Distinct functions of H-Ras and K-Ras in proliferation and survival of primary hepatocytes due to selective activation of ERK and PI3K

Ras proteins mediate signals both via extracellular signal-regulated kinase 1 and 2 (ERK), and phosphoinositide 3-kinase (PI3K). These signals are key events in cell protection and compensatory cell growth after exposure to cell damaging and pro-apoptotic stimuli, thus maintaining homeostasis. By transfection techniques, we found that both H-Ras and K-Ras were expressed and appeared functionally active in primary hepatocytes. We compared the ability of H-Ras and K-Ras homologues to preferentially activate one of the two pathways, thereby differentially controlling cell survival and growth. We found that ectopic expression of dominant negative (DN) H-RasN17, but not DN K-RasN17, efficiently inhibited both phosphorylation and translocation of ERK to the nuclear compartment, which are prerequisites for cell cycle progression. Furthermore, ectopic expression of constitutive active (CA) H-RasV12, but not CA K-RasV12, potentiated EGF-induced proliferation. We also found that expression of CA mutants of either H-Ras or K-Ras protected hepatocytes from transforming growth factor-beta1 (TGF-beta1)-induced apoptosis. However, H-Ras-induced survival was mediated by ERK/RSK as well as by PI3K, whereas K-Ras-induced survival was mediated by PI3K only. In conclusion, H-Ras and K-Ras had differential functions in proliferation and survival of primary hepatocytes. H-Ras was the major mediator of ERK-induced proliferation and survival, whereas H-Ras and K-Ras both mediated PI3K-induced survival.     

Ras activation induces expression of raet1 family NK receptor ligands

NK cells play a crucial role in innate immunity against tumors. In many human tumors, Ras is chronically active, and tumor cells frequently express ligands for the activating NK cell receptor NKG2D. In this study, we report that Ras activation upregulates the expression of Raet1 protein family members Rae1α and Rae1β in mouse and ULBP1-3 in human cells. In addition, Ras also induced MHC class I chain-related protein expression in some human cell lines. Overexpression of the constitutively active H-RasV12 mutant was sufficient to induce NKG2D ligand expression. H-RasV12-induced NKG2D ligand upregulation depended on Raf, MAPK/MEK, and PI3K, but not ATM or ATR, two PI3K-like kinases previously shown to induce NKG2D ligand expression. Analysis of the 5' untranslated regions of Raet1 family members suggested the presence of features known to impair translation initiation. Overexpression of the rate-limiting translation initiation factor eIF4E induced Rae1 and ULBP1 expression in a Ras- and PI3K-dependent manner. Upregulation of NKG2D ligands by H-RasV12 increased sensitivity of cells to NK cell-mediated cytotoxicity. In summary, our data suggest that chronic Ras activation is linked to innate immune responses, which may contribute to immune surveillance of H-Ras transformed cells.     

Apoptosis suppression by Raf-1 and MEK1 requires MEK- and phosphatidylinositol 3-kinase-dependent signals

Two Ras effector pathways leading to the activation of Raf-1 and phosphatidylinositol 3-kinase (PI3K) have been implicated in the survival signaling by the interleukin 3 (IL-3) receptor. Analysis of apoptosis suppression by Raf-1 demonstrated the requirement for mitochondrial translocation of the kinase in this process. This could be achieved either by overexpression of the antiapoptotic protein Bcl-2 or by targeting Raf-1 to the mitochondria via fusion to the mitochondrial protein Mas p70. Mitochondrially active Raf-1 is unable to activate extracellular signal-related kinase 1 (ERK1) and ERK2 but suppresses cell death by inactivating the proapoptotic Bcl-2 family member BAD. However, genetic and biochemical data also have suggested a role for the Raf-1 effector module MEK-ERK in apoptosis suppression. We thus tested for MEK requirement in cell survival signaling using the interleukin 3 (IL-3)-dependent cell line 32D. MEK is essential for survival and growth in the presence of IL-3. Upon growth factor withdrawal the expression of constitutively active MEK1 mutants significantly delays the onset of apoptosis, whereas the presence of a dominant negative mutant accelerates cell death. Survival signaling by MEK most likely results from the activation of ERKs since expression of a constitutively active form of ERK2 was as effective in protecting NIH 3T3 fibroblasts against doxorubicin-induced cell death as oncogenic MEK. The survival effect of activated MEK in 32D cells is achieved by both MEK- and PI3K-dependent mechanisms and results in the activation of PI3K and in the phosphorylation of AKT. MEK and PI3K dependence is also observed in 32D cells protected from apoptosis by oncogenic Raf-1. Additionally, we also could extend these findings to the IL-3-dependent pro-B-cell line BaF3, suggesting that recruitment of MEK is a common mechanism for survival signaling by activated Raf. Requirement for the PI3K effector AKT in this process is further demonstrated by the inhibitory effect of a dominant negative AKT mutant on Raf-1-induced cell survival. Moreover, a constitutively active form of AKT synergizes with Raf-1 in apoptosis suppression. In summary these data strongly suggest a Raf effector pathway for cell survival that is mediated by MEK and AKT.     

