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A novel neural Wiskott-Aldrich syndrome protein (N-WASP) binding protein, WISH, induces Arp2/3 complex activation independent of Cdc42.

Fukuoka M, Suetsugu S, Miki H, Fukami K, Endo T, Takenawa T - J. Cell Biol. (2001)

Bottom Line: WISH strongly enhanced N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization.Addition of WISH to extracts increased actin polymerization as Cdc42 did.These findings suggest that WISH activates Arp2/3 complex through N-WASP-dependent and -independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.

ABSTRACT
We identified a novel adaptor protein that contains a Src homology (SH)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif and termed this protein WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It bound Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhanced N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induced marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induced microspike formation when coexpressed with WISH. We also examined the contribution of WISH to a rapid actin polymerization induced by brain extract in vitro. Arp2/3 complex was essential for brain extract-induced rapid actin polymerization. Addition of WISH to extracts increased actin polymerization as Cdc42 did. However, WISH unexpectedly could activate actin polymerization even in N-WASP-depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP-dependent and -independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.

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WISH stimulates N-WASP–induced Arp2/3 complex activation. (A) Proteins used in actin polymerization assays. Purified proteins were subjected to SDS-PAGE and stained with Coomassie blue. (B) Effect of WISH on N-WASP–dependent Arp2/3 complex–induced actin polymerization. The pyrene-labeled actin assay was used to monitor the polymerization of 2.2 μM G-actin (2 μM unlabeled actin + 0.2 μM pyrene-labeled actin) in the presence of 60 nM Arp2/3 complex alone (actin+Arp2/3), Arp2/3 complex + 0.2 μM GST-VCA (VCA), Arp2/3 complex + 0.2 μM N-WASP (NW), Arp2/3 complex + N-WASP + 0.5 μM Cdc42 (NW+Cdc42), Arp2/3 complex + 0.2 μM WISH (WISH), Arp2/3 complex + N-WASP + WISH (NW+WISH), or Arp2/3 complex + N-WASP + WISH + Cdc42 (NW+WISH+Cdc42). Experiments were repeated three times. Results shown are means of triplicate measurements. (C) Effect of various SH3 domains on N-WASP–induced Arp2/3 complex activation. The pyrene-labeled actin assay was carried out with 0.2 μM WISH, Ash/Grb2 (Ash), myelin basic protein (MBP), or SH3s of WISH (SH3), Nck, Ash/Grb2 NH2 terminus (AshN), PI 3-kinase 85-kD subunit (p85), PLCγ1 (PLCγ), or Fyn in the presence of 60 nM Arp2/3 complex + 0.2 μM N-WASP (NW). Results shown are means of triplicate measurements. (D) Concentration-dependent effect on initial actin polymerization rate. Effects of Cdc42, WISH, WISH SH3 (SH3), Ash/Grb2 (Ash), Ash/Grb2 NH2-terminal SH3 (AshN), and Fyn SH3 (Fyn) in the presence of 60 nM Arp2/3 complex + 0.2 μM N-WASP (NW). Initial rates of actin polymerization were calculated by differentiating the plot curves. Error bars represent the standard deviation of three different experiments.
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Figure 5: WISH stimulates N-WASP–induced Arp2/3 complex activation. (A) Proteins used in actin polymerization assays. Purified proteins were subjected to SDS-PAGE and stained with Coomassie blue. (B) Effect of WISH on N-WASP–dependent Arp2/3 complex–induced actin polymerization. The pyrene-labeled actin assay was used to monitor the polymerization of 2.2 μM G-actin (2 μM unlabeled actin + 0.2 μM pyrene-labeled actin) in the presence of 60 nM Arp2/3 complex alone (actin+Arp2/3), Arp2/3 complex + 0.2 μM GST-VCA (VCA), Arp2/3 complex + 0.2 μM N-WASP (NW), Arp2/3 complex + N-WASP + 0.5 μM Cdc42 (NW+Cdc42), Arp2/3 complex + 0.2 μM WISH (WISH), Arp2/3 complex + N-WASP + WISH (NW+WISH), or Arp2/3 complex + N-WASP + WISH + Cdc42 (NW+WISH+Cdc42). Experiments were repeated three times. Results shown are means of triplicate measurements. (C) Effect of various SH3 domains on N-WASP–induced Arp2/3 complex activation. The pyrene-labeled actin assay was carried out with 0.2 μM WISH, Ash/Grb2 (Ash), myelin basic protein (MBP), or SH3s of WISH (SH3), Nck, Ash/Grb2 NH2 terminus (AshN), PI 3-kinase 85-kD subunit (p85), PLCγ1 (PLCγ), or Fyn in the presence of 60 nM Arp2/3 complex + 0.2 μM N-WASP (NW). Results shown are means of triplicate measurements. (D) Concentration-dependent effect on initial actin polymerization rate. Effects of Cdc42, WISH, WISH SH3 (SH3), Ash/Grb2 (Ash), Ash/Grb2 NH2-terminal SH3 (AshN), and Fyn SH3 (Fyn) in the presence of 60 nM Arp2/3 complex + 0.2 μM N-WASP (NW). Initial rates of actin polymerization were calculated by differentiating the plot curves. Error bars represent the standard deviation of three different experiments.

