Cortactin Is Involved in the Entry of Coxiella burnetii into Non-Phagocytic Cells

BACKGROUND: Cortactin is a key regulator of the actin cytoskeleton and is involved in pathogen-host cell interactions. Numerous pathogens exploit the phagocytic process and actin cytoskeleton to infect host cells. Coxiella burnetii, the etiologic agent of Q fever, is internalized by host cells through a molecular mechanism that is poorly understood. METHODOLOGY/PRINCIPAL FINDING: Here we analyzed the role of different cortactin motifs in the internalization of C. burnetii by non-phagocytic cells. C. burnetii internalization into HeLa cells was significantly reduced when the cells expressed GFP-cortactin W525K, which carries a mutation in the SH3 domain that renders the protein unable to bind targets such as N-WASP. However, internalization was unaffected when the cells expressed the W22A mutant, which has a mutation in the N-terminal acidic region that destroys the protein's ability to bind and activate Arp2/3. We also determined whether the phosphorylation status of cortactin is important for internalization. Expression of GFP-cortactin 3F, which lacks phosphorylatable tyrosines, significantly increased internalization of C. burnetii, while expression of GFP-cortactin 3D, a phosphotyrosine mimic, did not affect it. In contrast, expression of GFP-cortactin 2A, which lacks phosphorylatable serines, inhibited C. burnetii internalization, while expression of GFP-cortactin SD, a phosphoserine mimic, did not affect it. Interestingly, inhibitors of Src kinase and the MEK-ERK kinase pathway blocked internalization. In fact, both kinases reached maximal activity at 15 min of C. burnetii infection, after which activity decreased to basal levels. Despite the decrease in kinase activity, cortactin phosphorylation at Tyr421 reached a peak at 1 h of infection. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the SH3 domain of cortactin is implicated in C. burnetii entry into HeLa cells. Furthermore, cortactin phosphorylation at serine and dephosphorylation at tyrosine favor C. burnetii internalization. We present evidence that ERK and Src kinases play a role early in infection by this pathogen

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

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

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

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

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

Tyrosine phosphorylation of cortactin terminates its interaction with focal adhesion kinase (FAK) during cell spreading.

<p>(<b>A</b>) Coomassie staining of purified GST and GST-cortactin SH3 domain was scanned in the Odyssey system. (<b>B</b>) HeLa cells were detached with trypsin-EDTA, washed with trypsin inhibitor and kept in suspension (susp.) or allowed to spread for 3 h on fibronectin (FN)-treated 100-mm plates. RIPA cell lysates were used for pull-down experiments with GST or GST-SH3, which were analyzed by SDS-PAGE and WB with focal adhesion kinase (FAK) Ab, followed by labeling with a 800CW-conjugated goat rabbit Ab. (<b>C</b>) HeLa cells were transfected with ZipB-MycCortactin and empty vector (TF2) or with ZipB-MycCortactin and ZipA-HAΔSrc (TF3). After 20 h cells were detached with trypsin-EDTA, washed with trypsin inhibitor and allowed to spread on FN-coated 100-mm plates for 3 h. Cell lysates were subjected to immunoprecipitation with FAK MoAb. The immunoprecipitates were subjected to WB and probed in three steps: (1) with myc Ab to detect transfected cortactin, followed by a 680CW-labeled goat mouseAb (red); (2) with FAK Ab, followed by a 800CW-labeled goat rabbit Ab (green); and (3) with pY466 cortactin Ab, followed by a 800CW-labeled goat rabbit Ab. Transfected cortactin was immunoprecipitated by FAK (asterisk) only when the protein was not tyrosine-phosphorylated.</p

Contribution of Crk Adaptor Proteins to Host Cell and Bacteria Interactions

The Crk adaptor family of proteins comprises the alternatively spliced CrkI and CrkII isoforms, as well as the paralog Crk-like (CrkL) protein, which is encoded by a different gene. Initially thought to be involved in signaling during apoptosis and cell adhesion, this ubiquitously expressed family of proteins is now known to play essential roles in integrating signals from a wide range of stimuli. In this review, we describe the structure and function of the different Crk proteins. We then focus on the emerging roles of Crk adaptors during Enterobacteriaceae pathogenesis, with special emphasis on the important human pathogens Salmonella, Shigella, Yersinia, and enteropathogenic Escherichia coli. Throughout, we remark on opportunities for future research into this intriguing family of proteins

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