Signals from the Ras, Rac, and Rho GTPases converge on the Pak protein kinase in Rat-1 fibroblasts.
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Ras plays a key role in regulating cellular proliferation, differentiation, and transformation. Raf is the major effector of Ras in the Ras > Raf > Mek > extracellular signal-activated kinase (ERK) cascade. A second effector is phosphoinositide 3-OH kinase (PI 3-kinase), which, in turn, activates the small G protein Rac. Rac also has multiple effectors, one of which is the serine threonine kinase Pak (p65(Pak)). Here we show that Ras, but not Raf, activates Pak1 in cotransfection assays of Rat-1 cells but not NIH 3T3 cells. We tested agents that activate or block specific components downstream of Ras and demonstrate a Ras > PI 3-kinase > Rac/Cdc42 > Pak signal. Although these studies suggest that the signal from Ras through PI 3-kinase is sufficient to activate Pak, additional studies suggested that other effectors contribute to Pak activation. RasV12S35 and RasV12G37, two effector mutant proteins which fail to activate PI 3-kinase, did not activate Pak when tested alone but activated Pak when they were cotransfected. Similarly, RacV12H40, an effector mutant that does not bind Pak, and Rho both cooperated with Raf to activate Pak. A dominant negative Rho mutant also inhibited Ras activation of Pak. All combinations of Rac/Raf and Ras/Raf and Rho/Raf effector mutants that transform cells cooperatively stimulated ERK. Cooperation was Pak dependent, since all combinations were inhibited by kinase-deficient Pak mutants in both transformation assays and ERK activation assays. These data suggest that other Ras effectors can collaborate with PI 3-kinase and with each other to activate Pak. Furthermore, the strong correlation between Pak activation and cooperative transformation suggests that Pak activation is necessary, although not sufficient, for cooperative transformation of Rat-1 fibroblasts by Ras, Rac, and Rho. (+info)
Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity.
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We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells. (+info)
BLNK required for coupling Syk to PLC gamma 2 and Rac1-JNK in B cells.
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Signaling through the B cell receptor (BCR) is essential for B cell function and development. Despite the key role of Syk in BCR signaling, little is known about the mechanism by which Syk transmits downstream effectors. BLNK (B cell LiNKer protein), a substrate for Syk, is now shown to be essential in activating phospholipase C (PLC)gamma 2 and JNK. The BCR-induced PLC gamma 2 activation, but not the JNK activation, was restored by introduction of PLC gamma 2 membrane-associated form into BLNK-deficient B cells. As JNK activation requires both Rac1 and PLC gamma 2, our results suggest that BLNK regulates the Rac1-JNK pathway, in addition to modulating PLC gamma 2 localization. (+info)
Synergistic activation of JNK/SAPK by interleukin-1 and platelet-derived growth factor is independent of Rac and Cdc42.
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The c-Jun N-terminal kinases (JNKs) are activated strongly by inflammatory cytokines and environmental stresses, but only weakly by growth factors. Here we show that platelet-derived growth factor (PDGF) strongly potentiates activation of JNK by interleukin 1 (IL-1) in human fibroblasts and a pig aortic endothelial (PAE) cell line. This synergistic activation of JNK by IL-1 and PDGF was unaffected by bacterial toxins that inactivate Rho proteins and Ras. Since Rho proteins have been implicated in JNK activation, their possible involvement was investigated further using stably expressed, inducible N17 or V12 mutants in PAE cell lines. N17 Rac non-selectively reduced JNK activity by 30% in resting or stimulated cells (IL-1 alone, or with PDGF). N17 Cdc42 had no effect. V12 Rac weakly activated JNK and synergized with IL-1, but not with PDGF. V12 Cdc42 weakly activated JNK, but synergized with PDGF and not IL-1. Our results imply that Rho GTPases are not directly involved in mediating IL-1-induced JNK activation, or in the potentiation of this activation by PDGF. (+info)
Rac1 is required for the formation of three germ layers during gastrulation.
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The Rac1, a member of the Rho family proteins, regulates actin organization of cytoskeleton and cell adhesion. We used genetic analysis to elucidate the role of Rac1 in mouse embryonic development. The rac1 deficient embryos showed numerous cell deaths in the space between the embryonic ectoderm and endoderm at the primitive streak stage. Investigation of the primary epiblast culture isolated from rac1 deficient embryos indicated that Rac1 is involved in lamellipodia formation, cell adhesion and cell migration in vivo. These results suggest that Rac1-mediated cell adhesion is essential for the formation of three germ layers during gastrulation. (+info)
ARF6 requirement for Rac ruffling suggests a role for membrane trafficking in cortical actin rearrangements.
