Involvement of B-Raf in Ras-induced Raf-1 activation.

The mechanism of Ras-induced Raf-1 activation is not fully understood. Previously, we identified a 400-kDa protein complex as a Ras-dependent Raf-1 activator. In this study, we identified B-Raf as a component of this complex. B-Raf was concentrated during the purification of the activator. Immunodepletion of B-Raf abolished the effect of the activator on Raf-1. Furthermore, B-Raf and Ras-activated Raf-1 co-operatively, when co-transfected into human embryonic kidney 293 cells. On the other hand, Ras-dependent extracellular signal-regulated kinase/mitogen-activated protein kinase kinase stimulator (a complex of B-Raf and 14-3-3) failed to activate Raf-1 in our cell-free system. These results suggest that B-Raf is an essential component of the Ras-dependent Raf-1 activator.

Galphaq-dependent activation of mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase cascade.

G-protein-coupled receptors (GPCRs) typically activate c-Jun N-terminal kinase (JNK) through the G protein betagamma subunit (Gbetagamma), in a manner dependent on Rho family small GTPases, in mammalian cells. Here we show that JNK activation by the prototypic Gq-coupled alpha1B-adrenergic receptor is mediated by the alpha subunit of Gq (Galphaq), not by Gbetagamma, using a transient transfection system in human embryonic kidney cells. JNK activation by the alpha1B-adrenergic receptor/Galphaq was selectively mediated by mitogen-activated protein kinase kinase 4 (MKK4), but not MKK7. Also, MKK4 activation by the alpha1B-adrenergic receptor/Galphaq required c-Src and Rho family small GTPases. Furthermore, activation of the alpha1B-adrenergic receptor stimulated JNK activity through Src family tyrosine kinases and Rho family small GTPases in hamster smooth muscle cells that natively express the alpha1B-adrenergic receptor. Together, these results suggest that the alpha1B-adrenergic receptor/Galphaq may up-regulate JNK activity through a MKK4 pathway dependent on c-Src and Rho family small GTPases in mammalian cells.

Epidermal growth factor stimulation of the ACK1/Dbl pathway in a Cdc42 and Grb2-dependent manner.

The tyrosine kinase ACK1 phosphorylates and activates the guanine nucleotide exchange factor Dbl, which in turn directs the Rho family GTP-binding proteins. However, the regulatory mechanism of ACK1/Dbl signaling in response to extracellular stimuli remains poorly understood. Here we describe that epidermal growth factor stimulates the ACK1/Dbl pathway, leading to actin cytoskeletal rearrangements. The role of the two ACK1-binding proteins Cdc42 and Grb2 was assessed by overexpression of the Cdc42/Rac interactive binding domain and a dominant-negative Grb2 mutant, respectively. Specific inhibition of the interaction of ACK1 with Cdc42 or Grb2 by the use of these constructs diminished tyrosine phosphorylation of both ACK1 and Dbl in response to EGF. Therefore, the activation of ACK1 and subsequent downstream signaling require both Cdc42-dependent and Grb2-dependent processes within the cell. In addition, we show that EGF transiently induces formation of the focal complex and stress fibers when ACK1 was ectopically expressed. The induction of these structures was totally sensitive to the action of botulinum toxin C from Clostridium botulinum, suggesting a pivotal role of Rho. These results provide evidence that ACK1 acts as a mediator of EGF signals to Rho family GTP-binding proteins through phosphorylation and activation of GEFs such as Dbl.

Parallel regulation of mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 in Gq-signaling cascade.

Heterotrimeric G protein G(q) stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby alpha and betagamma subunits of G(q) activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Galpha(q) and Gbetagamma was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4. In addition, Galpha(q) and Gbetagamma stimulated MKK3 and MKK6 activities. The MKK3 and MKK6 activations by Galpha(q), but not by Gbetagamma, were dependent on phospholipase C and c-Src. Galpha(q) stimulated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent manner. On the other hand, Gbetagamma activated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-, Rac-, and Cdc42-dependent manner. Gbetagamma-induced MKK3 and MKK6 activations were dependent on a tyrosine kinase other than c-Src. These results suggest that Galpha(q) and Gbetagamma stimulate the activity of p38 MAPK by regulating MKK3 and MKK6 through parallel signaling pathways.

