Biology Reference
In-Depth Information
with the GTPase binding domain (GBD) located in the middle of the
polypeptide. This interaction prevents the VCA region from activating the
Arp2/3 complex. The Rho family GTPase Cdc42 and the membrane lipid
phosphatidylinositol(4,5)bisphosphate (PIP 2 ) act synergistically to overcome
the auto-inhibition of WASp and N-WASp by freeing VCA to interact
with the Arp2/3 complex. Other parts of WASp may contribute to
activation, since the full-length protein activated by Cdc42 and PIP 2 is 100
times more active than VCA alone. Alternatively, the SH3 proteins Grb2
and Nck can activate N-WASp together with PIP 2 (Carlier et al., 2000;
Rohatgi et al., 2001). The ability of multiple signalling molecules to
activate WASp allows the protein to be a coincidence detector for signals
flowing from diverse receptors (Prehoda et al., 2000).
Scar (also known as WAVE) lacks a GBD and is not regulated by auto-
inhibition. Instead, a complex of four other proteins interferes with the ability
of Scar to activate Arp2/3 complex (Eden et al., 2002). The GTPase Rac1 and
the adapter protein Nck overcome this regulatory complex and allow Scar to
activate the Arp2/3 complex (see also chapter by Blagg and Insall).
Activation of the Arp2/3 complex
Based on the crystal structure (Robinson et al., 2001) and our analysis of the
activation mechanism (Marchand et al., 2001), we proposed that nucleation-
promoting factors and actin filaments activate the Arp2/3 complex by
stabilizing a conformation with the Arps juxtaposed like two subunits in an
actin filament. We expect that the Arp2/3 complex visits this conformation
rarely even without activators. Accordingly, high (micromolar) concentrations
of purified Arp2/3 complex generate new actin filaments, but with a
stoichiometry of only 0.001 new barbed ends per complex (Mullins et al.,
1998a), reflecting the very low fraction of active complex. Therefore a small
fraction of the complex must be in the active conformation at any time.
Confirmation that nucleation-promoting factors stabilize an active conforma-
tion will require crystal structures or other biophysical probes of activated
Kinetic and thermodynamic analysis of the activation mechanism
(Marchand et al., 2001) showed that the VCA domains from WASp and
Scar bind actin and the Arp2/3 complex with submicromolar a nity and
that both reactions are rapidly reversible on a subsecond time scale. This
means that when WASp or Scar are activated, their VCA domains will
rapidly bind the micromolar concentrations of actin and Arp2/3 complex
diffusing in the cytoplasm. Given that VCA reacts faster and has higher
a nity for actin than the Arp2/3 complex, we propose that when freed from
Search WWH ::

Custom Search