Ligand-independent regulation of ErbB4 receptor phosphorylation by activated Ras

The ErbB family of receptor tyrosine kinases regulates cell growth, differentiation and survival. Activation of the receptors is induced by specific growth factors in an autocrine, paracrine or juxtacrine manner. The activated ErbB receptors turn on a large variety of signaling cascades, including the prominent Ras-dependent signaling pathways. The activated Ras can induce secretion of growth factors such as EGF and neuregulin, which activate their respective receptors. In the present study, we demonstrate for the first time that activated Ras can activate ErbB4 receptor in a ligand-independent manner. Expression of constitutively active H-Ras(12V), K-Ras(12V) or N-Ras(13V) in PC12-ErbB4 cells induced ErbB4-receptor phosphorylation, indicating that each of the most abundant Ras isoforms can induce receptor activation. NRG-induced phosphorylation of ErbB4 receptor was blocked by the soluble ErbB4 receptor, which had no effect on the Ras-induced receptor phosphorylation. Moreover, conditioned medium from H-Ras(12V)-transfected PC12-ErbB4 cells had no effect on receptor phosphorylation. It thus indicates that Ras induces ErbB4 phosphorylation in a ligand-independent manner. Each of the Ras effector domain mutants, H-Ras(12V)S35, H-Ras(12V)C40, and H-Ras(12V)G37, which respectively activate Raf1, PI3K, and RalGEF, induced a small but significant receptor phosphorylation. The PI3K inhibitor LY294002 and the MEK inhibitor PD98059 caused a partial inhibition of the Ras-induced ErbB4 receptor phosphorylation. Using a mutant ErbB4 receptor, which lacks kinase activity, we demonstrated that the Ras-mediated ErbB4 phosphorylation depends on the kinase activity of the receptor and facilitates ligand-independent neurite outgrowth in PC12-ErbB4 cells. These experiments demonstrate a novel mechanism controlling ErbB receptor activation. Ras induces ErbB4 receptor phosphorylation in a non-autocrine manner and this activation depends on multiple Ras effector pathways and on ErbB4 kinase activity.     

The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis

Farnesyltransferase inhibitors (FTIs) represent a novel class of anticancer drugs that exhibit a remarkable ability to inhibit malignant transformation without toxicity to normal cells. However, the mechanism by which FTIs inhibit tumor growth is not well understood. Here, we demonstrate that FTI-277 inhibits phosphatidylinositol 3-OH kinase (PI 3-kinase)/AKT2-mediated growth factor- and adhesion-dependent survival pathways and induces apoptosis in human cancer cells that overexpress AKT2. Furthermore, overexpression of AKT2, but not oncogenic H-Ras, sensitizes NIH 3T3 cells to FTI-277, and a high serum level prevents FTI-277-induced apoptosis in H-Ras- but not AKT2-transformed NIH 3T3 cells. A constitutively active form of AKT2 rescues human cancer cells from FTI-277-induced apoptosis. FTI-277 inhibits insulin-like growth factor 1-induced PI 3-kinase and AKT2 activation and subsequent phosphorylation of the proapoptotic protein BAD. Integrin-dependent activation of AKT2 is also blocked by FTI-277. Thus, a mechanism for FTI inhibition of human tumor growth is by inducing apoptosis through inhibition of PI 3-kinase/AKT2-mediated cell survival and adhesion pathway.     