Ral promotes anchorage-independent growth of a human fibrosarcoma, HT1080.

Ral has been shown to act downstream of Ras oncoprotein. However, the role of Ral in Ras-induced cellular transformation has not been fully understood. To test the involvement of Ral in Ras-induced anchorage-independent growth, we ectopically expressed Ral mutants in HT1080 cells, whose ability to grow in the absence of anchorage depends on the oncogenic mutation of N-ras. Expression of an activated mutant of Ral resulted in enhanced growth of HT1080 cells in soft agar, whereas a dominant-negative mutant of Ral inhibited their anchorage-independent growth. Moreover, the activated Ral mutant decreased the amount of p27(Kip1) in the absence of adhesion, while the dominant-negative mutant increased it. These results suggest that Ral is involved in the Ras-dependent anchorage-independent growth of HT1080 cells by regulating p27(Kip1).

LUCA15, a putative tumour suppressor gene encoding an RNA-binding nuclear protein, is down-regulated in ras-transformed Rat-1 cells.

BACKGROUND: The proliferation of mammalian cells is controlled by various intracellular mitogenic signalling pathways. In the intracellular pathways, Ras is involved in the activation of proto-oncogenes such as an immediate early gene c-fos. The somatic mutations of ras genes that elicit the constitutive activation of Ras have been found in tumours. Although these findings suggest that the constitutive activation of Ras-mediated pathways alters the expression of a set of genes involving tumorigenesis, these genes have not yet fully been studied. RESULTS: To study the up- or down-regulated genes in ras-transformed cells, we analysed Rat-1 transfectants expressing Ras(G12V) mutant protein in response to isopropyl-1-beta-thio-D-galactoside using a differential display. We found that the mRNA level of rat homologue of LUCA15, which has been cloned initially as a putative tumour suppressor gene mapped on human chromosome 3, was down-regulated by the expression of Ras(G12V). Epitope-tagged LUCA15 protein was localized in nuclei and had the ability to bind poly(G) RNA homopolymers in vitro. Moreover, ectopic expression of LUCA15 in human fibrosarcoma HT1080 cells suppressed the cell growth. CONCLUSION: These results demonstrate that LUCA15 is one of the down-regulated genes in ras-transformed cells, and suggests that LUCA15 may function as a negative regulator of cell proliferation by the alteration of its mRNA level.

MgcRacGAP is involved in the control of growth and differentiation of hematopoietic cells.

In a search for key molecules that prevent murine M1 leukemia cells from undergoing interleukin (IL)-6-induced differentiation into macrophages, we isolated an antisense complementary DNA (cDNA) that encodes full-length mouse MgcRac-GTPase-activating protein (GAP) through functional cloning. Forced expression of this antisense cDNA profoundly inhibited IL-6-induced differentiation of M1 cells into macrophage lineages. We also isolated a full-length human MgcRacGAP cDNA, which encodes an additional N-terminal polypeptide of 105 amino acid residues compared with the previously published human MgcRacGAP. In human HL-60 leukemic cells, overexpression of the full-length form of human MgcRacGAP alone induced growth suppression and macrophage differentiation associated with hypervacuolization and de novo expression of the myelomonocytic marker CD14. Analyses using a GAP-inactive mutant and 2 deletion mutants of MgcRacGAP indicated that the GAP activity was dispensable, but the myosin-like domain and the cysteine-rich domain were indispensable for growth suppression and macrophage differentiation. The present results indicated that MgcRacGAP plays key roles in controlling growth and differentiation of hematopoietic cells through mechanisms other than regulating Rac GTPase activity.

Involvement of Ras and Ral in chemotactic migration of skeletal myoblasts.

