Actin-binding protein-1 interacts with WASp-interacting protein to regulate growth factor-induced dorsal ruffle formation

Abstract

Growth factor stimulation induces the formation of dynamic actin structures known as dorsal ruffles. Mammalian actin-binding protein-1 (mAbp1) is an actin-binding protein that has been implicated in regulating clathrin-mediated endocytosis; however, a role for mAbp1 in regulating the dynamics of growth factor-induced actin-based structures has not been defined. Here we show that mAbp1 localizes to dorsal ruffles and is necessary for platelet-derived growth factor (PDGF)-mediated dorsal ruffle formation. Despite their structural similarity, we find that mAbp1 and cortactin have nonredundant functions in the regulation of dorsal ruffle formation. mAbp1, like cortactin, is a calpain 2 substrate and the preferred cleavage site occurs between the actin-binding domain and the proline-rich region, generating a C-terminal mAbp1 fragment that inhibits dorsal ruffle formation. Furthermore, mAbp1 directly interacts with the actin regulatory protein WASp-interacting protein (WIP) through its SH3 domain. Finally, we demonstrate that the interaction between mAbp1 and WIP is important in regulating dorsal ruffle formation and that WIP-mediated effects on dorsal ruffle formation require mAbp1. Taken together, these findings identify a novel role for mAbp1 in growth factor-induced dorsal ruffle formation through its interaction with WIP.

Figure 1.

mAbp1 localizes to PDGF-induced dorsal ruffles in fibroblast cells. NIH-3T3 cells were cultured on FN-coated coverslips, serum-starved, and stimulated with vehicle control or PDGF. Cells were fixed and stained with (A) rhodamine phalloidin and anti-mAbp1 antibody or (B) rhodamine phalloidin and anti-cortactin antibody. Bar, 10 μm.

Figure 2.

mAbp1 and cortactin are required for dorsal ruffle formation. (A) Representative Western blot of mAbp1 expression in NIH-3T3 cells stably expressing control or mAbp1 siRNA. Cell lysates were analyzed using an anti-mAbp1 antibody (top), anti-vinculin antibody as a loading control (middle), or anti-cortactin antibody (bottom). Western blot shown is representative of at least three independent experiments. Cells expressing control or mAbp1 siRNA were plated on FN-coated coverslips, serum-starved, and stimulated with PDGF. Cells were fixed and stained with rhodamine phalloidin and anti-cortactin antibody. Bar, 10 μm. Dorsal ruffles were quantified by counting the number of cells containing at least one dorsal ruffle after stimulation with PDGF. Greater than 100 cells were counted per condition, and each condition represents the average value from three independent experiments; error bars, SEM; *p < 0.01 (Student's t test), compared with Ctlsi. (B) Representative Western blot of cortactin expression in NIH-3T3 cells stably expressing control or cortactin siRNA. Cell lysates were analyzed using an anti-cortactin antibody (top), anti-vinculin antibody as a loading control (middle), or anti-mAbp1 antibody (bottom). Western blot shown is representative of at least three independent experiments. Cells expressing control or cortactin siRNA were plated on FN-coated coverslips, serum-starved, and stimulated with PDGF. Cells were fixed and stained with rhodamine phalloidin and anti-mAbp1 antibody. Dorsal ruffles were quantified by counting the number of cells containing at least one dorsal ruffle after stimulation with PDGF. Greater than 100 cells were counted per condition and each condition represents the average value from three independent experiments; error bars, SEM; *p < 0.01 (Student's t test), compared with Ctlsi.

Figure 3.

mAbp1 and cortactin have nonredundant functions in dorsal ruffle formation. NIH-3T3 cells stably expressing control, mAbp1, or cortactin siRNA were transiently transfected with GFP, GFP-mAbp1, or GFP-cortactin. Cells were plated on FN-coated coverslips, serum-starved, and stimulated with PDGF. Cells were fixed and stained with rhodamine phalloidin. Bar, 10 μm. Dorsal ruffles were quantified by counting the number of GFP-positive cells containing at least one dorsal ruffle after stimulation with PDGF. Greater than 50 cells were counted per condition, and each condition represents the average value from three independent experiments; error bars, SEM; *p < 0.01, compared with Ctlsi GFP, **p < 0.001 compared with Abpsi GFP, and ***p < 0.001 compared with Cortsi GFP by one-way ANOVA.

