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

N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis

The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-β-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR

Study of cortactin protein in cell adhesion and the family of CrK adaptor proteins in actin pedestal formation by enteropathogenic Escherichia coli

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Microbiología II, leída el 10-06-2015Cortactina es una proteína de unión a filamentos de actina que fue inicialmente identificada como sustrato de la proteína tirosina quinasa Src. Además, se ha descrito que cortactina está regulada por un mecanismo de acetilación y deacetilación en residuos de lisina. Sin embargo, el efecto de la fosforilación de cortactina en residuos de tirosina in vivo y su relación con la acetilación no había sido dilucidado. Por otra parte, la proteína adaptadora CrkII fue localizada mediante inmunofluorescencia en unas estructuras ricas en actina denominadas pedestales formados por Escherichia coli enteropatógena, EPEC. Posteriormente, nuestro grupo descubrió que la familia de proteínas adaptadoras Crk presentan un papel inhibitorio y redundante en la formación del pedestal de actina por EPEC. La principal ruta de polimerización de actina en los pedestales implica la interacción de la proteína bacteriana Tir fosforilada en residuos de tirosina con la proteína Nck que a su vez interacciona con NWASP, la cual activa al complejo Arp23. Objetivos Parte I Estudio de la fosforilación de cortactina en residuos de tirosina y su efecto en el spreading celular Para llevar a cabo este objetivo se ha empleado un sistema denominado Functional Interaction Trap, FIT, que promueve la fosforilación de cortactina en residuos de tirosina por la tirosina quinasa Src in vivo. El sistema FIT consiste en la expresión en las células de cortactina y de la tirosina quinasa Src, ambas fusionadas a unas hélices sintéticas complementarias. En este estudio empleamos el sistema FIT para dilucidar la relación entre la fosforilación en residuos de tirosina y la acetilación de cortactina. Además, utilizamos el sistema FIT para analizar el papel de la fosforilación de cortactina en residuos de tirosina en el spreading celular, así como su interacción con la tirosina quinasa FAK durante dicho proceso. Nuestros resultados han demostrado que el sistema FIT promueve la fosforilación de cortactina en residuos de tirosina por Src de forma eficiente y específica. Además, cortactina fosforilada en residuos de tirosina no se encuentra acetilada simultáneamente. Asimismo, cortactina promueve el spreading celular mientras que cortactina fosforilada en residuos de tirosina lo inhibe, impidiendo su interacción con la quinasa FAK. Finalmente encontramos que la interacción de cortactina y la quinasa FAK no está medida por NWASP. Parte II Estudio del papel inhibitorio y redundante de la familia de proteínas adaptadoras Crk en la formación de pedestales por EPEC El objetivo fue determinar el mecanismo por el cual la familia de proteínas adaptadoras Crk desempeñan una función inhibitoria y redundante en la formación de pedestales de actina inducidos por EPEC. Para ello se estudió la localización de diferentes mutantes de Crk durante la infección por EPEC. Además se analizó el estatus de fosforilación y la localización de las isoformas de Crk fosforiladas durante la infección por EPEC. Finalmente se analizó la interacción del dominio SH2 aislado de las proteínas CrkII y CrkL con la proteína efectora Tir. En conclusión, el dominio SH2 aislado de CrkII es suficiente para su localización en los pedestales. Además, este dominio es capaz de inhibir la formación de los pedestales, probablemente debido a que impide la localización de la proteína Nck en estas estructuras. Esto podría deberse a que CrkII y CrkL son capaces de interaccionar a través de su dominio SH2 con la proteína bacteriana Tir, para lo cual se requiere la fosforilación del residuo de tirosina 474 de Tir. Por otra parte, la infección por EPEC promueve la fosforilación del residuo de tirosina regulatorio de CrkII y CrkL en los pedestales, lo cual podría bloquear el efecto inhibitorio de estas proteínas adaptadoras en la formación de pedestales de actina por EPECDepto. de Farmacología, Farmacognosia y BotánicaFac. de FarmaciaTRUEunpu

Analysis of acetylation and tyrosine phosphorylation of transfected cortactin.

<p>(<b>A</b>) Lysates from various transfection combinations (lanes 1–4), treated or not with the deacetylase inhibitor Trichostatin A (TSA), were used to perform IPs using a myc MoAb that were examined by WB first with acetyl-cortactin Ab (in green) and second with myc MoAb (in red). The merge of both images is shown. After the membrane was gently stripped to remove the acetyl signal, it was blotted with pY466 Ab. The isotype control IP (Ctrl.) is also shown. (<b>B</b>) TSA-treated cell lysates from various transfection combinations (lanes 1–3) were subjected to parallel IP experiments with the myc MoAb and the generic pTyr MoAb. The IPs were blotted first with acetyl-cortactin Ab, and second with the myc MoAb; then the membranes were stripped and reprobed with pY466 Ab and myc MoAb. The asterisks denote non-specific bands. Quantification of the signals from cortactin immunoprecipitates showed a statistically significant inverse relationship between acetylation and tyrosine phosphorylation signals. a.u.: arbitrary units. *, p<0.05; **, p<0.01.</p

Tyrosine phosphorylation of cortactin affects cell spreading.

