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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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Bovine brain extract–induced rapid actin polymerization in vitro. Aliquots of the bovine brain extracts were treated with anti-WISH antibody, anti–N-WASP antibody, or GST-CA protein to deplete WISH, N-WASP, and Arp2/3 complex, respectively. As a control, mock-treated extracts were used. Actin polymerization activity was measured using pyrene-labeled actin in a total volume of 100 μl. In all assay systems, 100 μM GTPγS was added. When WISH and Cdc42 proteins were added, 4.5 μg of WISH and the equal amounts of WISH SH3, Fyn SH3, or 2 μg of Cdc42 were used. Results shown are means of triplicate measurements. (A) Western blots of WISH-depleted (WISH dep.), N-WASP–depleted (NW dep.), and Arp2/3 complex–depleted (Arp2/3 dep.) extracts. Degree of depletion was checked by scanning Western blots with a densitometer as described in the text. (B) Mock treatment. Mock-treated extract (MOCK), mock-treated extracts + 0.2 μM WISH (MOCK + WISH full), mock-treated extracts + 0.2 μM WISH SH3 (MOCK + WISH SH3), and mock-treated extracts + 0.2 μM Fyn SH3 (MOCK + Fyn SH3). Ba, Effect of Cdc42, WISH, WISH SH3, and Fyn SH3 on actin polymerization was examined using mock-treated bovine brain extracts. Bb, Initial rates of actin polymerization. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*). (C) Arp2/3 complex is essential for a rapid actin polymerization. Mock-treated extract (MOCK), mock-treated extracts + 0.2 μM WISH (MOCK + WISH full), mock-treated extracts + 0.5 μM Cdc42 (MOCK + Cdc42), Arp2/3 complex–depleted extracts (Arp2/3 dep.), Arp2/3 complex–depleted extracts + 0.5 μM Cdc42 (Arp2/3 dep. + Cdc42), and Arp2/3 complex–depleted extracts + WISH (Arp dep. + WISH full). Ca, actin polymerization curves; Cb, initial rates of actin polymerization. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*) and Arp2/3 complex–depleted extracts (**). (D) WISH depletion experiments. Mock-treated extracts (MOCK), mock-treated extracts + 0.2 μM WISH (MOCK + WISH), WISH-depleted extracts (WISH dep.), and WISH-depleted extracts + 0.2 μM WISH addback (WISH dep. + WISH full). Da, actin polymerization curves in WISH-depleted extracts; Db, initial rates of actin polymerization in WISH-depleted extracts. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*) and WISH-depleted extracts (**). (E) WISH activates Arp2/3 complex in N-WASP–depleted extracts. Mock-treated extracts (MOCK), N-WASP–depleted extracts (NW dep.), N-WASP–depleted extracts + 0.2 μM WISH (NW dep. + WISH full), N-WASP–depleted extracts + 0.5 μM Cdc42 (NW dep. + Cdc42), N-WASP–depleted extracts + 0.2 μM WISH SH3 (NW dep. + WISH SH3), and N-WASP–depleted extracts + Ash/Grb2 (NW dep. + Ash/Grb2). Ea, actin polymerization curves in N-WASP–depleted extracts; Eb, initial rates of actin polymerization in N-WASP–depleted extracts. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*) and N-WASP–depleted extracts (**).
