IDENTIFICATION OF A NOVEL HUMAN RHO-PROTEIN WITH UNUSUAL PROPERTIES -GTPASE DEFICIENCY AND IN-VIVO FARNESYLATION

We have identified a human Rho family protein, RhoE, which has unusual structural and biochemical properties that suggest a novel mechanism of regulation. Within a region that is highly conserved among small GT Pases, RhoE contains amino acid differences specifically at three posi tions that confer oncogenicity to Ras (12, 59, and 61). As predicted b y these substitutions, which impair GTP hydrolysis in Ras, RhoE binds GTP but lacks intrinsic GTPase activity and is resistant to Rho-specif ic GTPase-activating proteins. Replacing all three positions in RhoE w ith conventional amino acids completely restores GTPase activity. In v ivo, RhoE is found exclusively in the GTP-bound form, suggesting that unlike previously characterized small GTPases, RhoE may be normally ma intained in an activated state. Thus, amino acid changes in Ras that a re selected during tumorigenesis have evolved naturally in this Rho pr otein and have similar consequences for catalytic function. All previo usly described Rho family proteins are modified by geranylgeranylation , a lipid attachment required for proper membrane localization. In con trast, the carboxy-terminal sequence of RhoE predicts that, like Ras p roteins, RhoE is normally farnesylated. Indeed, we have found that Rho E is farnesylated in vivo and that this modification is required for a ssociation with the plasma membrane and with an unidentified cellular structure that may play a role in adhesion. Thus, two unusual structur al features of this novel Rho protein suggest a striking evolutionary divergence from the Rho family of GTPases.