Essential Role of Neural Wiskott-Aldrich Syndrome Protein in Neurite Extension in PC12 Cells and Rat Hippocampal Primary Culture Cells*
- From the Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan
Abstract
Neural Wiskott-Aldrich syndrome protein (N-WASP) is an actin-regulating protein that induces filopodium formation downstream of Cdc42. It has been shown that filopodia actively extend from the growth cone, a guidance apparatus located at the tip of neurites, suggesting their role in neurite extension. Here we examined the possible involvement of N-WASP in the neurite extension process. Since verprolin, cofilin homology andacidic region (VCA) of N-WASP is known to be required for the activation of Arp2/3 complex that induces actin polymerization, we prepared a mutant (Δcof) lacking four amino acid residues in the cofilin homology region. The corresponding residues in WASP had been reported to be mutated in some Wiskott-Aldrich syndrome patients. Expression of Δcof N-WASP suppressed neurite extension of PC12 cells. In support of this, the VCA region of Δcof cannot activate Arp2/3 complex enough compared with wild-type VCA. Furthermore, H208D mutant, which has been shown unable to bind to Cdc42, also works as a dominant negative mutant in neurite extension assay. Interestingly, the expression of H208D-Δcof double mutant has no significant dominant negative effect. Finally, the expression of the Δcof mutant also severely inhibited the neurite extension of primary neurons from rat hippocampus. Thus, N-WASP is thought to be a general regulator of the actin cytoskeleton indispensable for neurite extension, which is probably caused through Cdc42 signaling and Arp2/3 complex-induced actin polymerization.
Footnotes
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↵* This work was supported in part by a Grant-in-aid for Cancer Research from the Ministry of Education, Science, and Culture of Japan and by a Grant-in-aid for Research for the Future Program from the Japan Society for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan. Tel.: 81-3-5449-5510; Fax: 81-3-5449-5417; E-mail: takenawa@ims.u-tokyo.ac.jp.
- Abbreviations:
- N-WASP
-
neural Wiskott-Aldrich syndrome protein
- HS
-
horse serum
- NGF
-
nerve growth factor
- PBS
-
phosphate-buffered saline
- PCR polymerase chain reaction
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VCA
- verprolin-
-
cofilin homology andacidic region
- GST
-
glutathione S-transferase
- Bt2cAMP
-
dibutyryl cyclic AMP
- GFP
-
green fluorescent protein
- m.o.i.
-
multiplicity of infection
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- Received September 10, 1999.
- Revision received December 18, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
