Phosphatidylinositol 4,5-bisphosphate provides an alternative to guanine nucleotide exchange factors by stimulating the dissociation of GDP from Cdc42Hs

Abstract

Members of the Rho subfamily of Ras-related GTP-binding proteins play important roles in the organization of the actin cytoskeleton and in the regulation of cell growth. We have shown previously that the dbl oncogene product, which represents a prototype for a family of growth regulatory proteins, activates Rho subfamily GTP-binding proteins by catalyzing the dissociation of GDP from their nucleotide binding site. In the present study, we demonstrate that the acidic phospholipid, phosphatidylinositol 4,5- bisphosphate (PIP2), provides an alternative mechanism for the activation of Cdc42Hs. Among a variety of lipids tested, only PIP2 was able to stimulate GDP release from Cdc42Hs in a dose-dependent manner, with a half-maximum effect at ~50 μM. Unlike the Dbl oncoprotein, which requires the presence of (free) guanine nucleotide in the medium to replace the GDP bound to Cdc42Hs, PIP2 stimulates GDP release from Cdc42Hs in the absence of free guanine nucleotide. PIP2, when incorporated into phosphatidylcholine carrier vesicles, binds tightly to the guanine nucleotide-depleted form of Cdc42Hs and weakly to the GDP-bound form of the GTP-binding protein but does not bind to GTP-bound Cdc42Hs, similar to what was observed for the Dbl oncoprotein. However, mutational analysis of Cdc42Hs indicates that the site that is essential for the functional interaction between PIP2 and Cdc42Hs is distinct from the Dbl-binding site and is located at the positively charged carboxyl-terminal end of the GTP-binding protein. The GDP-releasing activity of PIP2 is highly effective toward Cdc42Hs and Rho (and is similar to the reported effects of PIP2 on Arf (Terui, T., Kahn, R. A., and Randazzo, P. A., (1994) J. Biol. Chem. 269, 2813028135)), is less effective with Rac, and is not observed with Ras, Rap1a, or Ran. The ability of PIP2 to activate Cdc42Hs (or Rho) and Arf provides a possible point of convergence for the biological pathways regulated by these different GTP-binding proteins and may be related to the synergism observed between Arf and Rho-subtype proteins in the stimulation of phospholipase D activity (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950).