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Fig. 6. Serine phosphorylation of cortactin is required for ECM degradation at invadopodia. (A) A375MM cells transfected with cortactin^S405,418D display a remarkably increased ability to degrade the ECM compared with control cells transfected with cortactin^WT. By contrast, transfection of cortactin^S405,418A markedly reduced ECM degradation. Data represent the mean ± s.d. of three independent experiments. Statistical significance evaluated by Student's t-test: wild type vs S405,418D, P<0.01; wild type vs S405,418A, P<0.005. (B) Representative image of A375MM cells transfected with cortactin^S405,418D 48 hours after cortactin depletion. Typically cortactin^S405,418D localizes to invadopodia. (C) A375MM cells transfected with the autoinhibitory domain of PAK (AID) display a reduced ability to degrade the matrix compared with mock-transfected cells. Data represent the mean ± s.d. of three independent experiments. Statistical significance was evaluated by Student's t-test: control vs autoinhibitory domain of PAK P<0.0001. (D) A375MM cells were transfected with cortactin^WT, nonphosphorylatable cortactin^S113A and pseudophosphorylated cortactin^S113D. The areas of degradation were then quantified. Data represent the mean ± s.d. of three independent experiments. Statistical significance was evaluated by Student's t-test: wild type vs S113A and S113D, P<0.004. (E) Representative immunofluorescence image of A375MM cells transfected with cortactin^S113D 48 hours after cortactin knockdown labeled with anti-FLAG antibodies. Cortactin^S113D clearly localizes to ECM degradation patches at invadopodia. Scale bars: 10 µm.

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