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Abstract
WASP (Wiskott-Aldrich syndrome protein) was identified as the gene product whose mutation causes the human hereditary disease Wiskott-Aldrich syndrome. WASP contains many functional domains and has been shown to induce the formation of clusters of actin filaments in a manner dependent on Cdc42. However, there has been no report investigating what domain(s) is(are) important for the function. Here we present for the first time the results of detailed analyses on the domain-function relationship of WASP. First, the C-terminal verprolin-cofilin-acidic domain was shown to be essential for the regulation of actin cytoskeleton. In addition, we found that the clustering of WASP itself is distinct from actin clustering. The partial protein containing the region from the N-terminal pleckstrin homology domain to the basic residue-rich region also clustered especially around the nucleus as wild type WASP without inducing actin clustering. Finally, we obtained the quite unexpected result that a WASP mutant deficient in binding to Cdc42 still induced actin cluster formation, indicating that direct interaction between Cdc42 and WASP is not required for the regulation of actin cytoskeleton. This result may explain why no Wiskott-Aldrich syndrome patients have been identified with a missense mutation in the Cdc42-binding site.
Highlights
WASP (Wiskott-Aldrich syndrome protein) was originally identified as the gene product whose mutation causes the human hereditary disease Wiskott-Aldrich syndrome [1]
Another WASP family protein, N-WASP, has the pleckstrin homology (PH) domain, GTPase binding domain (GBD)/ Cdc42 and Rac interactive binding (CRIB) motif, PR domain, verprolin homology (VPH) domain, and C-terminal acidic region [17]. This protein has been shown to play important roles in Cdc42-dependent filopodium formation [14], in structure it is very similar to WASP. Both of these proteins cause actin polymerization, but with different features when they are expressed in cells; WASP mainly localizes at perinuclear areas and causes actin clustering [3], but most N-WASP is present at plasma membranes and induces filopodium formation [14, 17]
Verprolin-Cofilin-Acidic Domain Is Essential for the Actin Clustering Induced by WASP—We first tried to determine which region of WASP is essential for the regulation of actin cytoskeleton
Summary
Antibodies—Polyclonal anti-WASP antibody was prepared in rabbits immunized with bacterially expressed recombinant protein (amino acids 149 –310). The polyclonal antibody specific for the Arp was prepared in rabbits according to the method of Welch et al [18]. ⌬PH [107– 502] was obtained by AccI digestion and excision of the cDNA fragment. WASP Mutagenesis—Construction of mutants of ⌬PHWI (deletion of amino acids 1–193), ⌬WI (deletion of amino acids 155–193), ⌬PRVCA (deletion of amino acids 311–502), and ⌬PR (deletion of amino acids 311– 413) was done by using the polymerase chain reaction.
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