D4-GDI
BACKGROUND:
Cdc42, a Rho-related small GTP binding protein, plays
pivotal roles in actin cytoskeletal
organization, Golgi vesicular trafficking, receptor endocytosis, and cell cycle progression. However, the
target/effectors mediating these cellular activities and, in particular, those
responsible for Cdc42-mediated cell growth regulation and transformation are
still being determined. In this study, we set out to examine how the regulatory
protein RhoGDI influences the cellular responses
elicited by activated Cdc42. RESULTS: X-ray crystallographic analysis of the
Cdc42-RhoGDI complex suggested that arginine 66 of
Cdc42 is essential for its interaction with RhoGDI.
Here we show that mutation of either arginine 66 or arginine 68 within the Switch II domain of Cdc42 completely
abolished the binding of Cdc42 to RhoGDI without
affecting the binding of other known regulators or target/effectors of this GTP
binding protein. Introduction of the RhoGDI
binding-defective mutation R66A within a constitutively active Cdc42(F28L)
background was accompanied by changes in cell shape and an accumulation of
Cdc42 in the Golgi when these cells were compared to
those expressing Cdc42(F28L). However, the most striking change was that unlike
Cdc42(F28L), which was able to induce the transformation of NIH 3T3 fibroblasts
as assayed by their growth in low serum or their ability to form colonies in
soft-agar, the Cdc42(F28L,R66A) mutant was transformation-defective. Likewise,
the introduction of RhoGDI siRNA
into Cdc42(F28L)-transfected
cells inhibited their transformation. CONCLUSIONS: Taken together, the results
reported here indicate that despite being a negative regulator of Cdc42
activation and GTP hydrolysis, RhoGDI plays an
essential role in Cdc42-mediated cellular transformation.
PMID: 12956948 [PubMed - indexed for MEDLINE]