In skeletal myoblasts, Ras has been considered to be a strong inhibitor of myogenesis. Here, we demonstrate that Ras is involved also in the chemotactic response of skeletal myoblasts. Expression of a dominant-negative mutant of Ras inhibited chemotaxis of C2C12 myoblasts in response to basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1), key regulators of limb muscle development and skeletal muscle regeneration. A dominant-negative Ral also decreased chemotactic migration by these growth factors, while inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase (MEK) showed no effect. Activation of the Ras-Ral pathway by expression of an activated mutant of either Ras, the guanine-nucleotide dissociation stimulator for Ral, or Ral resulted in increased motility of myoblasts. The ability of Ral to stimulate motility was reduced by introduction of a mutation which prevents binding to Ral-binding protein 1 or phospholipase D. These results suggest that the Ras-Ral pathway is essential for the migration of myoblasts. Furthermore, we found that Ras and Ral are activated in C2C12 cells by bFGF, HGF and IGF-1 and that the Ral activation is regulated by the Ras- and the intracellular Ca(2+)-mediated pathways. Taken together, our data indicate that Ras and Ral regulate the chemotactic migration of skeletal muscle progenitors.

Thrombin-induced inhibition of myoblast differentiation is mediated by Gbetagamma.

Thrombin has been shown to inhibit skeletal muscle differentiation. However, the mechanisms by which thrombin represses myogenesis remain unknown. Since the thrombin receptor couples to G(i), G(q/11) and G(12), we examined which subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (Galpha(i), Galpha(q/11), Galpha(12) or Gbetagamma) participate in the thrombin-induced inhibition of C2C12 myoblast differentiation. Galpha(i2) and Galpha(11) had no inhibitory effect on the myogenic differentiation. Galpha(12) prevented only myoblast fusion, whereas Gbetagamma inhibited both the induction of skeletal muscle-specific markers and the myotube formation. In addition, the thrombin-induced reduction of creatine kinase activity was blocked by the C-terminal peptide of beta-adrenergic receptor kinase, which is known to sequester free Gbetagamma. These results suggest that the thrombin-induced inhibition of muscle differentiation is mainly mediated by Gbetagamma.

Positive regulation of skeletal myogenesis by R-Ras.

Most of the proteins in the Ras-family proteins, including Ras, Rap and TC21, have been reported to be strong inhibitors of skeletal myogenesis. Here we show that R-Ras, another member of this family, promotes terminal differentiation of C2C12 skeletal myoblasts. In contrast to Ras, which induced a markedly transformed phenotype of C2C12 cells, an activated mutant of R-Ras (R-RasQ87L) did not exhibit any inhibitory effect on the differentiation of C2C12 cells, but enhanced the formation of multinucleated myotubes. Although R-RasQ87L showed little effect on induction of two muscle-specific proteins, creatine kinase and myogenin, it prevented cell death during myoblast differentiation, probably through Akt activation and Bcl-xL induction. Motility of C2C12 cells, which may be involved in fusion of myoblasts, was also stimulated by R-RasQ87L. Furthermore, we observed a transient activation of endogenous R-Ras during differentiation of C2C12 cells. The ectopic expression of R-Ras GAP inhibited the differentiation. These results suggest that R-Ras has a positive effect on the terminal differentiation of myoblasts and may be involved in the program of skeletal myogenesis.

G(i)-dependent activation of c-Jun N-terminal kinase in human embryonal kidney 293 cells.

Heterotrimeric G proteins stimulate the activities of two stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase in mammalian cells. In this study, we examined whether alpha subunits of G(i) family activate JNK using transient expression system in human embryonal kidney 293 cells. Constitutively activated mutants of Galpha(i1), Galpha(i2), and Galpha(i3) increased JNK activity. In contrast, constitutively activated Galpha(o) and Galpha(z) mutants did not stimulate JNK activity. To examine the mechanism of JNK activation by Galpha(i), kinase-deficient mutants of mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7), which are known to be JNK activators, were transfected into the cells. However, Galpha(i)-induced JNK activation was not blocked effectively by kinase-deficient MKK4 and MKK7. In addition, activated Galpha(i) mutant failed to stimulate MKK4 and MKK7 activities. Furthermore, JNK activation by Galpha(i) was inhibited by dominant-negative Rho and Cdc42 and tyrosine kinase inhibitors, but not dominant-negative Rac and phosphatidylinositol 3-kinase inhibitors. These results indicate that Galpha(i) regulates JNK activity dependent on small GTPases Rho and Cdc42 and on tyrosine kinase but not on MKK4 and MKK7.

