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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 enhances N-WASP–induced microspike formation in vivo. (A) Microspike formation in Cos7 cells expressing N-WASP or Myc-WISH. Transfected cells were treated with or without EGF for 5 min, and then fixed, immunostained with anti–N-WASP (N-WASP) or anti-Myc (WISH) antibodies, and stained with rhodamine-phalloidin (actin filaments). (B) Microspike formation in Cos7 cells expressing N-WASP and Myc-WISH. Transfected cells were treated with or without EGF for 5 min, and then fixed, immunostained with anti–N-WASP (N-WASP) and anti-Myc (WISH) antibodies, and stained with rhodamine-phalloidin (actin filaments). (C) Activation of N-WASP by WISH is independent of Cdc42. Cos7 cells expressing the N-WASP mutant H208D only or together with Myc-WISH were observed for microspike formation. Cells were incubated with or without EGF for 5 min, and then fixed, immunostained with anti–N-WASP (H208D) and anti-Myc (WISH) antibodies, and stained with rhodamine-phalloidin (actin filaments). (D) Real-time observation of microspike formation. Cos7 cells were microinjected with purified N-WASP and/or GST-WISH proteins. After microinjection, cells were observed with a phase–contrast microscope. Membrane protrusions (filopodia) are indicated with arrows. (E) Quantification of microspike formation. Transfected cells were serum-starved and then stimulated with or without EGF for 5 min. The percentage of cells forming microspikes among transfected cells was calculated. Error bars represent the SD of three different measurements. At least 50 cells were counted in each determination.
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Figure 7: WISH enhances N-WASP–induced microspike formation in vivo. (A) Microspike formation in Cos7 cells expressing N-WASP or Myc-WISH. Transfected cells were treated with or without EGF for 5 min, and then fixed, immunostained with anti–N-WASP (N-WASP) or anti-Myc (WISH) antibodies, and stained with rhodamine-phalloidin (actin filaments). (B) Microspike formation in Cos7 cells expressing N-WASP and Myc-WISH. Transfected cells were treated with or without EGF for 5 min, and then fixed, immunostained with anti–N-WASP (N-WASP) and anti-Myc (WISH) antibodies, and stained with rhodamine-phalloidin (actin filaments). (C) Activation of N-WASP by WISH is independent of Cdc42. Cos7 cells expressing the N-WASP mutant H208D only or together with Myc-WISH were observed for microspike formation. Cells were incubated with or without EGF for 5 min, and then fixed, immunostained with anti–N-WASP (H208D) and anti-Myc (WISH) antibodies, and stained with rhodamine-phalloidin (actin filaments). (D) Real-time observation of microspike formation. Cos7 cells were microinjected with purified N-WASP and/or GST-WISH proteins. After microinjection, cells were observed with a phase–contrast microscope. Membrane protrusions (filopodia) are indicated with arrows. (E) Quantification of microspike formation. Transfected cells were serum-starved and then stimulated with or without EGF for 5 min. The percentage of cells forming microspikes among transfected cells was calculated. Error bars represent the SD of three different measurements. At least 50 cells were counted in each determination.

Mentions: To examine whether WISH stimulates N-WASP–induced microspike formation, WISH and N-WASP were coexpressed in Cos7 cells. Expression of WISH or N-WASP alone did not cause any morphological changes in Cos7 cells (Fig. 7 A). In response to EGF stimulation, however, N-WASP–expressing cells formed microspikes, whereas WISH-expressing cells did not. When WISH and N-WASP were coexpressed, microspike formation was induced even in the absence of EGF (Fig. 7 B), though the structure of actin filaments was also altered. To clarify whether Cdc42 is necessary for N-WASP–induced microspike formation when WISH is present, we used an N-WASP mutant (H208D) that cannot bind Cdc42 (Miki et al. 1998a). H208D alone was not able to induce microspike formation even in the presence of EGF (Fig. 7 C, top panels). However, coexpression of H208D and WISH did induce microspike formation (Fig. 7 C, bottom panels). Real-time observation by phase–contrast microscopy revealed that these microspikes were filopodia but not retraction fibers (Fig. 7 D). The numbers of cells that formed microspikes were also increased by coexpression of WISH and N-WASP or WISH and H208D (Fig. 7 E). All these results indicate that WISH alone has an ability to activate N-WASP to induce microspike formation without the aid of Cdc42.


