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

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

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

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

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

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