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et al., 1999), and in VASP has been identified as an actin-binding motif
(Huttelmaier et al., 1999). The actin-binding activity of the EVH2 domain is
dependent upon the phosphorylation state of the protein at sites flanking the
proline-rich domain (Harbeck et al., 2000). Phosphorylation is necessary for
normal Ena/VASP function during random cell motility, but does not affect
the cellular location of the proteins (Loureiro et al., 2002). Harbeck et al.,
2000) showed that VASP phosphorylation negatively regulates actin
polymerization through inhibition of the interaction between the VASP
EVH2 domain and F-actin. The EVH1 domain located at the N-terminus
contributes to interactions with several focal adhesion proteins, such as
vinculin (Huttelmaier et al., 1998) and zyxin (Drees et al., 2000), serving to
recruit Ena/VASP proteins to focal adhesions, as well as the zyxin-related
Listeria protein ActA (Niebuhr et al., 1997).
VASP has an important role in actin polymerization during intracellular
motility of Listeria, being required for increased rates of bacterial movement
in reconstituted cell-free systems (Geese et al., 2002; Laurent et al., 1999;
Skoble et al., 2001). This suggests that VASP facilitates an increased rate of
actin polymerization. VASP over-expression, however, negatively regulates
fibroblast motility in a dose-dependent manner (Bear et al., 2000) suggesting
that the simple correlation between filament assembly and cell movement
observed with Listeria does not hold in mammalian cells (Machesky, 2002).
Recently, Bear and colleagues have deepened our understanding of the
cellular role of Ena/VASP proteins to resolve this apparent controversy (Bear
et al., 2002). They show that Ena/VASP proteins compete in an antagonistic
manner with capping proteins to promote filament elongation. VASP over-
expression reduces branched filament network formation as caused by Arp2/3
complex-dependent nucleation to affect production of lamellipodia (Bear et al.,
2002). This observation explains both the activity of VASP to allow increased
Listeria motility and its ability to slow cell motility when over-expressed, which
could be due to inhibiting formation of the actin network structure required for
membrane ruing whilst promoting the formation of longer filaments.
The function of Ena/VASP localization in focal adhesions and the tips of
stress fibres is not yet entirely clear, although several hypotheses are possible.
The proteins may have a molecular linker function through their ability to
bind to both F-actin and to anchor fibres to focal adhesion complexes through
interaction with vinculin and zyxin. As well as anchoring stress fibres it is
feasible that Ena/VASP proteins have a role in stress fibre formation through
promotion of filament elongation and bundling.
Conclusions
Clearly, many proteins work together to regulate actin dynamics in cells and
we have only touched on a few of these. Uncapping and severing of
