p27Kip1 promotes invadopodia turnover and invasion through the regulation of the PAK1/Cortactin pathway
- Cancer Research Center of Toulouse, France
- Université Toulouse III Paul Sabatier, France
- CNRS ERL5294, France
- Inserm U1016 Institut Cochin, Sorbonne Paris Cité, France
- National Institute of Radiological Sciences, Japan
Figures
Figure 1
p27 binds to Cortactin.
(A–B) Co-immunoprecipitation of p27 and Cortactin: (A) p27 was immunoprecipitated using rabbit anti-p27 (C19) antibodies from HEK293 lysates transfected with plasmids encoding p27, Myc-Cortactin …
Figure 2 with 2 supplements
p27 colocalizes with Cortactin in invadopodia.
(A–B) p27+/+ MEFs were seeded on gelatin-coated coverslips for 24 hr. Cells were permeabilized with digitonin prior to fixation. Cells were labeled with mouse anti p27 (SX53G8.5) in red (A–B) and …
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Figure 2—source data 1
Quantification of co-immunoprecipitation between p27 and Cortactin in MEF E6 (Figure 2C) and HeLa cells (Figure 2—figure supplement 2).
- https://doi.org/10.7554/eLife.22207.005
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Figure 2—source data 2
Statistical analyses for Figure 2C and Figure 2—figure supplement 2.
- https://doi.org/10.7554/eLife.22207.006
Figure 2—figure supplement 1
p27 colocalizes with Cortactin and Tks5 in different tumor cell lines.
(A–C) Twenty-four hr after seeding, A549 [A-B] or A375 cells [C] were permeabilized with digitonin prior to paraformaldehyde fixation. Cells were immunostained with mouse anti p27 (SX53G8.5) (red) …
Figure 2—figure supplement 2
p27/Cortactin interaction in HeLa cells after EGF stimulation.
(A) HeLa cells were starved with DMEM 0.1% FCS overnight, stimulated with 100 ng/ml EGF (Peprotech) for the indicated times and cell lysates were submitted to co-immunoprecipitation using rabbit …
Figure 3 with 2 supplements
p27 regulates invadopodia formation and matrix degradation.
(A) p27+/+, p27CK−/CK− and p27−/− immortalized MEFs were seeded on Oregon green-gelatin (gelatin-A488) for 16 hr. Cells were stained with rabbit anti-Tks5 (M-300) to visualize invadopodia. (B) The …
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Figure 3—source data 1
Quantification of cells with invadopodia (Figure 3B); quantification of degraded gelatin area per cell (Figure 3C); quantification of cells with invadopodia after p27 re-expression (Figure 3F) and quantification of degraded gelatin area per cell after p27 re-expression (Figure 3G).
- https://doi.org/10.7554/eLife.22207.010
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Figure 3—source data 2
Statistical analyses for Figure 3B,C,F and G.
- https://doi.org/10.7554/eLife.22207.011
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Figure 3—source data 3
Immunoblot scans of Figure 3E.
- https://doi.org/10.7554/eLife.22207.012
Figure 3—figure supplement 1
p27+/+, p27CK−/CK− and p27−/− MEFs form functional invadopodia.
HPV E6 immortalized p27+/+, p27CK−/CK− and p27−/− MEFs were seeded on Gelatin-A488 for 16 hr. Cells were fixed in paraformaldehyde and stained with rabbit anti-Tks5 (M-300; red) antibodies to …
Figure 3—figure supplement 2
p27 colocalizes with Tks5 at sites of gelatin degradation.
HPV E6 immortalized p27+/+ MEFs (A) or A549 lung adenocarcionma cells (B) were seeded on Gelatin-A488 and treated with 1 μM FRAX597 to stabilize invadopodia after 2 hr. After 72 hr, cells were …
Figure 4
p27 promotes cell migration and invasion.
(A) Representative images of scratch wound migration assays with p27+/+, p27CK−/CK− and p27−/− immortalized MEFs. Dark grey areas show the initial wound masks and dotted lines the migration fronts. …
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Figure 4—source data 1
quantification of relative wound density (Figure 4B); quantification of invasion (Figure 4D); and quantification of invasion rescue by p27 re-expression (Figure 4E).
- https://doi.org/10.7554/eLife.22207.016
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Figure 4—source data 2
Statistical analyses for Figure 4B,D and E.
- https://doi.org/10.7554/eLife.22207.017
Figure 5 with 1 supplement
p27 promotes binding of Cortactin to PAK1.
(A) Live p27+/+ and p27−/− immortalized MEFs expressing eGFP-Tks5 were imaged by videomicroscopy to measure invadopodia lifetime, using Tks5 as an invadopodia marker. The graph represents the …
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Figure 5—source data 1
Quantification of invadopodia lifetime (Figure 5A); quantification of co-immunoprecipitation between Cortactin and PAK1 in MEFs (Figure 5C); and quantification of co-immunoprecipitation between Cortactin and PAK1 after serum stimulation (Figure 5E).
- https://doi.org/10.7554/eLife.22207.019
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Figure 5—source data 2
Statistical analyses for Figure 5A.
- https://doi.org/10.7554/eLife.22207.020
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Figure 5—source data 3
Statistical analyses for Figure 5C.
- https://doi.org/10.7554/eLife.22207.021
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Figure 5—source data 4
Statistical analyses for Figure 5E.