Formation of the Ras dimer is essential for Raf-1 activation.

Although it is well established that Ras requires membrane localization for activation of its target molecule, Raf-1, the reason for this requirement is not fully understood. In this study, we found that modified Ras, which is purified from Sf9 cells, could activate Raf-1 in a cell-free system, when incorporated into liposome. Using a bifunctional cross-linker and a protein-fragmentation complementation assay, we detected dimer formation of Ras in the liposome and in the intact cells, respectively. These results suggest that dimerization of Ras in the lipid membrane is essential for activation of Raf-1. To support this, we found that, when fused to glutathione S-transferase (GST), unprocessed Ras expressed in Escherichia coli could bypass the requirement for liposome. A Ras-dependent Raf-1 activator, which we previously reported (Mizutani, S., Koide, H., and Kaziro, Y. (1998) Oncogene 16, 2781-2786), was still required for Raf-1 activation by GST-Ras. Furthermore, an enforced dimerization of unmodified oncogenic Ras mutant in human embryonic kidney (HEK) 293 cells, using a portion of gyrase B or estrogen receptor, also resulted in activation of Raf-1. From these results, we conclude that membrane localization allows Ras to form a dimer, which is essential, although not sufficient, for Raf-1 activation.

Induction of rac-guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) following phosphorylation by the nonreceptor tyrosine kinase Src.

Ras-GRF1/CDC25(Mm) has been implicated as a Ras-guanine nucleotide exchange factor (GEF) expressed in brain. Ras-GEF activity of Ras-GRF1 is augmented in response to Ca(2+) influx and G protein betagamma subunit (Gbetagamma) stimulation. Ras-GRF1 also acts as a GEF toward Rac, but not Rho and Cdc42, when activated by Gbetagamma-mediated signals. Tyrosine phosphorylation of Ras-GRF1 is critical for the induction of Rac-GEF activity as evidenced by inhibition by tyrosine kinase inhibitors. Herein, we show that the nonreceptor tyrosine kinase Src phosphorylates Ras-GRF1, thereby inducing Rac-GEF activity. Ras-GRF1 transiently expressed with v-Src was tyrosine-phosphorylated and showed significant GEF activity toward Rac, but not Rho and Cdc42, which was comparable with that induced by Gbetagamma. In contrast, Ras-GEF activity remained unchanged. The recombinant c-Src protein phosphorylated affinity-purified glutathione S-transferase-tagged Ras-GRF1 in vitro and thereby elicited Rac-GEF activity. Taken together, tyrosine phosphorylation by Src is sufficient for the induction of Rac-GEF activity of Ras-GRF1, which may imply the involvement of Src downstream of Gbetagamma to regulate Ras-GRF1.

Activation of the guanine nucleotide exchange factor Dbl following ACK1-dependent tyrosine phosphorylation.

Signals triggered by diverse receptors modulate the activity of Rho family proteins, although the regulatory mechanism remains largely unknown. On the basis of their biochemical activity as guanine nucleotide exchange factors (GEFs), Dbl family proteins are believed to be implicated in the regulation of Rho family GTP-binding proteins in response to a variety of extracellular stimuli. Here we show that GEF activity of full-length proto-Dbl is enhanced upon tyrosine phosphorylation. When transiently coexpressed with the activated form of the non-receptor tyrosine kinase ACK1, a downstream target of Cdc42, Dbl became tyrosine-phosphorylated. In vitro GEF activity of Dbl toward Rho and Cdc42 was augmented following tyrosine phosphorylation. Moreover, accumulation of the GTP-bound form of Rho and Rac within the cell paralleled ACK-1-dependent tyrosine phosphorylation of Dbl. Consistently, activation of c-Jun N-terminal kinase downstream of Rho family GTP-binding proteins was also enhanced when Dbl was tyrosine-phosphorylated. Collectively, these findings suggest that the tyrosine kinase ACK1 may act as a regulator of Dbl, which in turn activates Rho family proteins.

Analysis of Ras-dependent signals that prevent caspase-3 activation and apoptosis induced by cytokine deprivation in hematopoietic cells.