Oncogenic Ras leads to Rho activation by activating the mitogen-activated protein kinase pathway and decreasing Rho-GTPase-activating protein activity

Transformation by oncogenic Ras requires signaling through Rho family proteins including RhoA, but the mechanism(s) whereby oncogenic Ras regulates the activity of RhoA is (are) unknown. We examined the effect of Ras on RhoA activity in NIH 3T3 cells either stably transfected with H-Ras(V12) under control of an inducible promoter or transiently expressing the activated H-Ras. Using a novel method to quantitate enzymatically the GTP bound to Rho, we found that expression of the oncogenic Ras increased Rho activity approximately 2-fold. Increased Rho activity was associated with increased plasma membrane binding of RhoA and decreased activity of the Rho/Ras-regulated p21(WAF1/CIP1) promoter. RhoA activation by oncogenic Ras could be explained by a decrease in cytosolic p190 Rho-GAP activity and translocation of p190 Rho-GAP from the cytosol to a detergent-insoluble cytoskeletal fraction. Pharmacologic inhibition of the Ras/Raf/MEK/ERK pathway prevented Ras-induced activation of RhoA and translocation of p190 Rho-GAP; expression of constitutively active Raf-1 kinase or MEK was sufficient to induce p190 Rho-GAP translocation. We conclude that in NIH 3T3 cells oncogenic Ras activates RhoA through the Raf/MEK/ERK pathway by decreasing the cytosolic activity and changing the subcellular localization of p190 Rho-GAP.     

Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation.

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.     

v-Crk activates the phosphoinositide 3-kinase/AKT pathway by utilizing focal adhesion kinase and H-Ras

v-Crk, an oncogene product of avian sarcoma virus CT10, efficiently transforms chicken embryo fibroblasts (CEF). We have recently reported that constitutive activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway plays a critical role in the v-Crk-induced transformation of CEF. In the present study we investigated the molecular mechanism by which v-Crk activates the PI3K/AKT pathway. First, we found that v-Crk promotes the association of the p85 regulatory subunit of PI3K with focal adhesion kinase (FAK) by inducing the phosphorylation of the Y397 residue in FAK. This FAK phosphorylation needs activation of the Src family tyrosine kinase(s) for which the v-Crk SH2 domain is responsible. v-Crk was unable to activate the PI3K/AKT pathway in FAK-null cells, indicating the functional importance of FAK. In addition, we found that H-Ras is also required for the activation of the PI3K/AKT pathway. The v-Crk-induced activation of AKT was greatly enhanced by the overexpression of H-Ras or its guanine nucleotide exchange factor mSOS, which binds to the v-Crk SH3 domain, whereas a dominant-negative mutant of H-Ras almost completely suppressed this activation. Furthermore, we showed that v-Crk stimulates the interaction of H-Ras with the Ras binding domain in the PI3K p110 catalytic subunit. Our data indicated that the v-Crk-induced activation of PI3K/AKT pathway was cooperatively achieved by two distinct interactions. One is the interaction of p85 with tyrosine-phosphorylated FAK promoted by the v-Crk SH2 domain, and another is the interaction of p110 with H-Ras dictated by the v-Crk SH3 domain.     

Galectin-1 augments Ras activation and diverts Ras signals to Raf-1 at the expense of phosphoinositide 3-kinase

Ras proteins activate diverse effector molecules. Depending on the cellular context, Ras activation may have different biological consequences: induction of cell proliferation, senescence, survival, or death. Augmentation and selective activation of particular effector molecules may underlie various Ras actions. In fact, Ras effector-loop mutants interacting with distinctive effectors provide evidence for such selectivity. Interactions of active Ras with escort proteins, such as galectin-1, could also direct Ras selectivity. Here we show that in comparison with Ras transfectants, H-Ras/galectin-1 or K-Ras4B/galectin-1 co-transfectants exhibit enhanced and prolonged epidermal growth factor (EGF)-stimulated increases in Ras-GTP, Raf-1 activity, and active extracellular signal-regulated kinase. Galectin-1 antisense RNA inhibited these EGF responses. Conversely, Ras and galectin-1 co-transfection inhibited the EGF-stimulated increase in phosphoinositide 3-kinase (PI3K) activity. Galectin-1 transfection also inhibited Ras(G12V)-induced PI3K but not Raf-1 activity. Galectin-1 co-immunoprecipitated with Ras(G12V) or with Ras(G12V/T35S) that activate Raf-1 but not with Ras(G12V/Y40C) that activates PI3K. Thus, galectin-1 binds active Ras and diverts its signal to Raf-1 at the expense of PI3K. This demonstrates a novel mechanism controlling the duration and selectivity of the Ras signal. Ras gains selectivity when it is associated with galectin-1, mimicking the selectivity of Ras(T35S), which activates Raf-1 but not PI3K.     

