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generation of new barbed ends is necessary. It is thought that there are three
mechanisms for generation of new barbed ends: uncapping of pre-existing
filaments, severing of pre-existing filaments, and de novo nucleation of actin
core (Suetsugu et al., 2002; Condeelis, 2001; Wear et al., 2000). Among these
mechanisms, de novo nucleation appears to play the most important role in
leading edge formation (see also chapter by Condeelis). The recent progress in
our understanding of the process of actin network formation began with the
discovery of the Arp2/3 complex, a complex of seven proteins that is the only
known nucleator of new actin filaments. However, the Arp2/3 complex alone
can not enhance actin polymerization, suggesting that some other factor is
necessary for Arp2/3 complex activation.
In studies of the regulation of the Arp2/3 complex, pathogenic bacteria such
as Shigella and Listeria have played important roles. These bacteria are
known to form actin comets and move freely within infected cells. Actin comet
structures resemble those at the leading edge, which consist of filopodia and
lamellipodia (Cameron et al., 2001). The ActA surface protein from Listeria
was found to bind directly to the Arp2/3 complex, leading to its activation
(Welch et al., 1997). Surprisingly, the amino acid sequence of the ActA protein
is homologous to the VCA region of WASp family proteins. The VCA region
alone can activate the Arp2/3 complex, enhancing actin polymerization,
suggesting that the VCA region is the minimum essential region for Arp2/3
complex activation. The V region is a monomeric actin-binding region, and
the CA region is an Arp2/3 complex-binding region (Miki and Takenawa,
1998; Rohatgi et al., 1999). This appears to be the basis for the activation of
the actin nucleating activity of the Arp2/3 complex by the VCA region. Of the
WASp family proteins, only N-WASp has two V motifs in the VCA region.
However, the VCA regions of all WASp family proteins can activate the actin-
nucleating activity of the Arp2/3 complex and induce rapid polymerization of
actin (Yamaguchi et al., 2000). The N-WASp VCA region is the most potent
activator of Arp2/3 complex-induced-actin polymerization among WASp
family proteins. We previously reported that the tandem V motifs are
responsible for the strong activity of the N-WASp VCA region (Yamaguchi
et al., 2000). However, another group reported that a three-amino-acid stretch
in the acidic region is important for the strong activity of N-WASp VCA and
that the extra V motif has little effect on activity (Zalevsky et al., 2000). There
is a difference in the Arp2/3 complex-mediated actin polymerization activity
between the full-length WAVE1 and the free WAVE1 VCA fragment. Full-
length WAVE1 protein induces actin polymerization more effectively than
does the free WAVE1 VCA fragment (Yamaguchi et al., 2002), suggesting
that regions other than the VCA play important roles in the activity of VCA.
Thus, we added linker proteins to the WAVE1 VCA region. When fused to
GST, maltose binding protein (MBP) and WAVE1 proline-rich domain,
N-WASp VCA and WAVE1 VCA mutants containing two V motifs showed
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