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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 Ash/Grb2. (A) Ectopically expressed WISH associates with Ash/Grb2 in vivo. Myc-tagged full-length WISH expressing plasmid (Myc-WISH) and empty vector (Myc-vec) alone were transfected into Cos7 cells. Anti-Myc immunoprecipitates (left) and whole cell lysates (right) from these cells were immunoblotted with anti-Myc antibody (top) or anti-Ash/Grb2 antibody (bottom). (B) Endogenous WISH associates with Ash/Grb2 in vivo. N1E-115 cell lysates were immunoprecipitated with preimmune serum (Pre) or anti-WISH antibody (α-WISH). The immunoprecipitates (I.P.) and whole cell lysates were immunoblotted with anti-WISH antibody (top) or anti-Ash/Grb2 antibody (bottom). (C) In vitro association of WISH with Ash/Grb2. Binding of the His-tagged WISH proline-rich sequence (His-Pro) was investigated. GST fusion proteins of full-length Ash/Grb2 (GST-Ash), the NH2-terminal SH3 domain of Ash/Grb2 (GST-AshN), and the COOH-terminal SH3 of Ash/Grb2 (GST-AshC) were immobilized on glutathione-agarose beads and incubated with (right) or without (left) His-Pro. Bound proteins were analyzed by Western blotting with anti–His-tag antibody (α-His).
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Figure 3: Association of WISH with Ash/Grb2. (A) Ectopically expressed WISH associates with Ash/Grb2 in vivo. Myc-tagged full-length WISH expressing plasmid (Myc-WISH) and empty vector (Myc-vec) alone were transfected into Cos7 cells. Anti-Myc immunoprecipitates (left) and whole cell lysates (right) from these cells were immunoblotted with anti-Myc antibody (top) or anti-Ash/Grb2 antibody (bottom). (B) Endogenous WISH associates with Ash/Grb2 in vivo. N1E-115 cell lysates were immunoprecipitated with preimmune serum (Pre) or anti-WISH antibody (α-WISH). The immunoprecipitates (I.P.) and whole cell lysates were immunoblotted with anti-WISH antibody (top) or anti-Ash/Grb2 antibody (bottom). (C) In vitro association of WISH with Ash/Grb2. Binding of the His-tagged WISH proline-rich sequence (His-Pro) was investigated. GST fusion proteins of full-length Ash/Grb2 (GST-Ash), the NH2-terminal SH3 domain of Ash/Grb2 (GST-AshN), and the COOH-terminal SH3 of Ash/Grb2 (GST-AshC) were immobilized on glutathione-agarose beads and incubated with (right) or without (left) His-Pro. Bound proteins were analyzed by Western blotting with anti–His-tag antibody (α-His).

Mentions: To confirm the association between WISH and Ash/Grb2, we performed coimmunoprecipitation experiments. Myc-tagged full-length WISH expression plasmids (Myc-WISH) and empty vectors (Myc-vec) were transfected transiently into Cos7 cells, and interactions were detected by Western blot analysis after precipitation with anti-Myc antibody (Fig. 3 A). Only immunoprecipitates from Myc-tagged WISH-transfected cell lysates contained Ash/Grb2 (Fig. 3 A, lane 2). The positive signal was specific, as anti-Myc immunoprecipitates from cells transfected with empty vector did not give positive signal (Fig. 3 A, lane 1). The two bands above the Ash/Grb2 band in Fig. 3 A are the light chains of the anti-Myc antibody. We then examined whether endogenous WISH associates with Ash/Grb2 by immunoprecipitation with anti-WISH antibody using N1E-115 cell lysates. Fig. 3 B shows that endogenous WISH associated with Ash/Grb2 in N1E-115 cells as well.


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 Ash/Grb2. (A) Ectopically expressed WISH associates with Ash/Grb2 in vivo. Myc-tagged full-length WISH expressing plasmid (Myc-WISH) and empty vector (Myc-vec) alone were transfected into Cos7 cells. Anti-Myc immunoprecipitates (left) and whole cell lysates (right) from these cells were immunoblotted with anti-Myc antibody (top) or anti-Ash/Grb2 antibody (bottom). (B) Endogenous WISH associates with Ash/Grb2 in vivo. N1E-115 cell lysates were immunoprecipitated with preimmune serum (Pre) or anti-WISH antibody (α-WISH). The immunoprecipitates (I.P.) and whole cell lysates were immunoblotted with anti-WISH antibody (top) or anti-Ash/Grb2 antibody (bottom). (C) In vitro association of WISH with Ash/Grb2. Binding of the His-tagged WISH proline-rich sequence (His-Pro) was investigated. GST fusion proteins of full-length Ash/Grb2 (GST-Ash), the NH2-terminal SH3 domain of Ash/Grb2 (GST-AshN), and the COOH-terminal SH3 of Ash/Grb2 (GST-AshC) were immobilized on glutathione-agarose beads and incubated with (right) or without (left) His-Pro. Bound proteins were analyzed by Western blotting with anti–His-tag antibody (α-His).
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Related In: Results  -  Collection

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Figure 3: Association of WISH with Ash/Grb2. (A) Ectopically expressed WISH associates with Ash/Grb2 in vivo. Myc-tagged full-length WISH expressing plasmid (Myc-WISH) and empty vector (Myc-vec) alone were transfected into Cos7 cells. Anti-Myc immunoprecipitates (left) and whole cell lysates (right) from these cells were immunoblotted with anti-Myc antibody (top) or anti-Ash/Grb2 antibody (bottom). (B) Endogenous WISH associates with Ash/Grb2 in vivo. N1E-115 cell lysates were immunoprecipitated with preimmune serum (Pre) or anti-WISH antibody (α-WISH). The immunoprecipitates (I.P.) and whole cell lysates were immunoblotted with anti-WISH antibody (top) or anti-Ash/Grb2 antibody (bottom). (C) In vitro association of WISH with Ash/Grb2. Binding of the His-tagged WISH proline-rich sequence (His-Pro) was investigated. GST fusion proteins of full-length Ash/Grb2 (GST-Ash), the NH2-terminal SH3 domain of Ash/Grb2 (GST-AshN), and the COOH-terminal SH3 of Ash/Grb2 (GST-AshC) were immobilized on glutathione-agarose beads and incubated with (right) or without (left) His-Pro. Bound proteins were analyzed by Western blotting with anti–His-tag antibody (α-His).
Mentions: To confirm the association between WISH and Ash/Grb2, we performed coimmunoprecipitation experiments. Myc-tagged full-length WISH expression plasmids (Myc-WISH) and empty vectors (Myc-vec) were transfected transiently into Cos7 cells, and interactions were detected by Western blot analysis after precipitation with anti-Myc antibody (Fig. 3 A). Only immunoprecipitates from Myc-tagged WISH-transfected cell lysates contained Ash/Grb2 (Fig. 3 A, lane 2). The positive signal was specific, as anti-Myc immunoprecipitates from cells transfected with empty vector did not give positive signal (Fig. 3 A, lane 1). The two bands above the Ash/Grb2 band in Fig. 3 A are the light chains of the anti-Myc antibody. We then examined whether endogenous WISH associates with Ash/Grb2 by immunoprecipitation with anti-WISH antibody using N1E-115 cell lysates. Fig. 3 B shows that endogenous WISH associated with Ash/Grb2 in N1E-115 cells as well.

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