Figure 5.
PEAK1 recruits Shc1 to FAs. (A) GFP-PEAK1 was expressed in PEAK1-deficient GE11 cells to perform GFP-TRAP experiments followed by MS analysis of binding partners. Volcano plot shows proteins enriched in PEAK1-GFP over control (PEAK1 knockout cells) samples. The logarithmic ratio of protein LFQs were plotted against negative logarithmic P values of a two-sided two-sample t test. The hyperbolic curve separates significantly enriched proteins (indicated in green) from common binders (FDR: 0.05, n = 4). (B) GFP-PEAK1 WT or Y635E/Y635F/Y1188F mutants were expressed in PEAK1-deficient GE11 cells. Representative Western blots of GFP-TRAP experiments are shown. (C) Colocalization of GFP-PEAK1, Shc1 (green in merge), and Paxillin (magenta in merge) in PEAK1-deficient versus GFP-PEAK1 WT or Y1188F mutant–expressing GE11 cells. Scale bar, 10 μm; in magnified regions, 5 µm. (D) Quantifications of colocalization of Shc1 and phospho-Y118 Paxillin (calculated as a percentage of total Shc1) of immunofluorescence images as shown in C. n = 3, total number of cells analyzed: 52 (KO); 48 (PEAK1); 51 (Y1188F). Box plots range from the 25th to 75th percentile; central line indicates the median; whiskers show smallest to largest value. Mann–Whitney U test was performed to determine statistical significance. ****, P < 0.0001. (E) Migration of GE11 Tet-ON integrin β1 cells (WT or PEAK1-deficient with or without rescue with the indicated PEAK1-GFP constructs) treated with 1 μg/ml doxycycline for 48 h to induce β1 expression on fibronectin-coated plastic was analyzed in overnight videos. Kruskal–Wallis with Dunn’s multiple comparisons test was performed to determine statistical significance; error bars show SEM; ***, P < 0.0001; n = 93 (GE11), 105 (PEAK1 KO), 105 (PEAK1-GFP), 94 (PEAK1-Y635F), 94 (PEAK1-Y1188F) cells. (F) Model of PEAK1 and its binding partners. PEAK1 is connected to integrins by Tensin3, which binds to phospho-Y635 PEAK1 and the integrin β MP-NPxY motif. Shc1 binds phospho-Y1188 PEAK1. PEAK1 also interacts with proteins that were classified in different clusters based on their association rates with Shc1 upon EGF stimulation (Zheng et al., 2013), including the cluster 1 (C1) proteins Grb2 and Lrrk1 and C3 proteins Asap1/2, Dab2ip, Ppp1ca/cc, Prag1, and Rasal2. Source data are available for this figure: SourceData F5.

PEAK1 recruits Shc1 to FAs. (A) GFP-PEAK1 was expressed in PEAK1-deficient GE11 cells to perform GFP-TRAP experiments followed by MS analysis of binding partners. Volcano plot shows proteins enriched in PEAK1-GFP over control (PEAK1 knockout cells) samples. The logarithmic ratio of protein LFQs were plotted against negative logarithmic P values of a two-sided two-sample t test. The hyperbolic curve separates significantly enriched proteins (indicated in green) from common binders (FDR: 0.05, n = 4). (B) GFP-PEAK1 WT or Y635E/Y635F/Y1188F mutants were expressed in PEAK1-deficient GE11 cells. Representative Western blots of GFP-TRAP experiments are shown. (C) Colocalization of GFP-PEAK1, Shc1 (green in merge), and Paxillin (magenta in merge) in PEAK1-deficient versus GFP-PEAK1 WT or Y1188F mutant–expressing GE11 cells. Scale bar, 10 μm; in magnified regions, 5 µm. (D) Quantifications of colocalization of Shc1 and phospho-Y118 Paxillin (calculated as a percentage of total Shc1) of immunofluorescence images as shown in C. n = 3, total number of cells analyzed: 52 (KO); 48 (PEAK1); 51 (Y1188F). Box plots range from the 25th to 75th percentile; central line indicates the median; whiskers show smallest to largest value. Mann–Whitney U test was performed to determine statistical significance. ****, P < 0.0001. (E) Migration of GE11 Tet-ON integrin β1 cells (WT or PEAK1-deficient with or without rescue with the indicated PEAK1-GFP constructs) treated with 1 μg/ml doxycycline for 48 h to induce β1 expression on fibronectin-coated plastic was analyzed in overnight videos. Kruskal–Wallis with Dunn’s multiple comparisons test was performed to determine statistical significance; error bars show SEM; ***, P < 0.0001; n = 93 (GE11), 105 (PEAK1 KO), 105 (PEAK1-GFP), 94 (PEAK1-Y635F), 94 (PEAK1-Y1188F) cells. (F) Model of PEAK1 and its binding partners. PEAK1 is connected to integrins by Tensin3, which binds to phospho-Y635 PEAK1 and the integrin β MP-NPxY motif. Shc1 binds phospho-Y1188 PEAK1. PEAK1 also interacts with proteins that were classified in different clusters based on their association rates with Shc1 upon EGF stimulation (Zheng et al., 2013), including the cluster 1 (C1) proteins Grb2 and Lrrk1 and C3 proteins Asap1/2, Dab2ip, Ppp1ca/cc, Prag1, and Rasal2. Source data are available for this figure: SourceData F5.

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