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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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Association of WISH with N-WASP. (A) WISH binding proteins. Cell lysates from [35S]methionine-prelabeled N1E-115 cells were immunoprecipitated with anti-WISH antibody (α-WISH) or preimmune rabbit serum (Pre). The precipitates (I.P.) were subjected to SDS-PAGE and autoradiography. (B) Association of WISH with proline-rich proteins. N1E-115 cell lysates were immunoprecipitated as described in A. The immunoprecipitates and whole cell lysates were immunoblotted with anti-WISH antibody, anti-Sos antibody, anti–c-Cbl antibody, antisynaptojanin antibody (α-S.J.), anti-WAVE antibody, and anti–N-WASP antibody. (C) Various deletion constructs of WISH GST fusion proteins: full-length (Full), SH3 domain (SH3), proline-rich sequence (Pro), middle region (Mid), leucine-rich region (Leu), and COOH-terminal region (C). (D) WISH associates with N-WASP through its SH3 domain. The ability to bind GST fusion proteins of various deletion constructions of WISH was examined. These proteins were immobilized on glutathione-agarose beads and mixed with N-WASP protein. Bound proteins were immunoblotted with anti–N-WASP antibody. (E) WISH SH3 domain binds to the proline-rich region of N-WASP. GST fusion protein of the N-WASP proline-rich region (GST–NW-Pro) was immobilized on glutathione-agarose beads and mixed with His-tagged WISH SH3 (His-WISH–SH3). Bound proteins were analyzed by Western blotting with anti–His-tag antibody (α-His).
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Figure 4: Association of WISH with N-WASP. (A) WISH binding proteins. Cell lysates from [35S]methionine-prelabeled N1E-115 cells were immunoprecipitated with anti-WISH antibody (α-WISH) or preimmune rabbit serum (Pre). The precipitates (I.P.) were subjected to SDS-PAGE and autoradiography. (B) Association of WISH with proline-rich proteins. N1E-115 cell lysates were immunoprecipitated as described in A. The immunoprecipitates and whole cell lysates were immunoblotted with anti-WISH antibody, anti-Sos antibody, anti–c-Cbl antibody, antisynaptojanin antibody (α-S.J.), anti-WAVE antibody, and anti–N-WASP antibody. (C) Various deletion constructs of WISH GST fusion proteins: full-length (Full), SH3 domain (SH3), proline-rich sequence (Pro), middle region (Mid), leucine-rich region (Leu), and COOH-terminal region (C). (D) WISH associates with N-WASP through its SH3 domain. The ability to bind GST fusion proteins of various deletion constructions of WISH was examined. These proteins were immobilized on glutathione-agarose beads and mixed with N-WASP protein. Bound proteins were immunoblotted with anti–N-WASP antibody. (E) WISH SH3 domain binds to the proline-rich region of N-WASP. GST fusion protein of the N-WASP proline-rich region (GST–NW-Pro) was immobilized on glutathione-agarose beads and mixed with His-tagged WISH SH3 (His-WISH–SH3). Bound proteins were analyzed by Western blotting with anti–His-tag antibody (α-His).

Mentions: Glutathione S-transferase (GST)-Ash/Grb2 (Miki et al. 1994), GST-AshN, GST-AshC (Watanabe et al. 1995), GST-phosphatidylinositol 3-kinase (PI 3-kinase) 85-kD subunit (p85) SH3 (Shibasaki et al. 1993), PLCγ1 SH3 (Homma et al. 1990), GST-Nck SH3 (Matuoka and Takenawa 1998), GST-Fyn SH3 (Miki et al. 1999), and GST-CA (Miki and Takenawa 1998) were purified as described. Bacterial expression plasmids coding various GST fusion proteins of WISH were produced by in-frame insertion of fragments corresponding to each region as shown in Fig. 4 C into pGEX plasmids (Amersham Pharmacia Biotech). GST-SH3 (1–132 amino acids) expression plasmid was made by cutting the cDNA of mouse WISH, and inserting it into pGEX-4T-3. GST-Pro (132–268 amino acids), GST-Mid (240–442 amino acids), and GST-Leu (442–611 amino acids) were produced by in-frame insertion of a PCR-amplified fragment corresponding to each sequence into the BamHI-EcoRI site of pGEX-2T. GST-C (611–711 amino acids) was produced by in-frame insertion of a PCR-amplified fragment into the BamHI-XhoI site of pGEX-4T-1.