Figure 4.

The calpain 2–mediated proteolytic fragment of mAbp1 negatively regulates dorsal ruffle formation. (A) Immunoblot analysis of endogenous mAbp1. Lysates from NIH-3T3 fibroblasts expressing control or calpain 2–specific siRNA were blotted and probed using an antibody against mouse mAbp1. The arrow indicates the calpain-dependent cleavage fragment. Western blot shown is representative of at least three independent experiments. (B) In vitro cleavage of mAbp1-GST. mAbp1-GST bound to glutathione-Sepharose beads was incubated with increasing concentrations of purified calpain 2. The reaction mixture was immunoblotted with anti-GST antibody. The arrow indicates the band that was analyzed by N-terminal sequencing. Western blot shown is representative of at least three independent experiments. (C) Schematic of mAbp1. Arrows indicate the sites of calpain proteolysis identified by N-terminal sequencing. N- and C-terminal mAbp1 constructs were made, corresponding to the calpain 2 cleavage fragments. (D) Parental NIH-3T3 cells were transiently transfected with GFP, GFP-mAbp1, GFP-N-terminal-mAbp1, or GFP-C-terminal-mAbp1. Cells were plated on FN-coated coverslips, serum-starved, and stimulated with PDGF. Cells were fixed and stained with rhodamine phalloidin and anti-cortactin antibody. Bar, 10 μm. Dorsal ruffles were quantified by counting the number of GFP-positive cells containing at least one dorsal ruffle after stimulation with PDGF. Greater than 50 cells were counted per condition, and each condition represents the average value from three independent experiments; error bars, SEM; *p < 0.01, compared with GFP control by one-way ANOVA.

Figure 5.

mAbp localizes with WIP at dorsal ruffles. (A) GFP-mAbp1 and mCherry-WIP were transiently transfected into NIH-3T3 cells. Cells were plated on FN-coated, glass-bottomed dishes and serum-starved. Cells were stimulated with PDGF and subsequently analyzed by dual, time-lapse fluorescence microscopy. Montages are representative of at least three independent experiments. Bar, 10 μm. See accompanying Video 5. (B) GFP-WIP was transiently transfected into NIH-3T3 cells. Cells were plated on FN-coated coverslips, serum-starved, and stimulated with PDGF. Cells were fixed and stained with rhodamine phalloidin and anti-cortactin antibody (top panel) or anti-mAbp1 and anti-cortactin antibodies (bottom panel). Bar, 10 μm. (C) NIH-3T3 cells were transiently transfected with GFP, GFP-C-terminal mAbp1, or GFP-C-terminal mAbp1 and mCherry WIP. Cells were plated on FN-coated coverslips, serum-starved, and stimulated with PDGF. Cells were fixed and stained with anti-cortactin antibody. A representative image of a GFP-C-terminal mAbp1 coexpressing mCherry-WIP is shown (left). Bar, 10 μm. Dorsal ruffles were quantified by counting the number of transfected cells containing at least one dorsal ruffle after stimulation with PDGF. Greater than 50 cells were counted per condition, and each condition represents the average value from three independent experiments; error bars, SEM; *p < 0.01 compared with GFP control by one-way ANOVA.

Figure 6.

mAbp1 interacts with WIP through its SH3 domain. (A) Schematic of mAbp1. Arrow indicates the mutation, W415K, introduced into the SH3 domain of mAbp1. (B) HEK-293 cells were transfected with GFP, GFP-mAbp1, or GFP-mAbp1 containing a W415K mutation in the SH3 domain, in combination with Flag-WIP. Protein was immunoprecipitated with anti-GFP antibody, separated by SDS-PAGE, and probed with anti-GFP antibody to detect mAbp1 or anti-Flag antibody to detect WIP. Western blot shown is representative of at least three independent experiments. (C) HEK-293 cells were transfected with GFP, GFP-mAbp1, or GFP-mAbp1 containing a W415K mutation in the SH3 domain. Protein was immunoprecipitated with anti-GFP antibody, separated by SDS-PAGE, and probed with anti-GFP antibody (top). The immobilized, immunoprecipitated proteins were incubated with a FLAG-WIP probe and blotted with anti-FLAG antibody (bottom). Western blot and overlay data shown are representative of at least three independent experiments. (D) NIH-3T3 cells were transfected with Flag-WIP or vector control. Transfected cells were serum-starved and treated with vehicle control or PDGF. Protein was immunoprecipitated with anti-Flag agarose, separated by SDS-PAGE, and probed with anti-Flag antibody to detect WIP, anti-mAbp1, and anti-N-WASP antibodies. Levels of mAbp1 or N-WASP that coimmunoprecipitated with Flag-WIP were quantified. Western blot and quantification shown are representative of four independent experiments. (E) NIH-3T3 cells were serum-starved and treated with vehicle control or PDGF. Protein was immunoprecipitated with anti-mAbp1 antibody, separated by SDS-PAGE and probed with anti-mAbp1 and anti-WIP antibodies. Western blot shown is representative of two independent experiments.

