Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

Background: Cortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes. Methodology/Principal Findings: In the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation. Conclusions/Significance: Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results fro

Distinct phosphorylation requirements regulate cortactin activation by TirEPEC and its binding to N-WASP

<p>Abstract</p> <p>Background</p> <p>Cortactin activates the actin-related 2/3 (Arp2/3) complex promoting actin polymerization to remodel cell architecture in multiple processes (e.g. cell migration, membrane trafficking, invadopodia formation etc.). Moreover, it was called the Achilles' heel of the actin cytoskeleton because many pathogens hijack signals that converge on this oncogenic scaffolding protein. Cortactin is able to modulate N-WASP activation <it>in vitro </it>in a phosphorylation-dependent fashion. Thus Erk-phosphorylated cortactin is efficient in activating N-WASP through its SH3 domain, while Src-phosphorylated cortactin is not. This could represent a switch on/off mechanism controlling the coordinated action of both nucleator promoting factors (NPFs). Pedestal formation by enteropathogenic <it>Escherichia coli </it>(EPEC) requires N-WASP activation. N-WASP is recruited by the cell adapter Nck which binds a major tyrosine-phosphorylated site of a bacterial injected effector, Tir (translocated intimin receptor). Tir-Nck-N-WASP axis defines the current major pathway to actin polymerization on pedestals. In addition, it was recently reported that EPEC induces tyrosine phosphorylation of cortactin.</p> <p>Results</p> <p>Here we demonstrate that cortactin phosphorylation is absent on N-WASP deficient cells, but is recovered by re-expression of N-WASP. We used purified recombinant cortactin and Tir proteins to demonstrate a direct interaction of both that promoted Arp2/3 complex-mediated actin polymerization <it>in vitro</it>, independently of cortactin phosphorylation.</p> <p>Conclusion</p> <p>We propose that cortactin binds Tir through its N-terminal part in a tyrosine and serine phosphorylation independent manner while SH3 domain binding and activation of N-WASP is regulated by tyrosine and serine mediated phosphorylation of cortactin. Therefore cortactin could act on Tir-Nck-N-WASP pathway and control a possible cycling activity of N-WASP underlying pedestal formation.</p

Nck adaptors, besides promoting N-WASP mediated actin-nucleation activity at pedestals, influence the cellular levels of enteropathogenic <i>Escherichia coli</i> Tir effector

<div><p>Enteropathogenic <i>Escherichia coli</i> (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area. </p></div

Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic <i>Escherichia coli</i> (EPEC) by Binding to Tir Effector

<div><p>Infections by enteropathogenic <i>Escherichia coli</i> (EPEC) cause diarrhea linked to high infant mortality in developing countries. EPEC adheres to epithelial cells and induces the formation of actin pedestals. Actin polymerization is driven fundamentally through signaling mediated by Tir bacterial effector protein, which inserts in the plasma membrane of the infected cell. Tir binds Nck adaptor proteins, which in turn recruit and activate N-WASP, a ubiquitous member of the Wiskott-Aldrich syndrome family of proteins. N-WASP activates the Arp2/3 complex to promote actin polymerization. Other proteins aside from components of the Tir-Nck-N-WASP pathway are recruited to the pedestals but their functions are unknown. Here we investigate the function of two alternatively spliced isoforms of Crk adaptors (CrkI/II) and the paralog protein CrkL during pedestal formation by EPEC. We found that the Crk isoforms act as redundant inhibitors of pedestal formation. The SH2 domain of CrkII and CrkL binds to phosphorylated tyrosine 474 of Tir and competes with Nck to bind Tir, preventing its recruitment to pedestals and thereby inhibiting actin polymerization. EPEC infection induces phosphorylation of the major regulatory tyrosine in CrkII and CrkL, possibly preventing the SH2 domain of these proteins from interacting with Tir. Phosphorylated CrkII and CrkL proteins localize specifically to the plasma membrane in contact with EPEC. Our study uncovers a novel role for Crk adaptors at pedestals, opening a new perspective in how these oncoproteins regulate actin polymerization.</p></div

Down-regulation of CrkL expression by siRNA in CrkI/II-deficient MEFs potentiates pedestal formation.