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Figure 6: Bovine brain extract–induced rapid actin polymerization in vitro. Aliquots of the bovine brain extracts were treated with anti-WISH antibody, anti–N-WASP antibody, or GST-CA protein to deplete WISH, N-WASP, and Arp2/3 complex, respectively. As a control, mock-treated extracts were used. Actin polymerization activity was measured using pyrene-labeled actin in a total volume of 100 μl. In all assay systems, 100 μM GTPγS was added. When WISH and Cdc42 proteins were added, 4.5 μg of WISH and the equal amounts of WISH SH3, Fyn SH3, or 2 μg of Cdc42 were used. Results shown are means of triplicate measurements. (A) Western blots of WISH-depleted (WISH dep.), N-WASP–depleted (NW dep.), and Arp2/3 complex–depleted (Arp2/3 dep.) extracts. Degree of depletion was checked by scanning Western blots with a densitometer as described in the text. (B) Mock treatment. Mock-treated extract (MOCK), mock-treated extracts + 0.2 μM WISH (MOCK + WISH full), mock-treated extracts + 0.2 μM WISH SH3 (MOCK + WISH SH3), and mock-treated extracts + 0.2 μM Fyn SH3 (MOCK + Fyn SH3). Ba, Effect of Cdc42, WISH, WISH SH3, and Fyn SH3 on actin polymerization was examined using mock-treated bovine brain extracts. Bb, Initial rates of actin polymerization. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*). (C) Arp2/3 complex is essential for a rapid actin polymerization. Mock-treated extract (MOCK), mock-treated extracts + 0.2 μM WISH (MOCK + WISH full), mock-treated extracts + 0.5 μM Cdc42 (MOCK + Cdc42), Arp2/3 complex–depleted extracts (Arp2/3 dep.), Arp2/3 complex–depleted extracts + 0.5 μM Cdc42 (Arp2/3 dep. + Cdc42), and Arp2/3 complex–depleted extracts + WISH (Arp dep. + WISH full). Ca, actin polymerization curves; Cb, initial rates of actin polymerization. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*) and Arp2/3 complex–depleted extracts (**). (D) WISH depletion experiments. Mock-treated extracts (MOCK), mock-treated extracts + 0.2 μM WISH (MOCK + WISH), WISH-depleted extracts (WISH dep.), and WISH-depleted extracts + 0.2 μM WISH addback (WISH dep. + WISH full). Da, actin polymerization curves in WISH-depleted extracts; Db, initial rates of actin polymerization in WISH-depleted extracts. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*) and WISH-depleted extracts (**). (E) WISH activates Arp2/3 complex in N-WASP–depleted extracts. Mock-treated extracts (MOCK), N-WASP–depleted extracts (NW dep.), N-WASP–depleted extracts + 0.2 μM WISH (NW dep. + WISH full), N-WASP–depleted extracts + 0.5 μM Cdc42 (NW dep. + Cdc42), N-WASP–depleted extracts + 0.2 μM WISH SH3 (NW dep. + WISH SH3), and N-WASP–depleted extracts + Ash/Grb2 (NW dep. + Ash/Grb2). Ea, actin polymerization curves in N-WASP–depleted extracts; Eb, initial rates of actin polymerization in N-WASP–depleted extracts. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*) and N-WASP–depleted extracts (**).

Mentions: Addition of Cdc42 to extracts of neutrophils (Zigmond et al. 1998) or Xenopus oocytes (Ma et al. 1998) induces an increase in actin polymerization. In addition, WASP-dependent actin comets have also been shown to be formed in bovine brain extracts (Yarar et al. 1999). Thus, we further applied bovine brain extracts instead of purified proteins to examine the involvement of WISH in actin polymerization under a condition that more closely reflects the in vivo situation. Addition of bovine brain extracts induced actin polymerization in the presence of Cdc42. To address whether WISH, N-WASP, or Arp2/3 complex contributes to actin polymerization activity in brain extracts, WISH, N-WASP, and Arp2/3 complex were depleted with anti-WISH antibody, anti–N-WASP antibody, and GST-CA protein treatment, respectively (Fig. 6 A). In WISH-depleted extracts, 90% of WISH was removed, whereas N-WASP levels remained constant (Fig. 6 A). Approximately 98% of N-WASP or 95% of Arp2/3 complex was removed in the respective depleted extract. These depleted extracts were used to examine the roles of Arp2/3 complex, N-WASP, and WISH in brain extract–induced actin polymerization. Addition of Cdc42 or WISH to mock-treated extracts increased initial actin polymerization markedly, but Fyn SH3 did not (Fig. 6 B, a). On the other hand, the maximal actin polymerization levels that were attained up to 100 min were similar for Mock, Mock + Cdc42, and Mock + WISH (data not shown), suggesting that WISH activates the initial rate of actin polymerization. As expected, Cdc42, WISH, and WISH-SH3 activated the initial actin polymerization rate (Fig. 6 B, b). Arp2/3 complex was essential for brain extract–induced actin polymerization, because addition of Cdc42 or WISH to Arp2/3 complex–depleted extracts did not restore activity (Fig. 6 C, a and b). Adding back of Arp2/3 complex did restore polymerization activity (data not shown). WISH depletion did not affect basal activity without Cdc42 (Fig. 6 D, a and b), but addition of excess WISH to WISH-depleted extracts increased actin polymerization activity (Fig. 6, Da and Db). N-WASP depletion also did not affect basal actin polymerization activity, but addition of WISH to N-WASP–depleted extracts increased the activity significantly (Fig. 6 E, a and b), although addition of Cdc42 increased the activity only slightly. These findings suggest that WISH activates Arp2/3 complex not only in an N-WASP–dependent manner but also in an N-WASP–independent manner.