Mentions: Because WISH bound specifically to N-WASP, we examined whether WISH could further stimulate N-WASP–induced Arp2/3 complex activation in vitro. Actin polymerization can be monitored with pyrene-labeled actin, a fluorescent derivative of actin that exhibits higher fluorescence intensity when actin is assembled into filaments. Using a cell-free system, we measured the effect of N-WASP and WISH on Arp2/3 complex–induced actin polymerization. All proteins used in this experiment are shown in Fig. 5 A. The GST-VCA fusion protein of N-WASP (VCA) markedly activated Arp2/3 complex–induced actin polymerization (Fig. 5 B) as described previously (Rohatgi et al.., 1999). In contrast, full-length N-WASP was not as potent as VCA. Addition of GST-Cdc42 fusion protein (Cdc42) to N-WASP in this system increased Arp2/3 complex–induced actin polymerization to the level evoked by VCA in the presence of PIP2 (data not shown), but addition of Cdc42 without PIP2 did not increase activity to that of VCA (Fig. 5 B). Surprisingly, addition of GST-WISH fusion protein (WISH) to N-WASP increased the Arp2/3 complex–induced actin polymerization to the level induced by VCA even in the absence of Cdc42. Addition of Cdc42 did not increase activity any further (Fig. 5 B). WISH did not activate Arp2/3 complex directly without N-WASP (Fig. 5 B).


A novel neural Wiskott-Aldrich syndrome protein (N-WASP) binding protein, WISH, induces Arp2/3 complex activation independent of Cdc42.

Fukuoka M, Suetsugu S, Miki H, Fukami K, Endo T, Takenawa T - J. Cell Biol. (2001)

