types. They consist of an actin core with associated regulators, surrounded by an adhesive ring. Both fascin and cortactin are known constituents but the role of fascin actin bundling is still unclear and cortactin research rather focuses on its homologue hematopoietic lineage cell-specific protein-1 (HS1). A fascin nanobody (FASNb5) that inhibits actin bundling and a cortactin nanobody (CORNb2) specifically targeting its Src-homology 3 (SH3) domain were used as unique tools to study the function of these regulators in podosome dynamics in both THP-1 macrophages and dendritic cells (DC). Upon intracellular FASNb5 expression, the few podosomes present were aberrantly stable, long-living and large, suggesting a role for fascin actin bundling in podosome turnover and disassembly. Fascin modulates this by balancing the equilibrium between branched and bundled actin networks. In the presence of CORNb2, the few podosomes formed show disrupted structures but their dynamics were unaffected. This suggests a role of the cortactin SH3 domain in podosome assembly. Remarkably, both nanobody-induced podosome-losses were compensated for by focal adhesion structures. Furthermore, matrix degradation capacities were altered and migratory phenotypes were lost. In conclusion, the cortactin SH3 domain contributes to podosome assembly while fascin actin bundling is a master regulator of podosome disassembly in THP-1 macrophages and DC.

Two amino acid residues confer different binding affinities of Abelson family kinase SRC homology 2 domains for phosphorylated cortactin.

The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity "Arg-like" SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an "Abl-like" low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases.

The association of cortactin with profilin-1 is critical for smooth muscle contraction.

Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation.

Design of a bi-functional peptide for protein assays: observation of cortactin expression in human placenta.

A bi-functional peptide is designed to incorporate protein recognition and signal amplification functions into a single short peptide sequence.

The impact of either 4-R-hydroxyproline or 4-R-fluoroproline on the conformation and SH3m-cort binding of HPK1 proline-rich peptide.

SH3 domains are probably the most abundant molecular-recognition modules of the proteome. A common feature of these domains is their interaction with ligand proteins containing Pro-rich sequences. Crystal and NMR structures of SH3 domains complexes with Pro-rich peptides show that the peptide ligands are bound over a range of up to seven residues in a PPII helix conformation. Short proline-rich peptides usually adopt little or no ordered secondary structure before binding interactions, and consequently their association with the SH3 domain is characterized by unfavorable binding entropy due to a loss of rotational freedom on forming the PPII helix. With the aim to stabilize the PPII helix conformation into the proline-rich decapeptide PPPLPPKPKF (P2), we replaced some proline residues either with the 4(R)-4-fluoro-L-proline (FPro) or the 4(R)-4-hydroxy-L-proline (Hyp). The interactions of P2 analogues with the SH3 domain of cortactin (SH3(m-cort)) were analyzed by circular dichroism spectroscopy, while CD thermal transition experiments have been used to determine their propensity to adopt a PPII helix conformation. Results show that the introduction of three residues of Hyp efficiently stabilizes the PPII helix conformation, while it does not improve the affinity towards the SH3 domain, suggesting that additional forces, e.g., electrostatic interactions, are involved in the SH3(m-cort) substrate recognition.

Cortactin controls surface expression of the voltage-gated potassium channel K(V)10.1.

K(V)10.1 is a voltage-gated potassium channel aberrantly expressed in many cases of cancer, and participates in cancer initiation and tumor progression. Its action as an oncoprotein can be inhibited by a functional monoclonal antibody, indicating a role for channels located at the plasma membrane, accessible to the antibody. Cortactin is an actin-interacting protein implicated in cytoskeletal architecture and often amplified in several types of cancer. In this study, we describe a physical and functional interaction between cortactin and K(V)10.1. Binding of these two proteins occurs between the C terminus of K(V)10.1 and the proline-rich domain of cortactin, regions targeted by many post-translational modifications. This interaction is specific for K(V)10.1 and does not occur with K(V)10.2. Cortactin controls the abundance of K(V)10.1 at the plasma membrane and is required for functional expression of K(V)10.1 channels.

Cortactin is a substrate of activated Cdc42-associated kinase 1 (ACK1) during ligand-induced epidermal growth factor receptor downregulation.