In hematopoietic cells, Ras has been implicated in signaling pathways that prevent apoptosis triggered by deprivation of cytokines, such as interleukin-3 (IL-3). However, the mechanism whereby Ras suppresses cell death remains incompletely understood. We have investigated the role of Ras in IL-3 signal transduction by using the cytokine-dependent BaF3 cell line. Herein, we show that the activation of the pro-apoptotic protease caspase-3 upon IL-3 removal is suppressed by expression of activated Ras, which eventually prevents cell death. For caspase-3 suppression, the Raf/extracellular signal-regulated kinase (ERK)- or phosphatidylinositol 3-kinase (PI3-K)/Akt-mediated signaling pathway downstream of Ras was required. However, inhibition of both pathways did not block activated Ras-dependent suppression of cell death-associated phenotypes, such as nuclear DNA fragmentation. Thus, a pathway that is independent of both Raf/ERK and PI3-K/Akt pathways may function downstream of Ras, preventing activated caspase-3-initiated apoptotic processes. Conditional activation of c-Raf-1 also suppressed caspase-3 activation and subsequent cell death without affecting Akt activity, providing further evidence for a PI3-K/Akt-independent mechanism.

G protein betagamma subunits induce stress fiber formation and focal adhesion assembly in a Rho-dependent manner in HeLa cells.

In fibroblasts, the G protein alpha subunits Galpha(12) and Galpha(13) stimulate Rho-dependent stress fiber formation and focal adhesion assembly, whereas G protein betagamma subunits instead exert a disruptive influence. We show here that the latter can, however, stimulate the formation of stress fibers and focal adhesions in epithelial-like HeLa cells. Transient expression of beta(1) with gamma(2), gamma(5), gamma(7), and gamma(12) in quiescent HeLa cells induced stress fiber formation and focal adhesion assembly as did expression of the constitutively active Galpha(12). Co-expression of betagamma with Galpha(i2) and the C-terminal fragment of the beta-adrenergic receptor kinase, both of which are known to bind and sequester free betagamma, blocked betagamma-induced stress fiber and focal adhesion formation. Inhibition was also noted with co-expression of a dominant negative mutant of Rho. Botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho, and a Rho-associated protein kinase inhibitor, Y-27632, similarly inhibited betagamma-induced stress fiber and focal adhesion assembly. These results indicate that G protein betagamma subunits regulate Rho-dependent actin polymerization in HeLa cells.

Association of the proto-oncogene product dbl with G protein betagamma subunits.

The Rho family of GTP-binding proteins has been implicated in the regulation of various cellular functions including actin cytoskeleton-dependent morphological change. Its activity is directed by intracellular signals mediated by various types of receptors such as G protein-coupled receptors. However, the mechanisms underlying receptor-dependent regulation of Rho family members remain incompletely understood. The guanine nucleotide exchange factor (GEF) Dbl targets Rho family proteins thereby stimulating their GDP/GTP exchange, and thus is believed to be involved in receptor-mediated regulation of the proteins. Here, we show the association of Dbl with G protein betagamma subunits (Gbetagamma) in transient co-expression and cell-free systems. An amino-terminal portion conserved among a subset of Dbl family proteins is sufficient for the binding of Gbetagamma. In fact, Ost and Kalirin, which contain this Gbetagamma-binding motif, also associate with Gbetagamma. c-Jun N-terminal kinase was synergistically activated upon co-expression of Dbl and Gbeta in a dominant-negative Rho-sensitive manner. However, GEF activity of Dbl toward Rho as measured by in vitro GDP binding assays remained unaffected following Gbetagamma binding, suggesting that additional signals may be required for the regulation of Dbl.

The Src family tyrosine kinase is involved in Rho-dependent activation of c-Jun N-terminal kinase by Galpha12.