WISH stimulates N-WASP–induced Arp2/3 complex activation. (A) Proteins used in actin polymerization assays. Purified proteins were subjected to SDS-PAGE and stained with Coomassie blue. (B) Effect of WISH on N-WASP–dependent Arp2/3 complex–induced actin polymerization. The pyrene-labeled actin assay was used to monitor the polymerization of 2.2 μM G-actin (2 μM unlabeled actin + 0.2 μM pyrene-labeled actin) in the presence of 60 nM Arp2/3 complex alone (actin+Arp2/3), Arp2/3 complex + 0.2 μM GST-VCA (VCA), Arp2/3 complex + 0.2 μM N-WASP (NW), Arp2/3 complex + N-WASP + 0.5 μM Cdc42 (NW+Cdc42), Arp2/3 complex + 0.2 μM WISH (WISH), Arp2/3 complex + N-WASP + WISH (NW+WISH), or Arp2/3 complex + N-WASP + WISH + Cdc42 (NW+WISH+Cdc42). Experiments were repeated three times. Results shown are means of triplicate measurements. (C) Effect of various SH3 domains on N-WASP–induced Arp2/3 complex activation. The pyrene-labeled actin assay was carried out with 0.2 μM WISH, Ash/Grb2 (Ash), myelin basic protein (MBP), or SH3s of WISH (SH3), Nck, Ash/Grb2 NH2 terminus (AshN), PI 3-kinase 85-kD subunit (p85), PLCγ1 (PLCγ), or Fyn in the presence of 60 nM Arp2/3 complex + 0.2 μM N-WASP (NW). Results shown are means of triplicate measurements. (D) Concentration-dependent effect on initial actin polymerization rate. Effects of Cdc42, WISH, WISH SH3 (SH3), Ash/Grb2 (Ash), Ash/Grb2 NH2-terminal SH3 (AshN), and Fyn SH3 (Fyn) in the presence of 60 nM Arp2/3 complex + 0.2 μM N-WASP (NW). Initial rates of actin polymerization were calculated by differentiating the plot curves. Error bars represent the standard deviation of three different experiments.
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Figure 5: WISH stimulates N-WASP–induced Arp2/3 complex activation. (A) Proteins used in actin polymerization assays. Purified proteins were subjected to SDS-PAGE and stained with Coomassie blue. (B) Effect of WISH on N-WASP–dependent Arp2/3 complex–induced actin polymerization. The pyrene-labeled actin assay was used to monitor the polymerization of 2.2 μM G-actin (2 μM unlabeled actin + 0.2 μM pyrene-labeled actin) in the presence of 60 nM Arp2/3 complex alone (actin+Arp2/3), Arp2/3 complex + 0.2 μM GST-VCA (VCA), Arp2/3 complex + 0.2 μM N-WASP (NW), Arp2/3 complex + N-WASP + 0.5 μM Cdc42 (NW+Cdc42), Arp2/3 complex + 0.2 μM WISH (WISH), Arp2/3 complex + N-WASP + WISH (NW+WISH), or Arp2/3 complex + N-WASP + WISH + Cdc42 (NW+WISH+Cdc42). Experiments were repeated three times. Results shown are means of triplicate measurements. (C) Effect of various SH3 domains on N-WASP–induced Arp2/3 complex activation. The pyrene-labeled actin assay was carried out with 0.2 μM WISH, Ash/Grb2 (Ash), myelin basic protein (MBP), or SH3s of WISH (SH3), Nck, Ash/Grb2 NH2 terminus (AshN), PI 3-kinase 85-kD subunit (p85), PLCγ1 (PLCγ), or Fyn in the presence of 60 nM Arp2/3 complex + 0.2 μM N-WASP (NW). Results shown are means of triplicate measurements. (D) Concentration-dependent effect on initial actin polymerization rate. Effects of Cdc42, WISH, WISH SH3 (SH3), Ash/Grb2 (Ash), Ash/Grb2 NH2-terminal SH3 (AshN), and Fyn SH3 (Fyn) in the presence of 60 nM Arp2/3 complex + 0.2 μM N-WASP (NW). Initial rates of actin polymerization were calculated by differentiating the plot curves. Error bars represent the standard deviation of three different experiments.
Mentions: Because WISH bound specifically to N-WASP, we examined whether WISH could further stimulate N-WASP–induced Arp2/3 complex activation in vitro. Actin polymerization can be monitored with pyrene-labeled actin, a fluorescent derivative of actin that exhibits higher fluorescence intensity when actin is assembled into filaments. Using a cell-free system, we measured the effect of N-WASP and WISH on Arp2/3 complex–induced actin polymerization. All proteins used in this experiment are shown in Fig. 5 A. The GST-VCA fusion protein of N-WASP (VCA) markedly activated Arp2/3 complex–induced actin polymerization (Fig. 5 B) as described previously (Rohatgi et al.., 1999). In contrast, full-length N-WASP was not as potent as VCA. Addition of GST-Cdc42 fusion protein (Cdc42) to N-WASP in this system increased Arp2/3 complex–induced actin polymerization to the level evoked by VCA in the presence of PIP2 (data not shown), but addition of Cdc42 without PIP2 did not increase activity to that of VCA (Fig. 5 B). Surprisingly, addition of GST-WISH fusion protein (WISH) to N-WASP increased the Arp2/3 complex–induced actin polymerization to the level induced by VCA even in the absence of Cdc42. Addition of Cdc42 did not increase activity any further (Fig. 5 B). WISH did not activate Arp2/3 complex directly without N-WASP (Fig. 5 B).

Bottom Line: WISH strongly enhanced N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization.Addition of WISH to extracts increased actin polymerization as Cdc42 did.These findings suggest that WISH activates Arp2/3 complex through N-WASP-dependent and -independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.

ABSTRACT
We identified a novel adaptor protein that contains a Src homology (SH)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif and termed this protein WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It bound Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhanced N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induced marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induced microspike formation when coexpressed with WISH. We also examined the contribution of WISH to a rapid actin polymerization induced by brain extract in vitro. Arp2/3 complex was essential for brain extract-induced rapid actin polymerization. Addition of WISH to extracts increased actin polymerization as Cdc42 did. However, WISH unexpectedly could activate actin polymerization even in N-WASP-depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP-dependent and -independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.

Show MeSH
Related in: MedlinePlus