- https://doi.org/10.7554/eLife.22207.022
Figure 5—figure supplement 1
p27 does not affect Cortactin acetylation or the recruitment of c-Src, Arp2 and ERK1 to Cortactin.
(A) HEK 293 cells were transfected with either empty vector, p27 or p27CK- coding vectors. Lysates were used for immunoblot with rabbit anti-Acetyl-Cortactin (BD-Transduction Laboratories), …
Figure 6 with 1 supplement
p27 regulates invadopodia formation downstream of PAK1.
(A–B) p27+/+ and p27−/− E6 MEFs were seeded on gelatin-A488 and transfected with either control siRNAs or PAK1 siRNAs for 48 hr. Cells were stained with rabbit anti-Tks5 (M-300) to visualize …
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Figure 6—source data 1
Quantification of invadopodia forming cells (Figure 6A) and degraded gelatin area (Figure 6B) after PAK1 silencing; quantification of invadopodia forming cells (Figure 6D) and degraded gelatin area (Figure 6E) after FRAX597 treatment; quantification of invadopodia forming cells (Figure 6—figure supplement 1A) and degraded gelatin area (Figure 6—figure supplement 1B) after FRAX1036 and G-5555 treatment.
- https://doi.org/10.7554/eLife.22207.025
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Figure 6—source data 2
statistical analyses for Figure 6A,B,D and E and Figure 6—figure supplement 1A and B.
- https://doi.org/10.7554/eLife.22207.026
Figure 6—figure supplement 1
p27 regulates invadopodia formation in a PAK1 dependent manner.
(A–B) p27+/+ or p27−/− E6 MEFs were seeded for 48 hr on Gelatin-A488 and treated 1 hr after seeding with either DMSO,1 μM FRAX1036 or 1 μM G-5555, two PAK1-3 inhibitors. Cells were stained with …
Figure 7 with 1 supplement
p27 regulates invadopodia formation downstream of Rac1.
(A) Cells were seeded for 24 hr and Rac1-GTP levels measured by GTP pull-downs assays using GST-PAK1-CD beads. The amounts of Rac1-GTP bound to the beads and of total Rac1 in the extracts were …
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Figure 7—source data 1
Quantification of Rac1 GTP/Rac1 levels (Figure 7A); quantification of invadopodia forming cells (Figure 7B) and degraded gelatin area (Figure 7C) after silencing of Rac1; quantification of invadopodia forming cells (Figure 7E) and degraded gelatin area (Figure 7F) after NSC23766 treatment; quantification of invadopodia forming cells (Figure 7—figure supplement 1A) and degraded gelatin area (Figure 7—figure supplement 1B) after RhoA silencing; and quantification of invasion after Y27632 treatment (Figure 7—figure supplement 1D).
- https://doi.org/10.7554/eLife.22207.029
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Figure 7—source data 2
Statistical analyses for Figure 7A,B,C,E,F, and Figure 7—figure supplement 1A,B and D.
- https://doi.org/10.7554/eLife.22207.030
Figure 7—figure supplement 1
RhoA regulation by p27 is not involved in invadopodia formation.
(A–C) p27+/+ and p27−/− immortalized MEFs were transfected with siRNA control (ctl) or siRNA RhoA #1 or #2. After 3 days, cells were seeded on Gelatin-A488 for matrix degradation and for controlling …
Figure 8 with 2 supplements
Mimicking phosphorylation of Cortactin restores invadopodia dynamics in p27−/− cells.
(A–E) p27−/− E6 MEFs were infected with empty vector or with vectors encoding wild type Cortactin (WT), S113A-Cortactin (S113A) or S113D-Cortactin (S113D). (A) Cortactin levels after retroviral …
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Figure 8—source data 1
Quantification of cells forming invadopodia (Figure 8B–C) and degraded gelatin area (Figure 8D–E) after infection with S113 phospho-mutants of Cortactin; quantification of cells forming invadopodia (Figure 8G–H) and degraded gelatin area (Figure 8I–J) after infection with triple phospho-mutants of Cortactin; quantification of P-Ser Cortactin/Cortactin ratio (Figure 8—figure supplement 1B).
- https://doi.org/10.7554/eLife.22207.033
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Figure 8—source data 2
Statistical analyses for Figure 8.
- https://doi.org/10.7554/eLife.22207.034
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Figure 8—source data 3
Statistical analyses for Figure 8—figure supplement 1B.
- https://doi.org/10.7554/eLife.22207.035
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Figure 8—source data 4
Mascot search results for Cortactin for Figure 8—figure supplement 2.
- https://doi.org/10.7554/eLife.22207.036
Figure 8—figure supplement 1
Cortactin is phosphorylated on S113/S150 and/or S282 in vivo.
(A) HEK 293 cells were tranfected with Myc-tagged Cortactin for 24 hr or left untransfected (U) and 3 μM FRAX597 was added for 12 hr where indicated. Cortactin was immunoprecipitated with anti-Myc …
Figure 8—figure supplement 2
Cortactin is phosphorylated on S150 in vivo upon PAK activation.
MS/MS analysis of Cortactin immunoprecipitated from HEK 293 cells transfected either with Myc-Cortactin or Myc-Cortactin and Myr-SH3-2, the second SH3 domain of Nck1 fused to the myristoylation …