Gt12, a member of alpha subunit of heterotrimeric G protein G12 subfamily, has been shown to stimulate c-Jun N-terminal kinase (JNK) activity through the low molecular weight GTP-binding proteins Ras, Rac, and Cdc42. In this study using the transient expression of a constitutively activated mutant of Galpha12 (Galpha12Q229L) in human embryonic kidney (HEK) 293 cells, we found that Rho and Src family kinase are also involved in the Galpha12-induced activation of JNK. The activation of JNK by Galpha12Q229L was inhibited by dominant-negative RhoA(T19N), and botulinum C3 exoenzyme which specifically inactivates Rho. In addition, the expression of activated RhoA(G14V) elevated JNK activity in HEK 293 cells. The Galpha12Q229L-stimulated activation of JNK was blocked by a specific inhibitor of protein tyrosine kinases (PP2), and C-terminal Src kinase (Csk). Moreover, we observed that Galpha12Q229L stimulated Src family kinase activity and v-Src induced JNK activation. Interestingly, the v-Src-induced activation of JNK was inhibited by dominant-negative RhoA(T19N). In contrast, Csk did not inhibit the JNK activation by activated RhoA(G14V). These results suggest that Rho and Src family kinase are required for the Galpha12-induced JNK activation, and that Src family kinase acts upstream of Rho activation in the JNK pathway.

Activation of c-fos promoter by Gbetagamma-mediated signaling: involvement of Rho and c-Jun N-terminal kinase.

Several extracellular stimuli mediated by G protein-coupled receptors activate c-fos promoter. Recently, we and other groups have demonstrated that signals from G protein-coupled receptors stimulate mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. The activation of these three MAPKs is mediated in part by the G protein betagamma subunit (Gbetagamma). In this study, we characterized the signals from Gbetagamma to c-fos promoter using transient transfection of c-fos luciferase into human embryonal kidney 293 cells. Activation of m2 muscarinic acetylcholine receptor and overexpression of Gbetagamma, but not constitutively active Galphai2, stimulated c-fos promoter activity. The c-fos promoter activation by m2 receptor and Gbetagamma was inhibited by beta-adrenergic receptor kinase C-terminal peptide (betaARKct), which functions as a Gbetagamma antagonist. MEK1 inhibitor PD98059 and kinase-deficient mutant of JNK kinase, but not p38 MAPK inhibitor SB203580, attenuated the m2 receptor- and Gbetagamma-induced c-fos promoter activation. Activated mutants of Ras and Rho stimulated the c-fos promoter activity, and the dominant negative mutants of Ras and Rho inhibited the c-fos promoter activation by m2 receptor and Gbetagamma. Moreover, c-fos promoter activation by m2 receptor, Gbetagamma, and active Rho, but not active Ras, was inhibited by botulinum C3 toxin. These data indicated that both Ras- and Rho-dependent signaling pathways are essential for c-fos promoter activation mediated by Gbetagamma.

Anti-apoptotic function of Rac in hematopoietic cells.

The small GTP-binding protein Rac plays a pivotal role in the regulation of diverse physiological events including reorganization of the actin cytoskeleton, cell cycle progression, and transformation. Here we show an anti-apoptotic effect of Rac in interleukin-3-dependent murine hematopoietic BaF3 cells. Activated Rac(G12V), when ectopically expressed in BaF3 cells, rendered the cells resistant to apoptosis upon interleukin-3 deprivation, while activated mutants of Rho and Cdc42 displayed no significant anti-apoptotic effect. In contrast to activated Ras, which also supports cell survival in the absence of interleukin-3, Rac required fetal bovine serum for the prevention of cell death. The involvement of phosphatidylinositol 3-kinase downstream of Rac was demonstrated by the inhibition of Rac-induced cell survival by wortmannin and LY294002 and the presence of phosphatidylinositol kinase activity in the Rac immunoprecipitate. Furthermore, the serine/threonine kinase Akt was stimulated by activated Rac and fetal bovine serum in a synergistic manner. Rac-induced Akt activation was mediated by phosphorylation of threonine-308 and serine-473. In addition to the phosphatidylinositol 3-kinase/Akt pathway, the p38 mitogen-activated protein kinase pathway was crucial for Rac-dependent survival, whereas p38 mitogen-activated protein kinase nas not implicated in Ras-induced anti-apoptotic signaling. These findings provide evidence for the involvement of Rac in survival signaling of hematopoietic cells.