Biomedical Engineering Reference
In-Depth Information
FIGURE 18.2
Secramine inhibits intracellular membrane traffic. (
See insert for color rep-
resentation of the figure
.)
derived from PAK1, can be used to precipitate GTP-bound Cdc42. In the experiment,
Cdc42([
35
S]GTP
S
and PIP2 liposomes in a process catalyzed by the endogenous guanine nucleotide
exchange factor (GEF). Coprecipitation between Cdc42([
35
S]GTP
S) was made by mixing the
X. laevis
egg extract with [
35
S]GTP
S) and GST-PBD
was not observed in cases when secramine was either present in the
X. laevis
egg
extract or added to the pre-formed Cdc42([
35
S]GTP
S). The fact that the coprecipita-
tion between Cdc42([
35
S]GTP
S) and GST-PBD was detected when secramine was
added last in the assay suggested that secramine blocked the Cdc42([
35
S]GTP
S)
binding with GST-PBD indirectly in a noncompetitive manner.
To test if secramine inhibits the membrane association of Cdc42 before binding
its effectors [61], the compound's effects were examined through the membrane-
dependent nucleotide exchange activity of prenylated Cdc42. Secramine was found
to inhibit the exchange of GDP for [
35
S]GTP
S in a Cdc42(GDP)-RhoGDI1 com-
plex that was prepared with isolated PIP2 liposomes and the DH-PH domain of
