Fig 2: (A) The CAS SH3 binding motif based on the 14 unique sequences obtained from phage display. The x-axis shows the residue position relative to proline (position 0)64–66. (B) The CAS SH3 domain binding interaction partners with their respective binding motifs. Interactors with small differences in binding motif are in italics. References (R) are superscripted. (C–E) Isothermal titration calorimetry (ITC) data obtained for the interaction of CAS SH3 with four synthetic peptides.

Fig 3: PKN3 phosphorylates p130Cas and PKN3 activity correlates with p130Cas phosphorylation in human breast carcinomas. (A–C) Correlation statistics (graphs) of publicly available RNA-seq data of co-expression of (A) human p130Cas/BCAR1 and PKN3; (B) p130Cas/BCAR1 and Src; (C) p130Cas/BCAR1 and PKN2 or graph (D) showing positive linear dependency of protein phosphorylation levels (log ratio values) of PKN3 Thr860 and p130Cas/BCAR1 Ser428 in invasive human breast tumors. (E) Kinase reactions in vitro with precipitated Strep-PKN3 and GFP fusion p130Cas variants (shown as Cas). Reactions were carried out in the presence of ATP?S followed by alkylation with PNBM and detection with specific antithiophosphate esters (thioP ester) antibody. Combinations of PNBM (alkylation reagent) or ATP?S were facilitated to exclude false-positive signals. Antibodies anti-StrepII and GFP were used to detect PKN3 kinase or GFP-fused p130Cas variants, respectively. Representative blots are shown. (F) Lysates from p130Cas-/- MEFs re-expressing GFP-fused p130Cas variants with or without mCherry-PKN3 overexpression were run on SDS/PAGE using an acrylamide/bisacrylamide ratio of 30 : 0.2 followed by immunoblotting and detection by anti-p130Cas antibody. a-d refers to different GFP-p130Cas isoforms.

Fig 4: Src phosphorylation of CAS on Tyr12 inhibits CAS binding to FAK and Vinculin. Ten structures from molecular dynamics of phosphoTyr12 CAS SH3 with (A) PTP-PEST peptide (cyan) and (B) Vinculin peptide (green) are shown. These are compared with 40 NMR structures of unphosphorylated CAS SH3 complexes. (C) Co-precipitation analysis of the effect of Src activity on CAS binding capacity. CAS was immunoprecipitated from MEFs and from Src-transformed (SrcF) MEFs (in presence or absence of 5 µM saracatinib), and the amount of co-precipitated FAK and vinculin was analyzed. Numbers indicate the fold-change in ratio of FAK or Vinculin coimmunoprecipitated with CAS. (D) Immunoblot analysis of the effect of Src activity on CAS phosphorylation. (C,D) Blots were cropped from original full-size images (see Supplemental Material). (E) Quantification of phage-displayed peptides (clone 10 and clone 16) binding to CAS SH3 variants (WT, Y12F, Y12E) fused with GST or GST alone. The relative affinity represents the binding ratio relative to CAS-WT. The data are expressed as average ± standard deviation from three independent experiments. Statistical significance (*p < 0.05, ***p < 0.001) was determined on log-transformed data by one-way repeated-measures ANOVA followed by Tukey’s post-hoc test. (F) Representative curves of phage-displayed peptides (clone 10 and clone 16) binding to CAS SH3 variants (WT, Y12F, Y12E) fused to GST or GST. GST alone with phage clone 16 was used as a negative control.

Fig 5: PKN3 regulates cell migration in 3D environment of SrcF-transformed MEFs and tumor growth in vivo through interaction with p130Cas. (A) Representative tracking maps of SC or SCpkn3-/- cells stably expressing mCherry-PKN3 WT, mPR, or shRNA anti-PKN3 in Dox-dependent manner as indicated are shown (scale bar 100 µm). Cells migrated into collagen with or without Dox supplementation. (B) Quantification of cell velocity difference between induced and noninduced controls. Statistical significance was evaluated from 3 to 5 independent experiments by one-way ANOVA on ranks followed by Turkey post hoc test. (C) Gelatin degradation (Cy3-labeled) of SCpkn3-/- cells expressing Dox-inducible mCherry-PKN3. Representative images are shown (on the left). Scale bar represents 100 µm. Quantification calculated as % degraded area related to total cell area stained by phalloidin 488 (on the right). (D) Association of PKN3 with Src and Actin in p130Cas-dependent manner is shown by co-immunoprecipitations. SrcF-transformed cells with or without p130Cas were transfected by Strep-PKN3, and PKN3 was precipitated by Strep-Tactin® Superflow® resin followed by western blotting and detection by anti-StrepII antibody. Co-immunoprecipitated p130Cas, Src, and Actin were detected using appropriate antibodies (anti-p130Cas, anti-Src, anti-Actin). (E) Eight representative tumors of SC or SCpkn3-/- cells with Dox-induced mCherry, mCherry-PKN3, or mCherry-PKN3 mPR are shown with the same scale (units in cm). (F) Their respective quantification expressed as tumor mass weight at defined timescale as indicated. Statistics were evaluated by ANOVA on Ranks Dunn's Method. TL, total cell lysate; IP, immunoprecipitation; Ctrl, control sample prepared from SC cells.