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 enhances N-WASP–induced microspike formation in vivo. (A) Microspike formation in Cos7 cells expressing N-WASP or Myc-WISH. Transfected cells were treated with or without EGF for 5 min, and then fixed, immunostained with anti–N-WASP (N-WASP) or anti-Myc (WISH) antibodies, and stained with rhodamine-phalloidin (actin filaments). (B) Microspike formation in Cos7 cells expressing N-WASP and Myc-WISH. Transfected cells were treated with or without EGF for 5 min, and then fixed, immunostained with anti–N-WASP (N-WASP) and anti-Myc (WISH) antibodies, and stained with rhodamine-phalloidin (actin filaments). (C) Activation of N-WASP by WISH is independent of Cdc42. Cos7 cells expressing the N-WASP mutant H208D only or together with Myc-WISH were observed for microspike formation. Cells were incubated with or without EGF for 5 min, and then fixed, immunostained with anti–N-WASP (H208D) and anti-Myc (WISH) antibodies, and stained with rhodamine-phalloidin (actin filaments). (D) Real-time observation of microspike formation. Cos7 cells were microinjected with purified N-WASP and/or GST-WISH proteins. After microinjection, cells were observed with a phase–contrast microscope. Membrane protrusions (filopodia) are indicated with arrows. (E) Quantification of microspike formation. Transfected cells were serum-starved and then stimulated with or without EGF for 5 min. The percentage of cells forming microspikes among transfected cells was calculated. Error bars represent the SD of three different measurements. At least 50 cells were counted in each determination.
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Related In: Results  -  Collection

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Figure 7: WISH enhances N-WASP–induced microspike formation in vivo. (A) Microspike formation in Cos7 cells expressing N-WASP or Myc-WISH. Transfected cells were treated with or without EGF for 5 min, and then fixed, immunostained with anti–N-WASP (N-WASP) or anti-Myc (WISH) antibodies, and stained with rhodamine-phalloidin (actin filaments). (B) Microspike formation in Cos7 cells expressing N-WASP and Myc-WISH. Transfected cells were treated with or without EGF for 5 min, and then fixed, immunostained with anti–N-WASP (N-WASP) and anti-Myc (WISH) antibodies, and stained with rhodamine-phalloidin (actin filaments). (C) Activation of N-WASP by WISH is independent of Cdc42. Cos7 cells expressing the N-WASP mutant H208D only or together with Myc-WISH were observed for microspike formation. Cells were incubated with or without EGF for 5 min, and then fixed, immunostained with anti–N-WASP (H208D) and anti-Myc (WISH) antibodies, and stained with rhodamine-phalloidin (actin filaments). (D) Real-time observation of microspike formation. Cos7 cells were microinjected with purified N-WASP and/or GST-WISH proteins. After microinjection, cells were observed with a phase–contrast microscope. Membrane protrusions (filopodia) are indicated with arrows. (E) Quantification of microspike formation. Transfected cells were serum-starved and then stimulated with or without EGF for 5 min. The percentage of cells forming microspikes among transfected cells was calculated. Error bars represent the SD of three different measurements. At least 50 cells were counted in each determination.
Mentions: To examine whether WISH stimulates N-WASP–induced microspike formation, WISH and N-WASP were coexpressed in Cos7 cells. Expression of WISH or N-WASP alone did not cause any morphological changes in Cos7 cells (Fig. 7 A). In response to EGF stimulation, however, N-WASP–expressing cells formed microspikes, whereas WISH-expressing cells did not. When WISH and N-WASP were coexpressed, microspike formation was induced even in the absence of EGF (Fig. 7 B), though the structure of actin filaments was also altered. To clarify whether Cdc42 is necessary for N-WASP–induced microspike formation when WISH is present, we used an N-WASP mutant (H208D) that cannot bind Cdc42 (Miki et al. 1998a). H208D alone was not able to induce microspike formation even in the presence of EGF (Fig. 7 C, top panels). However, coexpression of H208D and WISH did induce microspike formation (Fig. 7 C, bottom panels). Real-time observation by phase–contrast microscopy revealed that these microspikes were filopodia but not retraction fibers (Fig. 7 D). The numbers of cells that formed microspikes were also increased by coexpression of WISH and N-WASP or WISH and H208D (Fig. 7 E). All these results indicate that WISH alone has an ability to activate N-WASP to induce microspike formation without the aid of Cdc42.

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