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

Cortesio CL, Perrin BJ, Bennin DA, Huttenlocher A - Mol. Biol. Cell (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706, USA.

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.

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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.
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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.

Mentions: Previous studies have demonstrated that calpain-mediated proteolysis of cortactin regulates the dynamics of actin-based structures including lamellipodia formation (Perrin et al., 2006) and invadopodia dynamics (Cortesio et al., 2008). Because cortactin and mAbp1 are structurally similar, we hypothesized that mAbp1 function may be similarly regulated by calpain-mediated proteolysis. mAbp1 proteolysis was observed in control NIH-3T3 cells with the generation of a fragment of ∼30 kDa. Calpain 2–deficient NIH-3T3 cells did not show significant mAbp1 proteolysis, suggesting that calpain 2 also cleaves mAbp1 (Figure 4A). To confirm calpain 2 proteolysis of mAbp1, we incubated purified mAbp1-GST with increasing concentrations of calpain 2. Consistent with the results in cultured fibroblasts, calpain 2 treatment in vitro generated a prominent cleavage fragment of a similar molecular weight (Figure 4B). Because calpain cleavage sites are not defined by a consensus primary amino acid sequence (Tompa et al., 2004), we mapped the calpain cleavage site in mAbp1, using N-terminal sequencing. Sequencing revealed two fragments that were generated by calpain 2 proteolysis at two separate sites within a nine-amino acid region that is located between the charged-helical F-actin–binding domain and the proline-rich domain (Figure 4C and Supplemental Figure 1A).


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

Cortesio CL, Perrin BJ, Bennin DA, Huttenlocher A - Mol. Biol. Cell (2009)

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.
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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.
Mentions: Previous studies have demonstrated that calpain-mediated proteolysis of cortactin regulates the dynamics of actin-based structures including lamellipodia formation (Perrin et al., 2006) and invadopodia dynamics (Cortesio et al., 2008). Because cortactin and mAbp1 are structurally similar, we hypothesized that mAbp1 function may be similarly regulated by calpain-mediated proteolysis. mAbp1 proteolysis was observed in control NIH-3T3 cells with the generation of a fragment of ∼30 kDa. Calpain 2–deficient NIH-3T3 cells did not show significant mAbp1 proteolysis, suggesting that calpain 2 also cleaves mAbp1 (Figure 4A). To confirm calpain 2 proteolysis of mAbp1, we incubated purified mAbp1-GST with increasing concentrations of calpain 2. Consistent with the results in cultured fibroblasts, calpain 2 treatment in vitro generated a prominent cleavage fragment of a similar molecular weight (Figure 4B). Because calpain cleavage sites are not defined by a consensus primary amino acid sequence (Tompa et al., 2004), we mapped the calpain cleavage site in mAbp1, using N-terminal sequencing. Sequencing revealed two fragments that were generated by calpain 2 proteolysis at two separate sites within a nine-amino acid region that is located between the charged-helical F-actin–binding domain and the proline-rich domain (Figure 4C and Supplemental Figure 1A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706, USA.

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.

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