Microinjection of Recombinant p2 lrh° Induces Rapid Changes in Cell Morphology

The rho proteins, p21 ~ , are ubiquitously expressed guanine nucleotide binding proteins with ,'o30% amino acid homology to p21'% but their biochemical function is unknown. We show here that microinjection of constitutively activated recombinant rho protein (Vall4rho) into subconfluent cells induces dramatic changes in cell morphology: 15-30 min after injection cells adopt a distinct and novel phenotype with a contracted cell body and finger-like processes still adherent to the substratum. Ribosylation of Vall4rho with the ADP-ribosyltransferase C3 from clostridium botulinum, before microinjection, renders the protein biologically inactive, but it has no effect on either its intrinsic biochemical properties or on its interaction with the GTPase activating protein, rho GAP. Microinjection of ribosylated normal rho, on the other hand, has a similar effect to injection of C3 transferase and induces complete rounding up of cells. We also report striking biochemical changes in actin filament organization when contact-inhibited quiescent 3T3 cells are injected with Vall4rho protein. The effects induced by activation or inactivation of p21 ~ described here, suggest that the biological function of this protein is to control some aspect of cytoskeletal organization. T HE guanine nucleotide binding proteins, p21 =~ have attracted a great deal of attention since they appear to be regulatory components of the proliferative response (Barbacid, 1987; Morris et al., 1989). However, it is now evident that the three ras proteins are members of a much larger superfamily of related proteins (Chardin, 1988). Six proteins, rapl (A and B), rap2, R-ras, and ral (A and B) have ~50% amino acid homology to ras and there is evidence that some of these may also be involved in the control of cell proliferation (Pizon et al., 1988; Lowe et al., 1987; Kitayama et al., 1989; Garrett et al., 1989). A large subgroup of ras-related proteins, rab, have less homology to ras (30%) and almost nothing is known of their function, though two S. cerevisiae proteins, YPT1 and SEC4, that belong to this group are involved in intracellular vesicle transport (Zahraoui et al., 1989; Segev et al., 1988; Salmien and Novick, 1987). The three mammalian rho proteins (A, B, and C) are "030% homologous to ras and are ubiquitously expressed (Madaule and Axel, 1985; Yeramian et al., 1987; Olofsson et al., 1988). Based on the resemblance of their carboxy termini to ras, they are presumed to function at the plasma membrane (Hancock et al., 1989), though significant amounts of rho can be found in cytoplasm (Narumiya et al., 1988). The RHO1 gene of S. cerevisiae is closely related to mammalian rho and deletion of the yeast gene is lethal (Madaule et al., 1987). It has been shown that an ADPribosyltransferase from clostridium botulinum, C3, can ribosylate rho on asparagine 41 (Narumiya et al., 1988; Aktories et al., 1989; Sekine et al., 1989), and that introduction of C3 into cells induces rounding up and the dissolution of actin microfilaments (Rubin et al., 1988; Chardin et al., 1989;). However, two additional C3 substrates, racl and rac2, have recently been identified (Didsbury et al., 1989), and the role of rho in the C3-induced effects is unclear. We have previously reported the purification of recombinant rho protein from an E. coli expression system (Garrett et al., 1989). The intrinsic biochemical properties of the protein were similar to those previously described for ras; i.e., a slow, Mg2+-dependent, guanine nucleotide exchange rate and a slow intrinsic GTP hydrolysis rate. In addition, we identified a 29-kD GTPase activating protein, rho GAP, that could stimulate the intrinsic GTPase activity of rho. Mutation of glycine to valine at codon 14 of rho had similar biochemical consequences to oncogenic codon 12 changes in ras; namely, it reduced the intrinsic GTPase activity and blocked rho GAP-stimulated GTP hydrolysis (Garrett et al., 1989). We reasoned that as with ras, the biological effects of a Vall4rho protein should represent those of a constitutively activated protein. We have, therefore, made use of the microinjection technique to examine both the biological function of rho and the effect of ribosylation on its biological and biochemical activities. Materials and Methods Expression and Purification of rho Protein Normal and mutant rhoA cDNAs were expressed under the control of the tryptophan promoter in E. coli as described earlier (Garrett et al., 1989). © The Rockefeller University Press, 0021-9525/90/09/1001/7 $2.00 The Journal of Cell Biology, Volume 111, September 199