Figure 4. The endocytic F-BAR protein CIP4 directly associates with paxillin and competes with vinculin for paxillin binding.

(A) Paxillin preferentially binds endocytic factors in neurons grown on soft substrates. Paxillin-associated complexes were immunoprecipitated (IP) in lysates made from E17.5 rat brain or from cortical neuronal cultures grown on different substrates using a specific paxillin antibody and were then detected by western blot analysis. Normal rabbit IgG (‘IgG’) served as a negative control. Histograms show the opposing binding preference of paxillin toward CIP4/CHC or vinculin when grown on soft (0.1 kPa or 1 kPa) versus rigid (20 kPa or glass) substrates. Data represent mean intensity ±SEM (n = 3 independent experiments; *p<0.05; t-test). (B) Western blot showing direct interaction of paxillin with CIP4. Bacterially expressed His-CIP4 was purified by fast protein liquid chromatography and subjected to a GST pull-down assay using GST-PXN^FL, GST-PXN^∆LIM3-4 or GST alone. Precipitants were analyzed by western blotting with antibodies specific to CIP4. Histograms summarize protein levels as determined by immunoblotting of full-length (His-CIP4) or F-BAR domain-deleted (His-ΔF-BAR) CIP4 pulled-down by GST-paxillin variants (±SEM, n = 3; normalized to the corresponding GST-PXN^FL or GST-PXN^∆LIM3-4 inputs; **p<0.01, t test). (C and D). Mapping of paxillin domains interacting with CIP4 or vinculin. (C) GST pull-down and immunoblotting of vinculin, myc-CIP4, and CHC in lysates of myc-CIP4-expressing HEK293T cells. Histograms reflect quantification of levels of proteins pulled-down by GST fusions of full-length (“FL“) or LIM domain- and/or LD motif-deleted forms of paxillin, all from experiments similar to those shown in top panels (±SEM, n ≥ 3 independent experiments; *p<0.05; **p<0.01; ***p<0.001; t test). (D) Schematic of GST fusion proteins used in c. Table summarizing relative CIP4 or vinculin (‘Vin’) binding by paxillin deletion mutants or full-length protein. Solid lines mark primary sites of interaction, and dashed lines mark accessory interaction motifs for strong binding to vinculin or CIP4. Binding strength relative to full-length paxillin indicated as: ‘++++' >75% > ‘+++' >50% > ‘++' >25% > '+' >5% > '+/-'. (E) In vitro protein interaction and competitive binding assays in HEK293T cells transfected with various amounts (1, 6, and 12 µg) of plasmids encoding myc-tagged CIP4 protein (myc-CIP4) and/or control vectors, as indicated. Cell lysates were subjected to a GST pull-down assay with GST-PXN^FL or GST alone, and immunoblotted with vinculin and myc antibodies. Line chart depicts averaged protein levels as determined by immunoblotting of CIP4 or vinculin pulled-down by GST-PXN^FL (±SEM, n = 4; normalized to band intensity of corresponding GST-paxillin variant). (F) In vivo protein interaction and competitive binding assays in HEK293T cells transfected with various amounts (7.5 μg and 15 μg) of plasmids encoding the F-BAR domain (‘F-BAR’) alone or F-BAR-domain-deleted (‘ΔN’-F-BAR’) CIP4 and/or control vectors, as indicated. Cell lysates were immunoprecipitated by paxillin antibodies and blotted with myc or vinculin antibodies. Histograms show relative protein levels as determined by immunoblotting of vinculin co-immunoprecipitated by paxillin antibodies (±SEM, n = 3; *p<0.05, t test).

Figure 4—figure supplement 1. Paxillin associates with the F-BAR domain of CIP4.

(A) Structures of the vinculin tail (PDB ID: 1QKR; top panel) and the CIP4 F-BAR domain (PDB ID: 2EFK; bottom panel) constructed by JMOL, version 14.4.4. Yellow marks indicate the predicted paxillin-binding subdomain (PBS) for vinculin (951K to 970Q) or CIP4 (35R to 55P). Secondary structures were predicted by the DSSP or STRIDE databases. (B) Mapping of paxillin domains interacting with CIP4 or vinculin. GST pull-down and immunoblotting of vinculin and myc-CIP4 in lysates of myc-CIP4-expressing HEK293T cells. Histograms reflect quantification of levels of proteins pulled-down by GST fusions of full-length ('FL') or LD motif-deleted forms of paxillin, all from experiments similar to those shown in top panels (±SEM, n ≥ 3 independent experiments; **p<0.01; ***p<0.001; multiple t test). Schematic of GST fusion proteins and table summarizing relative CIP4 or vinculin (‘Vin’) binding by paxillin deletion mutants or full-length protein shown in the top panel. Binding strength relative to full-length paxillin indicated as: ‘++++' >75% > ‘+++' >50% > ‘++' >25% > '+' >5% > '+/-'. Note that although CIP4 primarily associates with LIM domains, deletion of the paxillin LD1-3 domain reduced its affinity for CIP4. (C) Mapping of CIP4 domains interacting with paxillin. GST fusion proteins of full-length CIP4 (‘FL’) or its variants with F-BAR domain, HR1 and/or SH3 domain truncations were subjected to GST pull-down assays in HEK293T cell lysates, followed by immunoblotting for paxillin and actin. Histogram reflects quantitative measurement of relative protein levels (±SEM, n ≥ 3 independent experiments; compared to that of FL experiment; **p<0.01; t-test) pulled down by GST-CIP4, as indicated. (D) Schematic of GST fusion proteins used in B. Table summarizes relative paxillin binding by CIP4 deletion mutants or full-length protein. Binding strength relative to that of full-length CIP4 is represented as: ‘++++' >75% > ‘+++' >50% > ‘++' >25% > '+'. (E) In vivo protein interactions in HEK293T cells co-transfected with plasmids encoding paxillin-GFP and myc-tagged CIP4 deletion mutants or full-length protein, as indicated. Cell lysates were immunoprecipitated by myc antibody and blotted with paxillin or myc antibodies. Histograms show relative protein levels as determined by immunoblotting for paxillin co-immunoprecipitated by myc antibody. Data represents mean (±SEM from more than three independent experiments; compared to that of F-BAR experiment; *p<0.05; t-test). (F) In vitro protein interaction and competitive binding assays in HEK293T cells transfected with various amounts (1, 6, and 12 µg) of plasmids encoding myc-tagged CIP4 protein (myc-CIP4) and/or control vectors, as indicated. Cell lysates were subjected to a GST pull-down assay with GST-PXN^FL, GST-PXN^∆LD1, GST-PXN^∆LIM3-4 or GST alone, and immunoblotted with vinculin and myc antibodies. Line chart depicts averaged protein levels as determined by immunoblotting of CIP4 or vinculin pulled-down by GST-PXN variant (±SEM, n = 3–4; normalized to band intensity of corresponding GST-paxillin variant).