<p>(<b>A</b>) SYF and Rsrc cells were transfected for 20 h with empty vectors (not shown), ZipB-MycCortactin and empty vector (TF2), or ZipB-MycCortactin and ZipA-HAΔSrc (TF3). Cells were then trypsinized, replated on fibronectin-treated coverslips, and fixed at 1 and 3 h. Pictures were taken in a confocal microscope at 600× magnification. Immunofluorescence staining was done using myc MoAb (in green), pY466 cortactin Ab (in red) and Alexa Fluor 350-phalloidin (in blue). For each experimental condition, a representative image of a non-spread and spread cell is shown. * Denotes that spreading of Rsrc cells is incomplete. Images were merged using Leica software. Scale bars are shown. A total of 100 transfected cells were quantified and classified into two categories: spread or non-spread. Statistical analysis from 7 independent experiments at 1 and 3 h after replating Rsrc cells is shown for tranfections TF1 (empty vectors), TF2 (cortactin) and TF3 (phosphorylated cortactin). *, p<0.05; **, p<0.01; ***, p<0.001. (<b>B</b>) Inhibition of cortactin phosphorylation increases its acetylation during cell spreading. Rsrc cells were replated on fibronectin (FN)-coated coverslips and allowed to spread for 1 or 3 h. A third plate was allowed to spread for 1 h and then treated with PP2 for 2 h. The lysates were subjected to IPs using isotype control (Ctrl.) MoAb or 4F11 MoAb and were blotted first with acetyl-cortactin Ab and second with anti 4F11 MoAb. After gentle stripping, the membrane was incubated with pY466 cortactin Ab and 4F11 MoAb. Quantification of the ratio of acetyl:pY466 cortactin signals indicated a significantly higher ratio after PP2 treatment. a.u.: arbitrary units. **, p<0.01.</p

Efficient tyrosine phosphorylation of cortactin by Src in cells using the FIT system.

<p>SYF and Rsrc cells were transfected with different combinations of Src and cortactin FIT fusion vectors (lanes 1–8) or left untransfected (lane 9). Cell lysates were blotted for actin as a loading control and with different Abs, then blotted with the respective conjugated secondary antibodies and finally visualized with the Odyssey system. The lysates were blotted with (<b>A</b>) pY466 or (<b>B</b>) with pY421 cortactin Abs. In both cases, we observed a clear specific phosphorylation band (in green) when ZipA-HA-ΔSrc and ZipB-MycCortactin were cotransfected (transfection 5), and this band superimposes (asterisks) on the cortactin band detected with the 4F11 MoAb (in red). Sizes of the molecular weight markers (denoted M) are shown in kDa. A schematic cartoon of the FIT system is shown.</p

Analysis of acetylation and tyrosine phosphorylation of endogenous cortactin in WT and HDAC6-deficient MEFs.

<p>(<b>A</b>) Isotype control (Ctrl.) and 4F11 immunoprecipitates from cell lysates of WT and HDAC6-deficient MEFs (H) were blotted first with acetyl-cortactin Ab (in green) and second with the 4F11 cortactin MoAb (in red). The merge of both images is shown. After gentle stripping to remove the acetyl signal, the membrane was blotted with pY466 Ab and 4F11 MoAb. Quantification and statistical analysis of three independent 4F11 immunoprecipitates and the ratio of acetyl:pY466 cortactin signals are shown. a.u.: arbitrary units. *, p<0.05. (<b>B</b>) Immunoprecipitates obtained with acetyl-cortactin Ab were blotted with pY466 Ab and 4F11. The phosphorylation signal did not coincide with acetylated cortactin. (<b>C</b>) Blotting of WT and HDAC6-deficient cell lysates with HDAC6 Ab is shown as a control of cell phenotype.</p

Analysis of acetylation and tyrosine phosphorylation of transfected GFP-cortactin.

<p>(<b>A</b>) Tyrosine phosphorylation of cortactin is not required for acetylation of the protein. HeLa cells were transfected with vectors encoding GFP fused with WT cortactin or the Y421/466/482F non-phosphorylatable cortactin mutant (3F). Lysates were blotted with acetyl-cortactin Ab and GFP MoAb. Transfected cortactin was acetylated and no statistically significant difference was found in acetylation level between WT and 3F transfectants (data not shown). (<b>B</b>) Tyrosine phosphorylation of cortactin decreases acetylation of the protein. HeLa cells were transfected with a vector encoding GFP fused with WT cortactin. Transfectants were left untreated (-) or treated with pervanadate (PV), a generic phosphatase inhibitor, or with Thrichostatin A (TSA), a deacetylase inhibitor. Lysates were blotted with acetyl-cortactin Ab and with GFP MoAb. After stripping, the membrane was incubated with pY466 cortactin, which was merged with the GFP cortactin signal. The ratio of acetyl:pY466 cortactin is shown for untreated (-) and PV-treated cells. a.u.: arbitrary units. **, p<0.01.</p

Specificity of tyrosine phosphorylation in the FIT system.

<p>(<b>A</b>) Detection of the phosphorylation status of paxillin, another Src kinase substrate. SYF and Rsrc cells were transfected with FIT fusion vectors and the most relevant lysates (4 and 5) from two different experiments (FIT 8 and 9) were analyzed by WB with a rabbit Ab against phospho-paxillin (in green) and with a MoAb against actin (in red). As controls, cells were left untreated or treated with pervanadate (PV), a potent phosphatase inhibitor that induces the phosphorylation of paxillin. Rsrc cells showed a higher basal level of phospho-paxillin than did SYF cells, though in both cell lines, this basal level was enhanced by treatment with PV. The FIT system did not increase the basal level of phospho-paxillin. (<b>B</b>) Tyrosine phosphorylation of cortactin occurs on the expected tyrosines (Y421, Y466 and Y482). HeLa cell lysates were transfected with ZipA-HA-ΔSrc and ZipB-MycCortactin (lane 4) or with ZipA-HA-ΔSrc and ZipB-MycCortactin with the triple mutation Y421/466/482F (3F) (lane 5). Several control cotransfections were done (lanes 1–3). WB with generic pTyr MoAb demonstrated that only ZipB-Myc WT cortactin, and not the 3F mutant, was phosphorylated (in green). Cortactin was detected with a rabbit MoAb (in red). Actin is shown as a loading control.</p