Contribution of the ERK5/MEK5 pathway to Ras/Raf signaling and growth control.

The activity of the catalytic domain of the orphan MAP kinase ERK5 is increased by Ras but not Raf-1 in cells, which suggests that ERK5 might mediate Raf-independent signaling by Ras. We found that Raf-1 does contribute to Ras activation of ERK5 but in a manner that does not correlate with Raf-1 catalytic activity. A clue to the mechanism of action of Raf-1 on ERK5 comes from the observation that endogenous Raf-1 binds to endogenous ERK5, suggesting the involvement of regulatory protein-protein interactions. This interaction is specific because Raf-1 binds only to ERK5 and not ERK2 or SAPK. Finally, we demonstrate the ERK5/MEK5 pathway is required for Raf-dependent cellular transformation and that a constitutively active form of MEK5, MEK5DD, synergizes with Raf to transform NIH 3T3 cells. These observations suggest that ERK5 plays a large role in Raf-1-mediated signal transduction.     

The role of phosphatidylinositol 3-kinase, rho family GTPases, and STAT3 in Ros-induced cell transformation.

Using loss-of-function mutants of Ros and inducible epidermal growth factor receptor-Ros chimeras we investigated the role of various signaling pathways in Ros-induced cell transformation. Inhibition of the mitogen-activated protein kinase (MAPK) pathway with the MEK (MAP/extracellular signal-regulated kinase kinase) inhibitor PD98059 had little effect on the Ros-induced monolayer and anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells even though more than 70% of the MAPK was inhibited. In contrast, inhibiting the phosphatidylinositol 3-kinase (PI3K) pathway with the drug LY294002, a dominant negative mutant of PI3K, Deltap85, or the phosphatidylinositol phosphatase PTEN (phosphatase and tensin homologue deleted in chromosome ten) resulted in a dramatic reduction of v-Ros- and epidermal growth factor receptor-Ros-promoted anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells, respectively. Parallel and downstream components of PI3K signaling such as the Rho family GTPases (Rac, Rho, Cdc42) and the survival factor Akt were all shown to contribute to Ros-induced anchorage-independent growth, although Rac appeared to be less important for Ros-induced colony formation in NIH3T3 cells. Furthermore, the transformation-attenuated v-Ros mutants F419 and DI could be complemented by constitutively active mutants of PI3K and Akt. Finally, we found that overexpressing a constitutively active mutant of STAT3 (STAT3C) conferred a resistance to the inhibition of Ros-induced anchorage-independent growth by LY294002, suggesting a possible overlap of functions between PI3K and STAT3 signaling in mediating Ros-induced anchorage-independent growth.     

Activated Ras induces cytoplasmic vacuolation and non-apoptotic death in glioblastoma cells via novel effector pathways

