Limits...
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.

Show MeSH

Related in: MedlinePlus

Amino acid sequence of a novel N-WASP binding protein, WISH. (A) Sequence of WISH. The SH3 domain, proline-rich sequence, and leucine-rich sequences are boxed. The serine-rich sequence is underlined. The heptad repeat of hydrophobic residues in the leucine zipper-like motif is denoted by white-on-black. (B) Schematic structure of WISH. (C) Western blot analysis of ectopically expressed WISH and endogenous WISH. Western blot analyses were performed using cell lysates of Cos7 cells transfected with empty vector (vec) or WISH-expressing plasmid (ectopically expressed WISH) and rat brain. WISH (90 kD) is indicated by the arrow.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196001&req=5

Figure 1: Amino acid sequence of a novel N-WASP binding protein, WISH. (A) Sequence of WISH. The SH3 domain, proline-rich sequence, and leucine-rich sequences are boxed. The serine-rich sequence is underlined. The heptad repeat of hydrophobic residues in the leucine zipper-like motif is denoted by white-on-black. (B) Schematic structure of WISH. (C) Western blot analysis of ectopically expressed WISH and endogenous WISH. Western blot analyses were performed using cell lysates of Cos7 cells transfected with empty vector (vec) or WISH-expressing plasmid (ectopically expressed WISH) and rat brain. WISH (90 kD) is indicated by the arrow.

Mentions: The mouse skeletal muscle C2 myoblast cDNA expression library constructed in λZAPII was screened with GST-Ash/Grb2. Positive plaques were detected using anti-GST antibody (Amersham Pharmacia Biotech). Positive phage clone-inserted DNA fragments were excised into pBluescript II KS(−) (Stratagene) and sequenced. The clone encoding WISH (2,848 bases) included a single open reading frame of 711 amino acids as shown in Fig. 1 A.


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)

Amino acid sequence of a novel N-WASP binding protein, WISH. (A) Sequence of WISH. The SH3 domain, proline-rich sequence, and leucine-rich sequences are boxed. The serine-rich sequence is underlined. The heptad repeat of hydrophobic residues in the leucine zipper-like motif is denoted by white-on-black. (B) Schematic structure of WISH. (C) Western blot analysis of ectopically expressed WISH and endogenous WISH. Western blot analyses were performed using cell lysates of Cos7 cells transfected with empty vector (vec) or WISH-expressing plasmid (ectopically expressed WISH) and rat brain. WISH (90 kD) is indicated by the arrow.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196001&req=5

Figure 1: Amino acid sequence of a novel N-WASP binding protein, WISH. (A) Sequence of WISH. The SH3 domain, proline-rich sequence, and leucine-rich sequences are boxed. The serine-rich sequence is underlined. The heptad repeat of hydrophobic residues in the leucine zipper-like motif is denoted by white-on-black. (B) Schematic structure of WISH. (C) Western blot analysis of ectopically expressed WISH and endogenous WISH. Western blot analyses were performed using cell lysates of Cos7 cells transfected with empty vector (vec) or WISH-expressing plasmid (ectopically expressed WISH) and rat brain. WISH (90 kD) is indicated by the arrow.
Mentions: The mouse skeletal muscle C2 myoblast cDNA expression library constructed in λZAPII was screened with GST-Ash/Grb2. Positive plaques were detected using anti-GST antibody (Amersham Pharmacia Biotech). Positive phage clone-inserted DNA fragments were excised into pBluescript II KS(−) (Stratagene) and sequenced. The clone encoding WISH (2,848 bases) included a single open reading frame of 711 amino acids as shown in Fig. 1 A.

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