Signals from the Ras, Rac, and Rho GTPases Converge on the Pak Protein Kinase in Rat-1 Fibroblasts

Ras is one of the most commonly mutated oncogenes and is found activated in 20 to 30% of tumors (29). Ras encodes a small G protein that binds GTP and GDP and possesses intrinsic GTPase activity. In its oncogenic form, Ras acquires a point mutation that inactivates the GTPase activity and causes it to be locked into its activated GTP-bound state. Normally, Ras is activated by growth factor receptors through its guanine nucleotide exchange factors (GEF) (12). The major oncogenic signal from Ras utilizes the serine threonine kinase Raf as the effector (52, 54). GTP-bound Ras binds and activates Raf and simultaneously recruits it to the membrane. Upon activation, Raf phosphorylates and activates another kinase, Mek, which, in turn, activates extracellular signal-activated kinase (ERK) (mitogen-activated protein kinase [MAPK]). This Ras > Raf > Mek > ERK signal is usually referred to as the MAPK cascade (34). In recent years, Ras has also been shown to bind other effectors besides Raf and activate other signaling pathways that cooperate with the Raf > ERK signal (56). The other pathways are not as well defined as the Raf cascade. The three effectors that have been most widely studied are Rin, Ral GDS, and phosphoinositide 3-OH kinase (PI 3-kinase) (1, 39, 46). Each binds Ras-GTP and can, in some experimental systems, cooperate with partially activated Raf mutants to transform cells (Rin cooperates with Abl). Further evidence of the importance of these effectors in Ras signaling comes from new Ras point mutants, known as effector mutants, that bind and activate only subsets of Ras effectors (56). RasV12S35 binds and activates Raf, RasV12G37 binds and activates Ral GDS and Rin 1, and RasV12C40 binds and activates PI 3-kinase (21, 46, 56, 57). These mutants are deficient in signaling when tested alone but cooperate to transform cells when introduced together. Signals from Ras through the alternate effectors utilize other small G proteins. Ral GDS uses Ral, and PI 3-kinase uses the small G protein Rac (46, 57). Rho and two related proteins, Rac and Cdc42, are members of the Rho family of small G proteins. These proteins are about 50% identical to Ras and regulate the actin cytoskeleton. Rho induces stress fibers and focal adhesions (44), Rac induces accumulation of actin-rich ruffles or lamellipodia at the periphery of cells (45), and Cdc42 induces microspikes or filopodia (37). Each Rho family member also activates a kinase cascade that leads to transcriptional activation similar to the MAPK cascade but not as well defined. Rho activates the ternary complex factors, and Rac and Cdc42 activate the Jun N-terminal kinase cascade JNK(SAPK) (8, 15, 19, 35). Dominant negative mutants of Rac, Rho, and Cdc42 each inhibit Ras transformation, and activated mutants cooperate with Raf to transform cells (23, 40–42). These observations suggest that the signals through the Rho family of small G proteins play essential roles in Ras transformation. The signals through Rac are directly connected to Ras (4, 45). This is because Ras and Rac both cause membrane ruffling when microinjected into cells and a dominant negative Rac mutant inhibits Ras-induced ruffling. The signal from Ras to Rac is likely to be mediated by PI 3-kinase, since RasV12C40, which activates PI 3-kinase, and activated mutants of PI 3-kinase both induce ruffles (21, 46). The mechanism that PI 3-kinase uses to activate Rac probably involves stimulation of Rac GEF by PI 3-kinase products such as phosphatidylinositol-3,4,5-triphosphate (17, 36). The immediate effector downstream of Rac in Ras signal transduction to both the JNK and actin pathways has remained elusive. One candidate has been the serine threonine kinase p65Pak (32). Pak was first isolated as a protein that binds both Rac and Cdc42 in their GTP-bound forms. Pak is homologous to Ste20, a protein kinase in the yeast Saccharomyces cerevisiae regulated by Cdc42 (28, 47). Some of the activities of Pak resemble those of Ras and Rac. For example, microinjection of Pak into some cells causes ruffling and breaks up stress fibers and membrane targeting of Pak in PC12 cells induces extension of neurites (10, 31, 48). Although microinjection of Pak can cause membrane ruffling, ruffling does not require kinase activity (48). Moreover, direct signals from Ras to Pak have not been reported. Finally, RacV12H40, an effector mutant that does not bind to Pak, still cooperates with Raf to transform cells and causes membrane ruffling when it is microinjected (20, 26, 55). These studies suggest that there may be no role for Pak in Ras transformation or signaling and even question the existence of any direct signals from Ras to Pak. We recently reported that Pak mutants that lack kinase activity behave as dominant negative mutants and inhibit Ras transformation of Rat-1 cells and Schwann cells but not of NIH 3T3 cells (50, 51). Inhibition was not the result of Rac/Cdc42 sequestering, since kinase-deficient mutants that fail to bind Rac and Cdc42 also inhibited transformation. Studies of downstream effectors suggested that Pak mutants blocked the signal from Ras to ERK. More recently, Pak was shown to facilitate ERK kinase activation by phosphorylating Mek (14, 30). Together, these data suggest that there is a Ras > Rac > Pak > ERK signal that is essential to sustain transformation in some cells. We demonstrate here that Ras activates Pak in transfection assays of Rat-1 cells but not NIH 3T3 cells. The primary signal from Ras to Pak was mediated by PI 3-kinase to Rac and Cdc42. However, effector mutants of both Ras and Rac which could not activate Pak by themselves cooperated with each other and with Raf to activate Pak. All combinations of Ras, Rac, and Rho mutants that transformed cells were capable of activating Pak and were also inhibited by Pak dominant negative mutants. These studies developed an assay system for studying the signals from Ras to Pak and suggest that activation of Pak may be essential for transformation by Ras, Rac, and Rho.