Journal: PLoS ONE

Article Title: Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

doi: 10.1371/journal.pone.0033662

Figure Lengend Snippet: 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 ( A ) pY466 or ( 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.

Article Snippet: Mouse actin C4 MoAb was from MP Biomedicals, and rabbit cortactin MoAb was from Novus Biologicals.

Techniques: Transfection, Control, Phospho-proteomics, Molecular Weight

Journal: PLoS ONE

Article Title: Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

doi: 10.1371/journal.pone.0033662

Figure Lengend Snippet: ( A ) 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 ) 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.

Article Snippet: Mouse actin C4 MoAb was from MP Biomedicals, and rabbit cortactin MoAb was from Novus Biologicals.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Control

Journal: PLoS ONE

Article Title: Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

doi: 10.1371/journal.pone.0033662

Figure Lengend Snippet: ( A ) 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 ) 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.

Article Snippet: Mouse actin C4 MoAb was from MP Biomedicals, and rabbit cortactin MoAb was from Novus Biologicals.

Techniques: Transfection, Histone Deacetylase Assay, Membrane, Control, Phospho-proteomics

Journal: PLoS ONE

Article Title: Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

doi: 10.1371/journal.pone.0033662

Figure Lengend Snippet: ( A ) 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 ) 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.

Article Snippet: Mouse actin C4 MoAb was from MP Biomedicals, and rabbit cortactin MoAb was from Novus Biologicals.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Plasmid Preparation, Histone Deacetylase Assay, Stripping Membranes, Membrane, Incubation

Journal: PLoS ONE

Article Title: Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

doi: 10.1371/journal.pone.0033662

Figure Lengend Snippet: ( A ) 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 ) Immunoprecipitates obtained with acetyl-cortactin Ab were blotted with pY466 Ab and 4F11. The phosphorylation signal did not coincide with acetylated cortactin. ( C ) Blotting of WT and HDAC6-deficient cell lysates with HDAC6 Ab is shown as a control of cell phenotype.

Article Snippet: Mouse actin C4 MoAb was from MP Biomedicals, and rabbit cortactin MoAb was from Novus Biologicals.

Techniques: Control, Gentle, Stripping Membranes, Membrane, Phospho-proteomics

Journal: PLoS ONE

Article Title: Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

doi: 10.1371/journal.pone.0033662

Figure Lengend Snippet: Cell lysates of SYF and Rsrc MEFs were subjected to IPs using ( A ) isotype control Ab (Ctrl), 4F11 MoAb and generic phospho-tyrosine (pTyr) MoAb. These IPs were performed in parallel by probing first with acetyl-cortactin Ab (in green), after a gentle stripping, with pY466 and at last, the membrane was stripped and reprobed with cortactin Ab. Statistical analysis of the ratio of acetyl:pY466 cortactin signals is shown for 4F11 immunoprecipitates. a.u.: arbitrary units. **, p<0.01. Asterisks denote evidence that pTyr immunoprecipitates from Rsrc cell lysates contain phospho-cortactin but not acetyl-cortactin. ( B ) IPs with pY466 and isotype control Abs were probed with acetyl-cortactin Ab and 4F11 cortactin MoAb, and reprobed, after gentle stripping, with pY466 Ab and 4F11 MoAb.

Article Snippet: Mouse actin C4 MoAb was from MP Biomedicals, and rabbit cortactin MoAb was from Novus Biologicals.

Techniques: Control, Gentle, Stripping Membranes, Membrane

Journal: PLoS ONE

Article Title: Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

doi: 10.1371/journal.pone.0033662

Figure Lengend Snippet: ( A ) 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 ) 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.

Article Snippet: Mouse actin C4 MoAb was from MP Biomedicals, and rabbit cortactin MoAb was from Novus Biologicals.

Techniques: Transfection, Plasmid Preparation, Microscopy, Immunofluorescence, Staining, Software, Inhibition, Phospho-proteomics, Control, Gentle, Stripping Membranes, Membrane, Incubation

Journal: PLoS ONE

Article Title: Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

doi: 10.1371/journal.pone.0033662

Figure Lengend Snippet: SYF and Rsrc cells were transfected with empty vectors, with ZipB-MycCortactin and empty vector (TF2) or with ZipB-MycCortactin and ZipA-HAΔSrc (TF3). Cells were fixed and visualized by immunofluorescence using vinculin MoAb (in green) and myc Ab (in red). Photographs were taken using a Nikon Eclipse TE 200-U fluorescence microscope equipped with a Hamamatsu camera. Images were processed with Adobe Photoshop. A scale bar is shown.

Article Snippet: Mouse actin C4 MoAb was from MP Biomedicals, and rabbit cortactin MoAb was from Novus Biologicals.

Techniques: Transfection, Plasmid Preparation, Immunofluorescence, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

doi: 10.1371/journal.pone.0033662

Figure Lengend Snippet: ( A ) Coomassie staining of purified GST and GST-cortactin SH3 domain was scanned in the Odyssey system. ( 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. ( C ) 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.

Article Snippet: Mouse actin C4 MoAb was from MP Biomedicals, and rabbit cortactin MoAb was from Novus Biologicals.

Techniques: Staining, Purification, Suspension, SDS Page, Labeling, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: PLoS ONE

Article Title: Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

doi: 10.1371/journal.pone.0033662

Figure Lengend Snippet: Acetylated cortactin is inactive and probably has a closed conformation that masks the tyrosines targeted by Src. Upon appropriate stimulation, cortactin is deacetylated by HDAC6, exposing the tyrosines, which are then rapidly phosphorylated by Src. This phosphorylation keeps cortactin deacetylated. Whether this tyrosine-phosphorylated cortactin has an open or closed configuration is unknown (question mark).

Article Snippet: Mouse actin C4 MoAb was from MP Biomedicals, and rabbit cortactin MoAb was from Novus Biologicals.

Techniques: Phospho-proteomics