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Files in this Data Supplement:
Fig. S1. Catalytic activities of Abl variants and binding specificities of Crk and Nck1 SH3 domains to Abl PxxP motifs. (A). Schematic diagram of wild-type (wt) c-Abl, and Crk and Nck families of proteins. For Abl, wavy line represents myristoyl group; CAP, regulatory segment that suppresses catalytic activity; Proline-rich (red boxes), the cluster of four PxxP motifs, 1, 2, 3 and 4; NLS, nuclear localization signal; DNA, DNA binding domain; G and F, G- and F-actin binding domains. Mutations of Abl variants, SH2*, 1234, or SH2* 1234, are indicated by circle, triangles, or circle and triangles, respectively under the c-Abl diagram. For CrkII and CrkL, Y represents regulatory tyrosine phosphorylation site located at Y221 in human CrkII and Y207 in human CrkL. (B) Whole-cell lysates (WCL) of 293T cells transfected with various forms of Abl were subjected to immunoprecipitation (IP) with anti-Abl. Immunoprecipitated Abl was subjected for in vitro kinase assay using GST-CTD or GST-Crk-225 as a substrate. After reaction, the samples were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-Abl or phosphotyrosine (pTyr) antibody. (C) Residues of Abl PxxP motifs critical for SH3 binding are shown in color. NLS, nuclear localization signal. (D) WCL of 293T cells transfected with various forms of Abl were subjected to GST pulldown using Nck1- or CrkII-GST beads. The amount of Abl in WCL or GST pulldown was detected by western blotting. (E) WCL of 293T cells transfected with various forms of Abl were subjected to anti-Abl IP. Immunoprecipitated Abl was resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-Abl antibody, or GST fusion of Nck1 SH3 domains (Nck1-SH3s-GST), CrkII N-terminal SH3 (Crk-SH3-GST), or GST alone.
Fig. S2. Filopodium formation of NIH3T3 cells during attachment is influenced by overexpression of Crk and Nck adaptor proteins. (A,D) NIH3T3 cells were infected with retroviral vector expressing candidate Abl PxxP motif binding partners as marked. Expression levels of candidates were assayed by western blotting. (B,E) Serum-starved cells were plated on coverslips coated with 10 µg/ml fibronectin, and fixed at 20 minutes. The fixed cells were stained for DNA (blue) and F-actin (red) with Hoechst 33342 and Texas-Red-conjugated phalloidin, respectively. Infected cells express EGFP (green) as marker. Bars, 20 µm (EGFP and F-actin/DNA) and 10 µm (magnified). (C) and (F)The number of filopodia was counted from the fixed cells. Graph represents mean and/or s.e.m. of filopodia per cell or percentage of cells showing filopodia per total. The numbers above the bars indicate the number of cells examined. ‡P<0.05 when compared with NIH3T3 cells infected with control retrovirus.
Fig. S3. PxxP motifs enable c-Abl to phosphorylate Dok1 and CrkII during attachment. (A,B) NIH3T3 cells overexpressing various forms of Abl (A), NIH3T3 cells treated with or without STI-571 (B), and Abl DKO cells re-expressing various forms of Abl (C) were harvested in suspension or at 10 minutes after attachment on 10 µg/ml fibronectin-coated dish (see materials and methods for details). WCL were subjected to Dok1 IP (A) and Crk IP (B). Tyrosine phosphorylation of Dok1 and CrkII was detected by western blotting.
Fig. S4. Lack of p130Cas increases filopodium formation and decreases spreading speed and focal adhesion formation at the leading edge during attachment. (A). Whole cell lysates of serum-starved p130Cas knockout cells (p130Cas KO) with or without reexpression of p130Cas were subjected to immunoblotting for p130Cas expression. (B,C) p130Cas KO cells with or without re-expression of p130Cas were plated on coverslips coated with 10 µg/ml fibronectin, and fixed at 20 minutes. Fixed cells were stained as indicated. Bars, 20 µm (B). The number of filopodia counted from the fixed cells. ‡P<0.05 (C). (D,E) p130Cas KO cells with or without re-expression of Cas were plated on fibronectin-coated coverslips for 10 minutes and fixed. (D) Spreading cells were stained as indicated. Bar, 50 µm. (E) Cell areas for stained cells were measured using ImageJ software; ‡ indicates P<0.05. (F) p130Cas KO cells with or without re-expression of Cas were plated on coverslips coated with 10 µg/ml fibronectin for 10 minutes and fixed. Cells were stained for vinculin (green), DNA (blue), and Factin (red) with anti-vinculin/Alexa-fluor 488, Hoechst 33342, and Texas-Red-conjugated phalloidin, respectively. Bars, 20 µm (left three panels) and 10 µm (magnified).
Fig. S5. c-Abl and its PxxP motif interaction partners regulate pattern and extent of focal adhesion formation during attachment. (A-D) NIH3T3 cells overexpressing Grb2, Crk, Nck2, or c-Abl (A), Abl DKO cells re-expressing various forms of Abl (B), STI-571 treated or Nck family knockdown (C), or Crk family knockdown NIH3T3 cells (D) were plated on 10 µg/ml fibronectin-coated coverslips for 10 minutes and fixed. (A) Cells in A were stained for vinculin (green) and F-actin (red) with anti-vinculin/Alexa-fluor 647 and Texas-Red-conjugated phalloidin, respectively. Infected cells expressed EGFP (blue) as marker. Cells in B-D were stained as indicated. Bars, 20 µm (left three panels) and 10 µm (magnified).
Movie 1. Abl-double-knockout cells and Abl-double-knockout cells re-expressing wild-type Abl.
Movie 2. NIH3T3 cells overexpressing Abl, CrkII or Nck2.
Movie 3. NIH3T3 cells in which Crk family adaptor proteins have been knocked down.
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