BACKGROUND: Epidermal growth factor receptor (EGFR) internalization following ligand binding controls EGFR downstream pathway signaling activity. Internalized EGFR is poly-ubiquitinated by Cbl to promote lysosome-mediated degradation and signal downregulation. ACK1 is a non-receptor tyrosine kinase that interacts with ubiquitinated EGFR to facilitate EGFR degradation. Dynamic reorganization of the cortical actin cytoskeleton controlled by the actin related protein (Arp)2/3 complex is important in regulating EGFR endocytosis and vesicle trafficking. How ACK1-mediated EGFR internalization cooperates with Arp2/3-based actin dynamics during EGFR downregulation is unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that ACK1 directly binds and phosphorylates the Arp2/3 regulatory protein cortactin, potentially providing a direct link to Arp2/3-based actin dynamics during EGFR degradation. Co-immunoprecipitation analysis indicates that the cortactin SH3 domain is responsible for binding to ACK1. In vitro kinase assays demonstrate that ACK1 phosphorylates cortactin on key tyrosine residues that create docking sites for adaptor proteins responsible for enhancing Arp2/3 nucleation. Analysis with phosphorylation-specific antibodies determined that EGFR-induced cortactin tyrosine phosphorylation is diminished coincident with EGFR degradation, whereas ERK1/2 cortactin phosphorylation utilized in promoting activation of the Arp2/3 regulator N-WASp is sustained during EGFR downregulation. Cortactin and ACK1 localize to internalized vesicles containing EGF bound to EGFR visualized by confocal microscopy. RNA interference and rescue studies indicate that ACK1 and the cortactin SH3 domain are essential for ligand-mediated EGFR internalization. CONCLUSIONS/SIGNIFICANCE: Cortactin is a direct binding partner and novel substrate of ACK1. Tyrosine phosphorylation of cortactin by ACK1 creates an additional means to amplify Arp2/3 dynamics through N-WASp activation, potentially contributing to the overall necessary tensile and/or propulsive forces utilized during EGFR endocytic internalization and trafficking involved in receptor degradation.

ß1Integrin/FAK/cortactin signaling is essential for human head and neck cancer resistance to radiotherapy.

Integrin signaling critically contributes to the progression, growth, and therapy resistance of malignant tumors. Here, we show that targeting of ß1 integrins with inhibitory antibodies enhances the sensitivity to ionizing radiation and delays the growth of human head and neck squamous cell carcinoma cell lines in 3D cell culture and in xenografted mice. Mechanistically, dephosphorylation of focal adhesion kinase (FAK) upon inhibition of ß1 integrin resulted in dissociation of a FAK/cortactin protein complex. This, in turn, downregulated JNK signaling and induced cell rounding, leading to radiosensitization. Thus, these findings suggest that robust and selective pharmacological targeting of ß1 integrins may provide therapeutic benefit to overcome tumor cell resistance to radiotherapy.

Lysozyme contamination facilitates crystallization of a heterotrimeric cortactin-Arg-lysozyme complex.

Crystallization of contaminating proteins is a frequently encountered problem for macromolecular crystallographers. In this study, an attempt was made to obtain a binary cocrystal structure of the SH3 domain of cortactin and a 17-residue peptide from the Arg nonreceptor tyrosine kinase encompassing a PxxPxxPxxP (PxxP1) motif. However, cocrystals could only be obtained in the presence of trace amounts of a contaminating protein. A structure solution obtained by molecular replacement followed by ARP/wARP automatic model building allowed a 'sequence-by-crystallography' approach to discover that the contaminating protein was lysozyme. This 1.65 Å resolution crystal structure determination of a 1:1:1 heterotrimeric complex of Arg, cortactin and lysozyme thus provides an unusual `caveat emptor' warning of the dangers that underpurified proteins harbor for macromolecular crystallographers.

Cortical actin binding protein cortactin mediates ENaC activity via Arp2/3 complex.

Epithelial Na(+) channel (ENaC) activity is regulated, in part, by the cortical cytoskeleton. Here we demonstrate that cortactin is highly expressed in the kidney cortex and polarized epithelial cells, and is localized to the cortical collecting duct. Coexpression of cortactin with ENaC decreases ENaC activity, as measured in patch-clamp experiments. Biotinylation experiments and single-channel analysis reveal that cortactin decreases ENaC activity via affecting channel open probability (P(o)). Knockdown of cortactin in mpkCCD(c14) principal cells results in an increase in ENaC activity and sodium reabsorption. Coimmunoprecipitation analysis shows direct interactions between cortactin and all three ENaC subunits in cultured and native cells. To address the question of what mechanism underlies the action of cortactin on ENaC activity, we assayed the effects of various mutants of cortactin. The data show that only a cortactin mutant unable to bind Arp2/3 complex does not influence ENaC activity. Furthermore, inhibitor of the Arp2/3 complex CK-0944666 precludes the effect of cortactin. Depolymerization of the actin microfilaments and inhibition of the Arp2/3 complex does not result in the loss of association between ENaC and cortactin. Thus, these results indicate that cortactin is functionally important for ENaC activity and that Arp2/3 complex is involved in this mechanism.