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First published online 7 December 2004
doi: 10.1242/jcs.01586

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Cortactin regulates cell migration through activation of N-WASP

¹ Biological and Biomedical Sciences Program, Harvard Medical School, Boston, MA 02215, USA
² Cancer Biology Program Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, Boston, MA 02215, USA
³ Department of Cell Biology, Harvard Medical School, Boston, MA 02215, USA
⁴ Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, MA 02215, USA

View larger version (59K): [in a new window]   Fig. 1. Overexpression of full-length or C-terminal cortactin enhances migration of mouse SCp2 mammary epithelial cells. (A) Domain structure of cortactin variants used in this study. FL, full-length cortactin; Nt, N-terminal (1-326 amino acids) cortactin; Ct, C-terminal (324-546 amino acids) cortactin; CtW525L, C-terminus of cortactin with W525L mutation; SH3, SH3 domain; SH3W525L, SH3 domain with W525L mutation. (B) Expression of cortactin variants in SCp2 cells. Western blot showing expression of the HA-FL, HA-Ct and HA-CtW525L cortactin (upper blot) or endogenous cortactin (lower blot). Constructs were expressed at or below the level of endogenous cortactin. Arrowheads denote HA-FL, HA-Ct and HA-CtW525L bands. V, control vector expression. (C) Migration of SCp2 cells in a representative wounding experiment is shown for control vector only infected (V) or cortactin variant infected (HA-FL, HA-Ct, HA-CTW525L) cells with images at 0 and 15 hours. (D) Quantification of wound closure data from three to seven experiments. Fold change in wound area was calculated as described in Materials and Methods. Error bars indicate s.e.m. and asterisks indicate statistically significant differences from change in area calculated for control cells (P<0.05).

View larger version (50K): [in a new window]   Fig. 2. The SH3 domain of cortactin is sufficient to activate N-WASP. (A) Western blot (left) of GST pull-down assays showing association of GST-cortactin fusions with endogenous N-WASP. TCL, total cell lysate; 1/40th of the amount of lysate used in the pull down was used for TCL. Right panel shows comparative size of GST fusion proteins used in this study on Coomassie-stained gels. Values down the side of the gel show the positions of protein standards in kDa. (B-E) Pyrene Assays. 10 nM Arp2/3 complex, 1.5 µM pyrene-labeled G-actin and 25 nM GST-N-WASP or His6-N-WASP were incubated together with GST-tagged or untagged cortactin proteins at the specified concentrations. (B) Optimal activation of GST-N-WASP by GST-cortactin proteins (GST, 1.3 µM; GST-Ct, 1.3 µM; GST-SH3, 1 µM). (C) Fold activation of GST-N-WASP by GST, GST-Ct or GST-SH3 cortactin, or GST-Nck. All fold activation curves are a logarithmic `best fit' of the data points shown. Inset shows N-WASP binding to GST-Ct, GST-SH3 and GST-Nck beads. (D) GST tag on N-WASP is not required for N-WASP activation by cortactin. Sample polymerization curves are shown for each GST-cortactin protein at the concentration giving optimal N-WASP activation in the pyrene assay (GST, 250 nM; GST-Ct, 1 µM; GST-SH3, 250 nM). (E) GST tag is not required for N-WASP activation by cortactin C-terminus. Plots show fold activation of GST-N-WASP by GST, GST-Ct, untagged cortactin C-terminus (Ct) or buffer control (Buffer).

View larger version (39K): [in a new window]   Fig. 3. N-WASP activation requires an intact, accessible SH3 domain. (A,B) Peptide inhibition. Pyrene assays were performed with 10 nM Arp2/3 complex, 1.5 µM pyrene-labeled G-actin, 25 nM GST-N-WASP and 0.5 µM GST-SH3 in the presence of peptide inhibitors at the specified concentrations. (A) Activation of N-WASP by cortactin SH3 protein in the presence of 100 µM cortoptimal or 100 µM control peptides. The inset shows a western blot of competitive GST pull-down assays showing association of GST-SH3 cortactin with endogenous N-WASP in the presence of increasing concentrations (0, 25, 50, 100, 200, 500, 1000 µM, left to right) of cortoptimal or control peptide. (B) 100 µM cortoptimal and control peptides have no effect on N-WASP alone or on WA-mediated actin polymerization. (C,D) Mutation of the SH3 domain. Pyrene assays were performed with 10 nM Arp2/3 complex, 1.5 µM pyrene-labeled G-actin and 25 nM GST-N-WASP, in the presence of either GST-tagged or untagged cortactin SH3 domain. (C) Sample polymerization curves showing maximal GST-N-WASP activation by 2.5 µM SH3 or SH3W525L. Inset shows sample polymerization curves of maximal GST-N-WASP activation by 0.75 µM GST, GST-SH3 or GST-SH3W525L proteins. (D) Fold activation of GST-N-WASP in the presence of untagged cortactin SH3 and SH3W525L proteins over a range of concentrations. Curves are a logarithmic `best fit' of the data points shown. Inset shows GST pull-down assays for N-WASP binding performed in the presence of GST, GST-SH3 or GST-SH3W525L beads.

View larger version (58K): [in a new window]   Fig. 4. Maximal cortactin-enhanced migration requires N-WASP. (A) Western blot showing expression of the HA-FL, HA-Ct and HA-CtW525L cortactin (upper blot) or endogenous cortactin (lower blot) in NW–/– or rescued MEFs. Arrowheads indicate HA-FL cortactin. Expression of all HA-tagged constructs was equivalent to or less than endogenous cortactin levels. UI, uninfected cells. (B) HA-tagged, or in some assays GFP-tagged, cortactin-expressing cells were assayed for wound closure at 0, 2, 4 and 6 hours after wounding. Data are the average of four to five experiments at the 6-hour time point±s.e.m. Asterisks indicate statistically significant differences from wound closure results in control cells (P<0.05). (C) Representative results from wounding assay experiments are shown with images taken at 0 and 6 hours after wounding. The fold change in wound area is indicated underneath each figure.