<p>(<b>A</b>) WB with anti-CrkL Ab in WT and CrkI/II-deficient MEFs to show lower CrkL levels in cells treated using siRNA with an oligonucleotide against CrkL than in cells treated with a control oligonucleotide (lower bands). The levels of injected Tir effector were assessed by WB with anti-Tir MoAb. As a loading control, blots were probed with anti-actin MoAb. (<b>B</b>) Immunofluorescence images of WT and CrkI/II-deficient MEFs in which CrkL expression was inhibited by siRNA; cells were infected with preactivated EPEC for 3 h at an MOI of 3. Actin was stained red with TRITC-phalloidin, while EPEC was stained blue using anti-LPS Ab followed by Alexa 405-conjugated goat anti-mouse secondary Ab. Confocal images were merged using Leica software. Insets are 4× digital zoom images. (<b>C</b>) Quantitation of the number of pedestals and the ratio of pedestals to bacteria on WT and CrkI/II-deficient cells treated using siRNA with a scrambled control (Ctr.) oligonucleotide (white and dark-grey bars, respectively) or with an oligonucleotide to reduce CrkL expression (grey and black bars, respectively). The number of pedestals was quantified based on counts with 100 cells. The ratio of pedestals to bacteria was quantified by counting the number of adhered bacteria with or without pedestals on 50 cells; the resulting ratio was expressed as a percentage. The graph shows mean ± standard deviation (SD) for three independent experiments. The indicated groups differed significantly based on Student's <i>t</i>-test. *, p<0.05; **, p<0.01.</p

EPEC infection induces tyrosine phosphorylation of Crk adaptor proteins.

<p>HeLa cells were serum-starved for 16 h prior to infection with preactivated EPEC for 1, 2 or 3 h at an MOI of 45. The basal level of phosphorylation of Crk proteins was visualized in uninfected cells (- EPEC). (<b>A</b>) CrkII or IgG isotype control immunoprecipitates were probed by WB with a phosphospecific Ab against phospho-Tyr221 in CrkII (phospho-CrkII) to show induction of phosphorylation. The blot was also probed with anti-CrkI/II MoAb to show total levels of CrkII. The ratio of phospho-CrkII to non-phosphorylated CrkII in a representative experiment is shown. (<b>B</b>) Statistical analysis of the ratio of phospho-CrkII to CrkII signals with respect to the basal level in uninfected cells using one-way ANOVA with Dunnett test. The graph shows mean ± SD for four independent experiments. a.u.: arbitrary units. ***, p<0.001. (<b>C</b>) WBs with a phosphospecific Ab against phospho-Tyr207 in CrkL to show the induction of phosphorylation, and with anti-CrkL Ab to show total levels of CrkL. The ratio of the phospho-CrkL to CrkL in a representative experiment is shown. (<b>D</b>) Statistical analysis of the ratio of phospho-CrkL to CrkL levels with respect to the basal level at 0 h using one-way ANOVA with Dunnett test. The graph shows mean ± SD for four independent experiments. a.u.: arbitrary units. *, p<0.05. (<b>E</b>) Immunofluorescence images of HeLa cells that were starved for 16 h prior to infection with preactivated EPEC for 3 h at an MOI of 15. Immunofluorescence staining was done using phosphospecific Abs against Tyr221 in CrkII (phospho-CrkII) or Tyr207 in CrkL (phospho-CrkL) followed by Alexa 488-conjugated goat anti-rabbit secondary Ab (green). Actin was stained red using TRITC-phalloidin; bacteria were stained blue using DAPI. Pictures were taken on a confocal microscope and images from one section are shown, together with 4× digital zoom images (insets). Images were merged using Leica software.</p

Pedestal formation in cells expressing Crk mutants.