Figure 7.

The mAbp1 W415K mutant is unable to rescue dorsal ruffle formation in mAbp1 knockdown cells. (A) NIH-3T3 cells stably expressing control or mAbp1 siRNA were transiently transfected with GFP, WT GFP-mAbp1, or GFP-mAbp1-W415K and plated on FN-coated coverslips, serum-starved, and stimulated with PDGF. Cells were fixed and stained with anti-cortactin antibody. Bar, 10 μm. Dorsal ruffles of GFP-positive cells were quantified by counting the number of cells containing at least one dorsal ruffle after stimulation with PDGF. Greater than 50 cells were counted per condition, and each condition represents the average value from three independent experiments; error bars, SEM; *p < 0.01 compared with Ctlsi GFP by one-way ANOVA. (B) Parental NIH-3T3 cells were transiently transfected with GFP, GFP-C-terminal-mAbp1 or GFP-C-terminal-mAbp1-W415K. Cells were plated on FN-coated coverslips, serum-starved, and stimulated with PDGF. Cells were fixed and stained with rhodamine phalloidin. Bar, 10 μm. Dorsal ruffles were quantified by counting the number of GFP-positive cells containing at least one dorsal ruffle after stimulation with PDGF. Greater than 50 cells were counted per condition, and each condition represents the average value from three independent experiments; error bars, SEM; *p < 0.01, compared with GFP control by one-way ANOVA.

Figure 8.

A WIP mutant deficient in mAbp1 binding is unable to induce dorsal ruffle formation. (A) Schematic of wild-type WIP and a mutant WIP, Δ110-170, where the solid line represents a deletion of 60 amino acids in the proline-rich domain. (B). HEK-293 cells were transfected with GFP-WIP or GFP-WIP Δ110-170. Protein was immunoprecipitated with anti-GFP antibody, separated by SDS-PAGE, and probed with anti-GFP antibody (left). The immobilized, immunoprecipitated proteins were incubated with purified His-mAbp1-GST protein and blotted with anti-GST antibody (middle). Western blot and overlay data shown are representative of at least three independent experiments. The purified probe, His-mAbp1-GST (2 μg), was analyzed by Coomassie stain (right). (C) NIH-3T3 cells were transiently transfected with GFP, GFP-WIP, or GFP-WIP Δ110-170 and plated on FN-coated coverslips, serum-starved, and stimulated with PDGF. Cells were fixed and stained with rhodamine phalloidin and anti-cortactin antibody. Bar, 10 μm. Dorsal ruffles of GFP-positive cells were quantified by counting the number of cells containing at least one dorsal ruffle after stimulation with PDGF. Greater than 50 cells were counted per condition, and each condition represents the average value from three independent experiments; error bars, SEM; *p < 0.01 compared with GFP control by one-way ANOVA.

Figure 9.

WIP-mediated effects on dorsal ruffles require mAbp1. NIH-3T3 cells stably expressing control, mAbp1, or cortactin siRNA were transiently transfected with GFP or GFP-WIP. Cells were plated on FN-coated coverslips, serum-starved, and stimulated with PDGF. Cells were fixed and stained with rhodamine phalloidin. Bar, 10 μm. Dorsal ruffles were quantified by counting the number of GFP-positive cells containing at least one dorsal ruffle after stimulation with PDGF. Greater than 50 cells were counted per condition, and each condition represents the average value from three independent experiments; error bars, SEM; *p < 0.05, compared with Ctlsi GFP, **p < 0.01 compared with Cortsi GFP.