Expression of activated H-Ras induces a unique form of non-apoptotic cell death in human glioblastoma cells and other specific tumor cell lines. The major cytopathological features of this form of death are the accumulation of large phase-lucent, LAMP1-positive, cytoplasmic vacuoles. In this study we sought to determine if induction of cytoplasmic vacuolation a) depends on Ras farnesylation, b) is specific to H-Ras, and c) is mediated by signaling through the major known Ras effector pathways. We find that the unusual effects of activated H-Ras depend on farnesylation and membrane association of the GTPase. Both H-Ras(G12V) and K-Ras4B(G12V) stimulate vacuolation, but activated forms of Cdc42 and RhoA do not. Amino acid substitutions in the Ras effector domain, which are known to selectively impair its interactions with Raf kinase, class-I phosphatidylinositide 3-kinase (PI3K), or Ral nucleotide exchange factors, initially pointed to Raf as a possible mediator of cell vacuolation. However, the MEK inhibitor, PD98059, did not block the induction of vacuoles, and constitutively active Raf-Caax did not mimic the effects of Ras(G12V). Introduction of normal PTEN together with H-Ras(G12V) into U251 glioblastoma cells reduced the PI3K-dependent activation of Akt, but had no effect on vacuolation. Finally, co-expression of H-Ras(G12V) with a dominant-negative form of RalA did not suppress vacuolation. Taken together, the observations indicate that Ras activates non-conventional and perhaps unique effector pathways to induce cytoplasmic vacuolation in glioblastoma cells. Identification of the relevant signaling pathways may uncover specific molecular targets that can be manipulated to activate non-apoptotic cell death in this type of cancer.     

Oncogenic H-Ras enhances DNA repair through the Ras/phosphatidylinositol 3-kinase/Rac1 pathway in NIH3T3 cells. Evidence for association with reactive oxygen species

This study investigated the role of oncogenic H-Ras in DNA repair capacity in NIH3T3 cells. Expression of dominant-positive H-Ras (V12-H-Ras) enhanced the host cell reactivation of luciferase activity from UV-irradiated and cisplatin-treated plasmids and also increased the unscheduled DNA synthesis following cisplatin or UV treatment of cells. This observed enhancement of DNA repair capacity was inhibited by transient transfection with dominant-negative H-Ras (N17-H-Ras) or Rac1 (N17-Rac1) plasmids. Moreover, stable transfection of dominant-positive Rac1 (V12-Rac1) further enhanced DNA repair capacity. Because reactive oxygen species (ROS) are known to be a downstream effector of oncogenic Ras, we examined the role of ROS in DNA repair capacity. We found that ROS production by V12-H-Ras expression was mediated by the Ras/phosphatidylinositol 3-kinase (PI3K)/Rac1/NADPH oxidase-dependent pathway and that pretreatment of V12-H-Ras-transformed cells with an antioxidant (N-acetylcysteine) and an NADPH oxidase inhibitor (diphenyleneiodonium) decreased DNA repair capacity. Similarly, treatment with PI3K inhibitors (wortmannin and LY294002) inhibited the ability of oncogenic H-Ras to enhance DNA repair capacity. Furthermore, inhibition of the Ras/PI3K/Rac1/NADPH oxidase pathway resulted in increased sensitivity to cisplatin and UV in V12-H-Ras-expressing NIH3T3 cells. Taken together, these results provide evidence that oncogenic H-Ras activates DNA repair capacity through the Ras/PI3K/Rac1/NADPH oxidase-dependent pathway and that increased ROS production via this signaling pathway is required for enhancement of the DNA repair capacity induced by oncogenic H-Ras.     

Signals from the Ras, Rac, and Rho GTPases converge on the Pak protein kinase in Rat-1 fibroblasts

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.     

Hdm2 nuclear export, regulated by insulin-like growth factor-I/MAPK/p90Rsk signaling, mediates the transformation of human cells

Insulin-like growth factor (IGF)-I receptor activation leads to enhanced proliferation and cell survival via the MAP kinase and phosphatidylinositol 3-kinase-signaling pathways. Upon stimulation by IGF-I, the Hdm2 oncoprotein is phosphorylated by AKT, leading to its rapid nuclear translocation and subsequent inhibition of p53. We now show that IGF-I stimulation regulates the nuclear export of Hdm2 and p53 via the MAP kinase pathway. Inhibition of p38 MAPK or MEK via pharmacological means or expression of dominant negative proteins inhibited the cytoplasmic accumulation of Hdm2 and increased Hdm2 and p53 protein levels, whereas constitutively active p90Rsk promoted the nuclear export of Hdm2. Expression of constitutively active p90Rsk with E1A, oncogenic H-Ras, and hTERT resulted in the anchorage-independent growth of normal human fibroblasts. Our findings link p90Rsk-mediated modulation of Hdm2 nuclear to cytoplasmic shuttling with the diminished ability of p53 to regulate cell cycle checkpoints that ultimately leads to transformation.