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)

Association of WISH with N-WASP. (A) WISH binding proteins. Cell lysates from [35S]methionine-prelabeled N1E-115 cells were immunoprecipitated with anti-WISH antibody (α-WISH) or preimmune rabbit serum (Pre). The precipitates (I.P.) were subjected to SDS-PAGE and autoradiography. (B) Association of WISH with proline-rich proteins. N1E-115 cell lysates were immunoprecipitated as described in A. The immunoprecipitates and whole cell lysates were immunoblotted with anti-WISH antibody, anti-Sos antibody, anti–c-Cbl antibody, antisynaptojanin antibody (α-S.J.), anti-WAVE antibody, and anti–N-WASP antibody. (C) Various deletion constructs of WISH GST fusion proteins: full-length (Full), SH3 domain (SH3), proline-rich sequence (Pro), middle region (Mid), leucine-rich region (Leu), and COOH-terminal region (C). (D) WISH associates with N-WASP through its SH3 domain. The ability to bind GST fusion proteins of various deletion constructions of WISH was examined. These proteins were immobilized on glutathione-agarose beads and mixed with N-WASP protein. Bound proteins were immunoblotted with anti–N-WASP antibody. (E) WISH SH3 domain binds to the proline-rich region of N-WASP. GST fusion protein of the N-WASP proline-rich region (GST–NW-Pro) was immobilized on glutathione-agarose beads and mixed with His-tagged WISH SH3 (His-WISH–SH3). Bound proteins were analyzed by Western blotting with anti–His-tag antibody (α-His).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196001&req=5

Figure 4: Association of WISH with N-WASP. (A) WISH binding proteins. Cell lysates from [35S]methionine-prelabeled N1E-115 cells were immunoprecipitated with anti-WISH antibody (α-WISH) or preimmune rabbit serum (Pre). The precipitates (I.P.) were subjected to SDS-PAGE and autoradiography. (B) Association of WISH with proline-rich proteins. N1E-115 cell lysates were immunoprecipitated as described in A. The immunoprecipitates and whole cell lysates were immunoblotted with anti-WISH antibody, anti-Sos antibody, anti–c-Cbl antibody, antisynaptojanin antibody (α-S.J.), anti-WAVE antibody, and anti–N-WASP antibody. (C) Various deletion constructs of WISH GST fusion proteins: full-length (Full), SH3 domain (SH3), proline-rich sequence (Pro), middle region (Mid), leucine-rich region (Leu), and COOH-terminal region (C). (D) WISH associates with N-WASP through its SH3 domain. The ability to bind GST fusion proteins of various deletion constructions of WISH was examined. These proteins were immobilized on glutathione-agarose beads and mixed with N-WASP protein. Bound proteins were immunoblotted with anti–N-WASP antibody. (E) WISH SH3 domain binds to the proline-rich region of N-WASP. GST fusion protein of the N-WASP proline-rich region (GST–NW-Pro) was immobilized on glutathione-agarose beads and mixed with His-tagged WISH SH3 (His-WISH–SH3). Bound proteins were analyzed by Western blotting with anti–His-tag antibody (α-His).
Mentions: Glutathione S-transferase (GST)-Ash/Grb2 (Miki et al. 1994), GST-AshN, GST-AshC (Watanabe et al. 1995), GST-phosphatidylinositol 3-kinase (PI 3-kinase) 85-kD subunit (p85) SH3 (Shibasaki et al. 1993), PLCγ1 SH3 (Homma et al. 1990), GST-Nck SH3 (Matuoka and Takenawa 1998), GST-Fyn SH3 (Miki et al. 1999), and GST-CA (Miki and Takenawa 1998) were purified as described. Bacterial expression plasmids coding various GST fusion proteins of WISH were produced by in-frame insertion of fragments corresponding to each region as shown in Fig. 4 C into pGEX plasmids (Amersham Pharmacia Biotech). GST-SH3 (1–132 amino acids) expression plasmid was made by cutting the cDNA of mouse WISH, and inserting it into pGEX-4T-3. GST-Pro (132–268 amino acids), GST-Mid (240–442 amino acids), and GST-Leu (442–611 amino acids) were produced by in-frame insertion of a PCR-amplified fragment corresponding to each sequence into the BamHI-EcoRI site of pGEX-2T. GST-C (611–711 amino acids) was produced by in-frame insertion of a PCR-amplified fragment into the BamHI-XhoI site of pGEX-4T-1.

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