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)

Bovine brain extract–induced rapid actin polymerization in vitro. Aliquots of the bovine brain extracts were treated with anti-WISH antibody, anti–N-WASP antibody, or GST-CA protein to deplete WISH, N-WASP, and Arp2/3 complex, respectively. As a control, mock-treated extracts were used. Actin polymerization activity was measured using pyrene-labeled actin in a total volume of 100 μl. In all assay systems, 100 μM GTPγS was added. When WISH and Cdc42 proteins were added, 4.5 μg of WISH and the equal amounts of WISH SH3, Fyn SH3, or 2 μg of Cdc42 were used. Results shown are means of triplicate measurements. (A) Western blots of WISH-depleted (WISH dep.), N-WASP–depleted (NW dep.), and Arp2/3 complex–depleted (Arp2/3 dep.) extracts. Degree of depletion was checked by scanning Western blots with a densitometer as described in the text. (B) Mock treatment. Mock-treated extract (MOCK), mock-treated extracts + 0.2 μM WISH (MOCK + WISH full), mock-treated extracts + 0.2 μM WISH SH3 (MOCK + WISH SH3), and mock-treated extracts + 0.2 μM Fyn SH3 (MOCK + Fyn SH3). Ba, Effect of Cdc42, WISH, WISH SH3, and Fyn SH3 on actin polymerization was examined using mock-treated bovine brain extracts. Bb, Initial rates of actin polymerization. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*). (C) Arp2/3 complex is essential for a rapid actin polymerization. Mock-treated extract (MOCK), mock-treated extracts + 0.2 μM WISH (MOCK + WISH full), mock-treated extracts + 0.5 μM Cdc42 (MOCK + Cdc42), Arp2/3 complex–depleted extracts (Arp2/3 dep.), Arp2/3 complex–depleted extracts + 0.5 μM Cdc42 (Arp2/3 dep. + Cdc42), and Arp2/3 complex–depleted extracts + WISH (Arp dep. + WISH full). Ca, actin polymerization curves; Cb, initial rates of actin polymerization. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*) and Arp2/3 complex–depleted extracts (**). (D) WISH depletion experiments. Mock-treated extracts (MOCK), mock-treated extracts + 0.2 μM WISH (MOCK + WISH), WISH-depleted extracts (WISH dep.), and WISH-depleted extracts + 0.2 μM WISH addback (WISH dep. + WISH full). Da, actin polymerization curves in WISH-depleted extracts; Db, initial rates of actin polymerization in WISH-depleted extracts. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*) and WISH-depleted extracts (**). (E) WISH activates Arp2/3 complex in N-WASP–depleted extracts. Mock-treated extracts (MOCK), N-WASP–depleted extracts (NW dep.), N-WASP–depleted extracts + 0.2 μM WISH (NW dep. + WISH full), N-WASP–depleted extracts + 0.5 μM Cdc42 (NW dep. + Cdc42), N-WASP–depleted extracts + 0.2 μM WISH SH3 (NW dep. + WISH SH3), and N-WASP–depleted extracts + Ash/Grb2 (NW dep. + Ash/Grb2). Ea, actin polymerization curves in N-WASP–depleted extracts; Eb, initial rates of actin polymerization in N-WASP–depleted extracts. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*) and N-WASP–depleted extracts (**).
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Figure 6: Bovine brain extract–induced rapid actin polymerization in vitro. Aliquots of the bovine brain extracts were treated with anti-WISH antibody, anti–N-WASP antibody, or GST-CA protein to deplete WISH, N-WASP, and Arp2/3 complex, respectively. As a control, mock-treated extracts were used. Actin polymerization activity was measured using pyrene-labeled actin in a total volume of 100 μl. In all assay systems, 100 μM GTPγS was added. When WISH and Cdc42 proteins were added, 4.5 μg of WISH and the equal amounts of WISH SH3, Fyn SH3, or 2 μg of Cdc42 were used. Results shown are means of triplicate measurements. (A) Western blots of WISH-depleted (WISH dep.), N-WASP–depleted (NW dep.), and Arp2/3 complex–depleted (Arp2/3 dep.) extracts. Degree of depletion was checked by scanning Western blots with a densitometer as described in the text. (B) Mock treatment. Mock-treated extract (MOCK), mock-treated extracts + 0.2 μM WISH (MOCK + WISH full), mock-treated extracts + 0.2 μM WISH SH3 (MOCK + WISH SH3), and mock-treated extracts + 0.2 μM Fyn SH3 (MOCK + Fyn SH3). Ba, Effect of Cdc42, WISH, WISH SH3, and Fyn SH3 on actin polymerization was examined using mock-treated bovine brain extracts. Bb, Initial rates of actin polymerization. