The small GTP‐binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP‐bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP‐bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N‐terminus, followed by a coiled‐coil structure approximately 600 amino acids long, and a cysteine‐rich zinc finger‐like motif and a pleckstrin homology region in the C‐terminus. The N‐terminus region including a kinase domain and a part of coiled‐coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co‐expressed with RhoA in COS cells, p160 was co‐precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo.

Abstract. Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA-and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPAinduced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.

The Rho guanosine 5'-triphosphatase (GTPase) cycles between the active guanosine triphosphate (GTP)-bound form and the inactive guanosine diphosphate-bound form and regulates cell adhesion and cytokinesis, but how it exerts these actions is unknown. The yeast two-hybrid system was used to clone a complementary DNA for a protein (designated Rhophilin) that specifically bound to GTP-Rho. The Rho-binding domain of this protein has 40 percent identity with a putative regulatory domain of a protein kinase, PKN. PKN itself bound to GTP-Rho and was activated by this binding both in vitro and in vivo. This study indicates that a serine-threonine protein kinase is a Rho effector and presents an amino acid sequence motif for binding to GTP-Rho that may be shared by a family of Rho target proteins.

Using a mouse embryo cDNA library, we conducted a two-hybrid screening to identify new partners for the small GTPase Rho. One clone obtained by this procedure contained a novel cDNA of 291 base pairs and interacted strongly with RhoA and RhoC, weakly with RhoB, and not at all with Rac1 and Cdc42Hs. Full-length cDNAs were then isolated from a mouse brain library. While multiple splicing variants were common, we identified three cDNAs with an identical open reading frame encoding a 61-kDa protein that we named rhotekin (from the Japanese "teki," meaning target). The N-terminal part of rhotekin, encoded by the initial cDNA and produced in bacteria as a glutathione S-transferase fusion protein, exhibited in vitro binding to 35S-labeled guanosine 5'-3-O-(thio)triphosphate-bound Rho, but not to Rac1 or Cdc42Hs in ligand overlay assays. In addition, this peptide inhibited both endogenous and GTPase-activating protein-stimulated Rho GTPase activity. The amino acid sequence of this region shares approximately 30% identity with the Rho-binding domains of rhophilin and a serine/threonine kinase, PKN, two other Rho target proteins that we recently identified (Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). Thus, not only is rhotekin a novel partner for Rho, but it also belongs to a wide family of proteins that bear a consensus Rho-binding sequence at the N terminus. To our knowledge, this is the first conserved sequence for Rho effectors, and we have termed this region Rho effector motif class 1.

Sand dollar eggs were microinjected with botulinum C3 exoenzyme, an ADP-ribosyltransferase from Clostridium botulinum that specifically ADP-ribosylates and inactivates rho proteins. C3 exoenzyme microinjected during nuclear division interfered with subsequent cleavage furrow formation. No actin filaments were detected in the equatorial cortical layer of these eggs by rhodamine-phalloidin staining. When microinjected into furrowing eggs, C3 exoenzyme rapidly disrupted the contractile ring actin filaments and caused regression of the cleavage furrows. C3 exoenzyme had no apparent effect on nuclear division, however, and multinucleated embryos developed from the microinjected eggs. By contrast, C3 exoenzyme did not affect the organisation of cortical actin filaments immediately after fertilisation. Only one protein (molecular weight 22000) was ADP-ribosylated by C3 exoenzyme in the isolated cleavage furrow. This protein co-migrated with ADP-ribosylated rhoA derived from human platelets when analysed by two-dimensional gel electrophoresis. These results strongly suggest that a rho-like, small GTP-binding protein is selectively involved in the organisation and maintenance of the contractile ring.

Ahstract Using the yeast two hybrid system and overlay assays we identified a putative rholrac effector, citron, which interacts with the GTP-bound forms of rho and racl, but not with cdc42. Extensive homologies to known proteins were not observed. This 183 kDa protein contains a C~H2 zinc finger, a PH domain, and a long coiled-coil forming region including 4 leucine zippers and the rholrac binding site. We recently identified three others putative rho effeetors characterized by a common rho binding motif. Citron does not share this motif and displays a distinctive protein organization, thus defining a separate class of rho partners.

Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.

Abstract. Botulinum C3 exoenzyme specifically ADPribosylates a group of ms-related small molecular weight GTP-binding proteins, rho, and inhibits their biological activity. Using this enzyme, we examined the function of rho in PMA-induced activation of lymphocyte function-associated antigen-1 (LFA-1) in a B lymphoblastoid cell line, JY. Northern blot analysis revealed that among the three rho genes, rhoA mRNA was predominantly expressed in JY cells. Consistently, only one [32p]ADP-ribosylated band was found when the lysate of the cells was subjected to ADP ribosylation by C3 exoenzyme. When the cells were cultured with C3 exoenzyme, this substrate was ADPribosylated in situ in a time-and concentrationdependent manner. Concomitant with this ADP ribosylation, PMA-induced LFA-1/intercellular adhesion molecule (ICAM)-l-dependent aggregation of JY cells was inhibited. This inhibition was blocked by prior treatment of the enzyme with an anti-C3 monoclonal antibody, and overcome by stimulation with higher concentrations of PMA. The C3 exoenzymeinduced inhibition was not affected by shaking of the cell suspension, while inhibition of aggregation by cytochalasin B was abolished by this procedure, suggesting that the inhibitory effect of the C3 exoenzyme treatment was not due to decrease in cell motility. The C3 exoenzyme treatment affected neither protein phosphorylation in JY cells before and after PMA stimulation, nor affected surface expression of LFA-1 and ICAM-1. These results suggest that rhoA protein works downstream of protein kinase C activation linking PMA stimulation to LFA-1 activation and aggregation in JY cells. R as and ras-related genes constitute a gene family encoding a series of closely related proteins with guanine nucleotide-binding activities. To date, ~40 ras and ras-related GTP-binding proteins are known, and they are divided into four subfamilies: ms, rho, tab, and others (Hall, 1990;Bourne et al., 1991). These proteins exist in two interconvertible functional states; one in an inactive GDPbound form and the other in an active GTP-bound form. In resting cells, they are present in an inactive GDP-bound form, and upon cell stimulation, converted to the active GTP-bound form and work as molecular switches linking external stimuli to various cellular responses such as growth, differentiation, and secretion. Among the ras-related GTPbinding proteins, rho proteins are believed to be involved in organization of cytoskeleton and maintenance of cell shape. There are at least three members in this family in human, which are called rhoA, B, and C (Yeramian et al., 1987;Chardin et al., 1988). rho proteins are unique among the small GTP-binding proteins in being substrates for ADP ribosylation by the exoenzyme C3 from Clostridium botulinum (Aktories et al., 1987;Morii et al., 1988;Narumiya et al., 1988;Kikuchi et al., 1988). This enzyme ADPribosylates the proteins at an asparagine residue in the putative effector domain and inhibits their biological activities presumably by interfering with their interaction ...