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stronger activities than wild-type WAVE1 VCA with one V motif, indicating
the importance of two V motifs in strong VCA activity. In contrast, WAVE1
VCA fragment tagged with six histidine (His) residues showed markedly
reduced activity in comparison with GST-fused VCA, whereas His-tagged
N-WASp VCA showed similar activity. Furthermore, an additional V motif
failed to enhance activity of the His-tagged form of WAVE1 VCA. Thus, free
WAVE1 VCA may assume an unfavourable conformation for activation of
the Arp2/3 complex, suggesting that there are structural differences between
WAVE1 and N-WASp VCA in addition to the number of V motifs.
Mechanism of activation of N-WASp, WAVE1, and WAVE2
Although the VCA region of N-WASp is the strongest activator among the
VCA regions of WASp and WAVE family members, full-length N-WASp
did not cause remarkable activation of the Arp2/3 complex, suggesting that
the N-WASp molecule assumes a tertiary structure that masks the VCA
region. Indeed, the VCA region of N-WASp and WASp was found to be
masked by intramolecular interactions (Rohatgi et al., 1999; Miki et al., 1998;
Higgs and Pollard, 2000; Suetsugu et al., 2001). This auto-inhibition is
released when signalling molecules bind to N-WASp. Cdc42 and phospha-
tidylinositol (4,5)-bisphosphate (PIP
2
) bind to N-WASp through the GDB/
CRIB motif and WH1 and basic regions respectively and activate the Arp2/3
complex to a level similar to that of the VCA region. Furthermore, proteins
with SH3 domains such as WISH (Fukuoka et al., 2001), Ash/Grb2 (Carlier et
al., 2000), Nck (Rohatgi et al., 2001), and profilin (Yang et al., 2000; Suetsugu
et al., 1998), bind to the proline-rich region of N-WASp and completely or
partially release the auto-inhibition, thereby activating N-WASp. Taken
together, these data indicate that molecules that bind to N-WASp or WASp
activate them, although the potencies of these binding molecules are different.
WAVE1, 2 and 3 also have VCA regions similar to those of WASp and N-
WASp (Suetsugu et al., 1999). Thus, a similar mechanism of activation is
likely for WAVEs. However, it was unclear how WAVEs are activated
because WAVE proteins are already active when purified. WAVE1 is localized
at membrane ru
es, and a dominant negative WAVE1, which has the
verprolin-homology region (V region) deleted, inhibited Rac-induced forma-
tion of membrane ru
es, suggesting that WAVEs might function downstream
of Rac (Miki et al., 1998). Yeast two-hybrid system analyses yielded an
adapter protein, IRSp53, that associates with WAVE2 (Miki et al., 2000).
IRSp53 binds to the proline-rich region of WAVE2 through the SH3 domain
present in its C-terminus. IRSp53 also associates with activated Rac through a
Rac-binding (RCB) domain in the N-terminus, linking the Rac signal to Arp2/3
complex-mediated actin polymerization and formation of lamellipodia (Miki