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*). (C) Arp2/3 complex is essential for a rapid actin polymerization. Mock-treated extract (MOCK), mock-treated extracts + 0.2 μM WISH (MOCK + WISH full), mock-treated extracts + 0.5 μM Cdc42 (MOCK + Cdc42), Arp2/3 complex–depleted extracts (Arp2/3 dep.), Arp2/3 complex–depleted extracts + 0.5 μM Cdc42 (Arp2/3 dep. + Cdc42), and Arp2/3 complex–depleted extracts + WISH (Arp dep. + WISH full). Ca, actin polymerization curves; Cb, initial rates of actin polymerization. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*) and Arp2/3 complex–depleted extracts (**). (D) WISH depletion experiments. Mock-treated extracts (MOCK), mock-treated extracts + 0.2 μM WISH (MOCK + WISH), WISH-depleted extracts (WISH dep.), and WISH-depleted extracts + 0.2 μM WISH addback (WISH dep. + WISH full). Da, actin polymerization curves in WISH-depleted extracts; Db, initial rates of actin polymerization in WISH-depleted extracts. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*) and WISH-depleted extracts (**). (E) WISH activates Arp2/3 complex in N-WASP–depleted extracts. Mock-treated extracts (MOCK), N-WASP–depleted extracts (NW dep.), N-WASP–depleted extracts + 0.2 μM WISH (NW dep. + WISH full), N-WASP–depleted extracts + 0.5 μM Cdc42 (NW dep. + Cdc42), N-WASP–depleted extracts + 0.2 μM WISH SH3 (NW dep. + WISH SH3), and N-WASP–depleted extracts + Ash/Grb2 (NW dep. + Ash/Grb2). Ea, actin polymerization curves in N-WASP–depleted extracts; Eb, initial rates of actin polymerization in N-WASP–depleted extracts. Results shown are the ratio to the mock. Mean values from three experiments are indicated. Error bars represent SD. Asterisks indicate significant difference by Student's t test (P < 0.01) from mock-treated extracts (*) and N-WASP–depleted extracts (**).
Mentions: Addition of Cdc42 to extracts of neutrophils (Zigmond et al. 1998) or Xenopus oocytes (Ma et al. 1998) induces an increase in actin polymerization. In addition, WASP-dependent actin comets have also been shown to be formed in bovine brain extracts (Yarar et al. 1999). Thus, we further applied bovine brain extracts instead of purified proteins to examine the involvement of WISH in actin polymerization under a condition that more closely reflects the in vivo situation. Addition of bovine brain extracts induced actin polymerization in the presence of Cdc42. To address whether WISH, N-WASP, or Arp2/3 complex contributes to actin polymerization activity in brain extracts, WISH, N-WASP, and Arp2/3 complex were depleted with anti-WISH antibody, anti–N-WASP antibody, and GST-CA protein treatment, respectively (Fig. 6 A). In WISH-depleted extracts, 90% of WISH was removed, whereas N-WASP levels remained constant (Fig. 6 A). Approximately 98% of N-WASP or 95% of Arp2/3 complex was removed in the respective depleted extract. These depleted extracts were used to examine the roles of Arp2/3 complex, N-WASP, and WISH in brain extract–induced actin polymerization. Addition of Cdc42 or WISH to mock-treated extracts increased initial actin polymerization markedly, but Fyn SH3 did not (Fig. 6 B, a). On the other hand, the maximal actin polymerization levels that were attained up to 100 min were similar for Mock, Mock + Cdc42, and Mock + WISH (data not shown), suggesting that WISH activates the initial rate of actin polymerization. As expected, Cdc42, WISH, and WISH-SH3 activated the initial actin polymerization rate (Fig. 6 B, b). Arp2/3 complex was essential for brain extract–induced actin polymerization, because addition of Cdc42 or WISH to Arp2/3 complex–depleted extracts did not restore activity (Fig. 6 C, a and b). Adding back of Arp2/3 complex did restore polymerization activity (data not shown). WISH depletion did not affect basal activity without Cdc42 (Fig. 6 D, a and b), but addition of excess WISH to WISH-depleted extracts increased actin polymerization activity (Fig. 6, Da and Db). N-WASP depletion also did not affect basal actin polymerization activity, but addition of WISH to N-WASP–depleted extracts increased the activity significantly (Fig. 6 E, a and b), although addition of Cdc42 increased the activity only slightly. These findings suggest that WISH activates Arp2/3 complex not only in an N-WASP–dependent manner but also in an N-WASP–independent manner.

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