ROCKs, or Rho kinases, are serine/threonine kinases that are involved in many aspects of cell motility, from smooth-muscle contraction to cell migration and neurite outgrowth. Recent experiments have defined new functions of ROCKs in cells, including centrosome positioning and cell-size regulation, which might contribute to various physiological and pathological states.

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Scatter factor/hepatocyte growth factor (SF/HGF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of cell colonies followed by disruption of cell-cell junctions and subsequent cell scattering. These responses are accompanied by changes in the actin cytoskeleton, including increased membrane ruffling and lamellipodium extension, disappearance of peripheral actin bundles at the edges of colonies, and an overall decrease in stress fibers. The roles of the small GTP-binding proteins Ras, Rac, and Rho in regulating responses to SF/HGF were investigated by microinjection. Inhibition of endogenous Ras proteins prevented SF/HGF-induced actin reorganization, spreading, and scattering, whereas microinjection of activated H-Ras protein stimulated spreading and actin reorganization but not scattering. When a dominant inhibitor of Rac was injected, SF/HGF- and Ras-induced spreading and actin reorganization were prevented, although activated Rac alone did not stimulate either response. Microinjection of activated Rho inhibited spreading and scattering, while inhibition of Rho function led to the disappearance of stress fibers and peripheral bundles but did not prevent SF/HGF-induced motility. We conclude that Ras and Rac act downstream of the SF/HGF receptor p190Met to mediate cell spreading but that an additional signal is required to induce scattering.

Lysophosphatidic acid (LPA) and bombesin rapidly stimulate the formation of focal adhesions and actin stress fibres in serum‐starved Swiss 3T3 fibroblasts, a process regulated by the small GTP binding protein Rho. To investigate further the signalling pathways leading to these responses, we have tested the roles of three intracellular signals known to be induced by LPA: activation of protein kinase C (PK‐C), Ca2+ mobilization and decreased cAMP levels. Neither PK‐C activation nor increased [Ca2+]i, alone or in combination, induced stress fibre formation, and in fact activators of PK‐C inhibited this response to LPA and bombesin. The G(i)‐mediated decrease in cAMP was not required for the response to LPA, and increased cAMP levels did not prevent stress fibre formation. In contrast, the tyrosine kinase inhibitor genistein inhibited the formation of stress fibres induced by both extracellular factors and microinjected Rho protein. Genistein also inhibited the Rho‐dependent clustering of phosphotyrosine‐containing proteins at focal adhesions, and the increased tyrosine phosphorylation of several proteins including pp125FAK, induced by LPA and bombesin. This suggests a model where Rho‐induced activation of a tyrosine kinase is required for the formation of stress fibres.

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The actin cytoskeleton is regulated by Rho family proteins: in fibroblasts, Rho mediates the formation of actin stress fibers, whereas Rac regulates lamellipodium formation and Cdc42 controls filopodium formation. We have cloned the mouse RhoE gene, whose product is a member of the Rho family that shares (except in one amino acid) the conserved effector domain of RhoA, RhoB, and RhoC. RhoE is able to bind GTP but does not detectably bind GDP and has low intrinsic GTPase activity compared with Rac. The role of RhoE in regulating actin organization was investigated by microinjection in Bac1.2F5 macrophages and MDCK cells. In macrophages, RhoE induced actin reorganization, leading to the formation of extensions resembling filopodia and pseudopodia. In MDCK cells, RhoE induced the complete disappearance of stress fibers, together with cell spreading. However, RhoE did not detectably affect the actin bundles that run parallel to the outer membranes of cells at the periphery of colonies, which are known to be dependent on RhoA. In addition, RhoE induced an increase in the speed of migration of hepatocyte growth factor/scatter factor-stimulated MDCK cells, in contrast to the previously reported inhibition produced by activated RhoA. The subcellular localization of RhoE at the lateral membranes of MDCK cells suggests a role in cell-cell adhesion, as has been shown for RhoA. These results suggest that RhoE may act to inhibit signalling downstream of RhoA, altering some RhoA-regulated responses, such as stress fiber formation, but not affecting others, such as peripheral actin bundle formation.Rho family proteins consist of 11 mammalian members, in addition to many homologs in other species, and form a subgroup of the Ras GTPases (40). Over the past few years, members of the Rho family have been implicated in many different cellular events, including actin organization, cell adhesion, membrane trafficking, and transcriptional regulation (52). Like all members of the Ras superfamily, they function as molecular switches, cycling between an active GTP-bound form and an inactive GDP-bound form (recently reviewed in reference 52). This property is determined by five primary sequence motifs that are highly conserved evolutionarily among members of the Ras superfamily (6). The activity of Rho GTPases is determined by the ratio of their GTP-bound and GDP-bound states and is regulated by the opposing effects of guanine nucleotide exchange factors, which enhance the exchange of bound GDP for bound GTP, and the GTPaseactivating proteins (GAPs), which increase the intrinsic rate of hydrolysis of bound GTP. In addition, guanine nucleotide dissociation inhibitors can inhibit both the exchange of nucleotides and the hydrolysis of bound GTP (4).Rho, Rac, and Cdc42 are three members of the Rho family known to be involved in regulating the organization of the actin cytoskeleton. In Swiss 3T3 fibroblasts, Rho regulates the formation of actin stress fibers, whereas Rac regulates lamellipodium formation and Cdc42 regulates filopodium formatio...

to rac-dependent actin polymerization at the plasma membrane, and membrane ruffling. The product of the breakpoint cluster region gene bcr, rho GTPase accelerating protein (rhoGAP) and rasGAP-associated p190 share structurally related rho GAP domains, and possess GAP activity for rho family members in vitro. We have directly compared the activities of the isolated GAP domains of these three proteins in regulating different rho family GTPases, both by in vitro assays and by microinjection, to address their possible physiologic functions. We show that bcr accelerates the GTPase activity of rac, but not rho in vitro, and inhibits rac-mediated membrane ruffling, but not rho-mediated stress fibre formation, after microinjection into Swiss 3T3 fibroblasts. In vitro, rhoGAP has a striking preference for G25K as a substrate, whilst p190GAP has marked preferential activity for rho. Furthernore, p190 preferentially inhibits rho-mediated stress fibre formation in vivo. Our data suggest that p190, rhoGAP and bcr play distinct roles in signalling pathways mediated through different rho family GTPases.

The cellular responses to ras and nuclear oncogenes were investigated in purified populations of rat Schwann cells. v‐Ha‐ras and SV40 large T cooperate to transform Schwann cells, inducing growth in soft agar and allowing proliferation in the absence of added mitogens. Expression of large T alone reduces their growth factor requirements but is insufficient to induce full transformation. In contrast, expression of v‐Ha‐ras leads to proliferation arrest in Schwann cells expressing a temperature‐sensitive mutant of large T at the restrictive temperature. Cells arrest in either the G1 or G2/M phases of the cell cycle, and can re‐enter cell division at the permissive temperature even after prolonged periods at the restrictive conditions. Oncogenic ras proteins also inhibit DNA synthesis when microinjected into Schwann cells. Adenovirus E1a and c‐myc oncogenes behave similarly to SV40 large T. They cooperate with Ha‐ras oncogenes to transform Schwann cells, and prevent ras‐induced growth arrest. Thus nuclear oncogenes fundamentally alter the response of Schwann cells to a ras oncogene from cell cycle arrest to transformation.