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Fig. 8. Rho1 is a substrate of RhoGEF2 and binds and activates Dia. (A) Release of
tritium-loaded GDP from Rho1 and Rac1 GST fusion proteins by a fusion protein
of GST and the GEF domain from RhoGEF2. Bound GDP (expressed as counts of
radioactivity per minute; cpm) plotted against incubation time. (B)
Specificity of GDP release. Tritium loaded GST fusion proteins were incubated
with GST fusion proteins of the GEF domains of RhoGEF2 and Trio (domain 1) or
GST for 20 minutes. Bound GDP is indicated as the quotient of
[GDP](t=0 minutes) and [GDP](t=20 minutes). The protein
T1544A has a single point mutation in the GEF domain of RhoGEF2. (C) GST,
GST-Rho1, GST-Rho1Q63L, GST-Rac1, (loaded with GDP or GMP-PNP) bound to
glutathione Sepharose were incubated with reticulocyte lysate containing
35S-labelled Dia
N318 or DiaC464. Rho1Q63L has no
GTPase activity. Bound and unbound fractions were analysed by SDS-PAGE and
autoradiography. (D) Actin polymerisation induced by Dia. Time course of
fluorescence (relative units) of pyrene-labelled actin (3 µM) and the
indicated components. Dia FH, ZZ-DiaN519, Dia FH,
ZZ-DiaC518, Rho1, GST-Rho1 loaded with GTP
S. Rho1 loaded with
GDP or GMP-PNP or Rho1Q63L released autoinhibition to a similar degree.
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