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The ARF6 GTPase regulates a novel endosomal-plasma membrane recycling pathway and influences cortical actin remodeling. Here we examined the relationship between ARF6 and Rac1, a Rho family GTPase, implicated in cortical actin rearrangements. Endogenous Rac1 colocalized with ARF6 at the plasma membrane and on the ARF6 recycling endosome in untransfected HeLa and primary human fibroblast cells. In transfected HeLa cells Rac1 and ARF6 also colocalized. Cells expressing wild-type ARF6 or Rac1 formed actin-containing surface protrusions and membrane ruffles, respectively, upon treatment with the G protein activator aluminum fluoride. Aluminum fluoride-treatment of cells transfected with equivalent amounts of plasmid resulted in enhanced membrane ruffling, with protrusions appearing as Rac expression was lowered. Co-expression of the dominant negative, GTP binding-defective ARF6 T27N mutant inhibited the aluminum fluoride-induced ruffling observed in cells expressing Rac1, and the constitutive ruffling observed in cells expressing the activated Rac1 Q61L mutant. In contrast, co-expression of the GTP-binding-defective, T17N mutant of either Rac1 or Cdc42 with ARF6 did not inhibit the aluminum fluoride-induced surface protrusions, nor did inactivation of Rho with C3-transferase. These observations suggest that ARF6, a non-Rho family GTPase, can, by itself, alter cortical actin and can influence the ability of Rac1 to form lamellipodia, in part, by regulating its trafficking to the plasma membrane. (+info)
Myelin and collapsin-1 induce motor neuron growth cone collapse through different pathways: inhibition of collapse by opposing mutants of rac1.
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Precise growth cone guidance is the consequence of a continuous reorganization of actin filament structures within filopodia and lamellipodia in response to inhibitory and promoting cues. The small GTPases rac1, cdc42, and rhoA are critical for regulating distinct actin structures in non-neuronal cells and presumably in growth cones. Collapse, a retraction of filopodia and lamellipodia, is a typical growth cone behavior on contact with inhibitory cues and is associated with depolymerization and redistribution of actin filaments. We examined whether small GTPases mediate the inhibitory properties of CNS myelin or collapsin-1, a soluble semaphorin, in chick embryonic motor neuron cultures. As demonstrated for collapsin-1, CNS myelin-evoked growth cone collapse was accompanied by a reduction of rhodamine-phalloidin staining most prominent in the growth cone periphery, suggesting actin filament disassembly. Specific mutants of small GTPases were capable of desensitizing growth cones to CNS myelin or collapsin-1. Adenoviral-mediated expression of constitutively active rac1 or rhoA abolished CNS myelin-induced collapse and allowed remarkable neurite extension on a CNS myelin substrate. In contrast, expression of dominant negative rac1 or cdc42 negated collapsin-1-induced growth cone collapse and promoted neurite outgrowth on a collapsin-1 substrate. These findings suggest that small GTPases can modulate the signaling pathways of inhibitory stimuli and, consequently, allow the manipulation of growth cone behavior. However, the fact that opposite mutants of rac1 were effective against different inhibitory stimuli speaks against a universal signaling pathway underlying growth cone collapse. (+info)
Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense.
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In mammals, the Rho family GTPase Rac2 is restricted in expression to hematopoietic cells, where it is coexpressed with Rac1. Rac2-deficient mice were created to define the physiological requirement for two near-identical Rac proteins in hematopoietic cells. rac2-/- neutrophils displayed significant defects in chemotaxis, in shear-dependent L-selectin-mediated capture on the endothelial substrate Glycam-1, and in both F-actin generation and p38 and, unexpectedly, p42/p44 MAP kinase activation induced by chemoattractants. Superoxide production by rac2-/- bone marrow neutrophils was significantly reduced compared to wild type, but it was normal in activated peritoneal exudate neutrophils. These defects were reflected in vivo by baseline neutrophilia, reduced inflammatory peritoneal exudate formation, and increased mortality when challenged with Aspergillus fumigatus. Rac2 is an essential regulator of multiple specialized neutrophil functions. (+info)