<p>(<b>A</b>) Expression of GFP alone (GFP) or GFP-tagged CrkII SH2 domain (SH2-GFP) in transfectants was assessed by WB with anti-GFP Ab. Blots were probed with anti-actin MoAb as a loading control. (<b>B</b>) Fluorescence images of HeLa cells transfected with GFP or SH2-GFP and then infected with preactivated EPEC for 2 h at an MOI of 3. GFP-tagged constructs appear green; actin, red (TRITC-phalloidin) and EPEC, blue (DAPI). The merged images shown were generated using AxioVision software. Insets are 4× digital zoom images. (<b>C</b>) Quantitation of the number of pedestals on cells expressing GFP or SH-GFP. Quantitation was done by counting the number of pedestals on cells expressing the constructs in six experiments. (<b>D, E</b>) HeLa cells were transfected with Myc-tagged empty vector or with plasmids encoding Myc-CrkII-Y221F or CrkL-Y207F phosphorylation-deficient mutants, and then infected with preactivated EPEC at an MOI of 3. Pedestal number was compared in cells expressing Myc and Myc-CrkII-Y221F or between Mock-transfected cells and CrkL-Y207F transfected cells in three different experiments. (<b>F</b>) WB of Nck1/2-deficient MEFs transfected with Flag-Nck2 plasmid as well as a plasmid encoding either GFP alone or GFP-tagged CrkII SH2. (<b>G</b>) Immunofluorescence staining of transfected Nck1/2-deficient MEFs after infection with EPEC at an MOI of 225. Cells were stained with anti-Flag MoAb followed by Alexa 568-conjugated goat anti-mouse Ab. F-actin was visualized with Alexa 350 Phalloidin. GFP expression is shown in green. (<b>H</b>) The number of cells in three different experiments showing Flag staining at pedestals was counted among cells expressing empty GFP or GFP-tagged CrkII SH2 and normalized to 100. The graph shows mean ± standard deviation (SD) for three independent experiments. The differences among the groups were statistically significant based on Student's <i>t</i>-test; *, p<0.05, **, p<0.01, ***, p<0.001.</p

Down-regulation of CrkI/II expression by siRNA in CrkL-deficient MEFs enhances pedestal formation.

<p>(<b>A</b>) WB with anti-CrkII Ab in WT and CrkL-deficient MEFs to show lower CrkII levels in cells treated using siRNA with an oligonucleotide against CrkI/II than in cells treated with control (Ctr.) oligonucleotide. The blot was probed with anti-CrkL Ab as a control for cell genotype, and with anti-actin MoAb as a loading control. The levels of Tir effector are shown. (<b>B</b>) Fluorescence images of WT and CrkL-deficient MEFs in which CrkI/II expression was inhibited by siRNA; cells were infected with preactivated EPEC at an MOI of 3. Actin was stained red with TRITC-phalloidin, while EPEC was stained blue using DAPI. Images were merged using Leica software. Insets are 4× digital zoom images. (<b>C</b>) Quantitation of the number of pedestals on WT and CrkL-deficient cells treated for siRNA using a scrambled control (Ctr.) oligonucleotide (white bar) or an oligonucleotide to reduce CrkI/II expression (black bar). Quantitation was done by counting the number of pedestals on 100 cells. The graph shows mean ± standard deviation (SD) for three independent experiments. The indicated groups differed significantly based on Student's <i>t</i>-test. **, p<0.01.</p

Pedestal formation in HeLa cells treated with siRNA to reduce CrkI/II and CrkL expression.

<p>(<b>A</b>) Schematic representation of Crk proteins under investigation. (<b>B</b>) WB of HeLa cell extracts using anti-CrkI/II MoAb and anti-CrkL Ab and the Odyssey imaging system to show expression levels of the corresponding proteins, or (<b>C</b>) to show decreased Crk expression in infected cells pretreated with siRNA. As a loading control the blots were probed with anti-actin monoclonal Ab (MoAb) or anti-tubulin Ab (upper bands). (<b>D</b>) Confocal fluorescence images of HeLa cells pretreated with siRNA against CrkI/II and CrkL or control siRNA and infected with preactivated EPEC for 2 h at an MOI of 3. Actin was stained with TRITC-phalloidin (red), while bacteria were stained with DAPI (blue). Arrows point at pedestals and bacteria. The scale bar represents 20 μm. (<b>E</b>) Quantitation of the number of pedestals on infected HeLa cells pretreated using siRNA with two oligonucleotides against CrkI/II and CrkL (black bar) compared to control oligonucleotide treated cells (white bar). Quantitation was done by counting the number of pedestals on 100 cells. Data in the graph show mean ± standard deviation (SD) for three independent experiments. The difference between groups was statistically significant based on Students <i>t</i>-test analysis; **, p<0.01.</p

Model of the mechanism of action of Crk adaptor proteins in pedestal formation.

<p>(<b>A</b>) The functionally redundant Crk adaptor proteins inhibit pedestal formation, probably by competing with Nck for binding to Tir. Interestingly, Abl kinase, which phosphorylates CrkII, CrkL and Tir, has also been shown to associate with Tir through its SH3 domain. (<b>B</b>) Phosphorylation of Crk proteins by Abl, or the absence of Crk proteins, would leave more Tir molecules available for interacting with Nck, which would in turn promote actin polymerization by N-WASP and the Arp2/3 complex.</p