Ras signals principally via Erk in G1 but cooperates with PI3K/Akt for Cyclin D induction and S-phase entry

Numerous studies exploring oncogenic Ras or manipulating physiological Ras signalling have established an irrefutable role for Ras as driver of cell cycle progression. Despite this wealth of information the precise signalling timeline and effectors engaged by Ras, particularly during G1, remain obscure as approaches for Ras inhibition are slow-acting and ill-suited for charting discrete Ras signalling episodes along the cell cycle. We have developed an approach based on the inducible recruitment of a Ras-GAP that enforces endogenous Ras inhibition within minutes. Applying this strategy to inhibit Ras stepwise in synchronous cell populations revealed that Ras signaling was required well into G1 for Cyclin D induction, pocket protein phosphorylation and S-phase entry, irrespective of whether cells emerged from quiescence or G2/M. Unexpectedly, Erk, and not PI3K/Akt or Ral was activated by Ras at mid-G1, albeit PI3K/Akt signalling was a necessary companion of Ras/Erk for sustaining cyclin-D levels and G1/S transition. Our findings chart mitogenic signaling by endogenous Ras during G1 and identify limited effector engagement restricted to Raf/MEK/Erk as a cogent distinction from oncogenic Ras signalling.     

Regulation of Ras signaling specificity by protein kinase C

Ras proteins have the capacity to bind to and activate at least three families of downstream target proteins: Raf kinases, phosphatidylinositol 3 (PI 3)-kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). We have previously shown that the Ras/Ral-GEF and Ras/Raf pathways oppose each other upon nerve growth factor stimulation, with the former promoting proliferation and the latter promoting cell cycle arrest. Moreover, the pathways are not activated equally. While the Ras/Raf/Erk signaling pathway is induced for hours, the Ras/Ral-GEF/Ral signaling pathway is induced for only minutes. Here we show that this preferential down-regulation of Ral signaling is mediated, at least in part, by protein kinase C (PKC). In particular, we show that PKC activation by phorbol ester treatment of cells blocks growth factor-induced Ral activation while it enhances Erk activation. Moreover, suppression of growth factor-induced PKC activation enhances and prolongs Ral activation. PKC does not influence the basal activity of the Ral-GEF designated Ral-GDS but suppresses its activation by Ras. Interestingly, Ras binding to the C-terminal Ras binding domain of Ral-GDS is not affected by PKC activity. Instead, suppression of Ral-GDS activation occurs through the region N terminal to the catalytic domain, which becomes phosphorylated in response to phorbol ester treatment of cells. These findings identify a role for PKC in determining the specificity of Ras signaling by its ability to differentially modulate Ras effector protein activation.     

Enhanced clonogenic survival induced by protein tyrosine phosphatase (PTP) inhibition after Cr(VI) exposure is mediated by c-Raf and Ras activity

Our recent studies showed that maintenance of protein tyrosine phosphorylation by PTP inhibition enhanced cell growth, clonogenic survival, and mutagenesis after a single low-level Cr(VI) exposure, thereby suggesting that tyrosine phosphorylation-dependent signaling may govern inappropriate survival in human lung fibroblasts (HLFs). Our goal is to identify specific phospho-tyrosine regulator(s)/ downstream effectors involved in enhanced survival after Cr(VI) exposure and PTP inhibition. Phosphotyrosine profiling array showed that PTP inhibition following Cr(VI) exposure increased tyrosine phosphorylation of specific proteins, such as FGR and ABL, which are upstream regulators of both Erk and Akt pathways. To explore the roles of these pathways in the PTP-induced increase in clonogenic survival after Cr(VI) exposure, we examined the effect of combined Akt1 and Erk1/2 knockdown via siRNA technology. Akt1 and/or Erk1/2 silencing had no effect on the PTP inhibitor-induced increase in survival following Cr(VI) exposure, suggesting the presence of non-Akt/non-Erk-mediated survival signaling. Interestingly, geldanamycin, an HSP90 inhibitor and non-specific Raf inhibitor, abrogated the PTP inhibitor-mediated increase in survival following Cr(VI) exposure and abolished the expression/activity of c-Raf and activity of Mek. These findings prompted us to explore upstream regulators of Erk, i.e., Ras, c-Raf and Mek for their potential roles in clonogenic survival. GW5074, a specific c-Raf kinase inhibitor did not alter the effect of the PTP inhibitor but decreased Cr(VI)-mediated clonogenic lethality, potentially though Mek hyperactivation. A genetic approach with a c/a Mek1 mutant also showed that Mek activity was not directly associated with the PTP inhibitor effect. Finally, a genetic approach with d/n or c/a Ras and c-Raf mutants, showed that Ras and c-Raf activities play a substantive role in enhancing clonogenic survival by PTP inhibition following Cr(VI) insult. In conclusion, these studies highlight a novel pro-survival mechanism for clonogenic survival in the face of genotoxic stress in the presence of PTP inhibition via an Erk/Mek-independent and Ras/c-Raf-dependent regulation in normal human lung fibroblasts.     

“RAF” neighborhood: Protein–protein interaction in the Raf/Mek/Erk pathway

The Raf/Mek/Erk signaling pathway, activated downstream of Ras primarily to promote proliferation, represents the best studied of the evolutionary conserved MAPK cascades. The investigation of the pathway has continued unabated since its discovery roughly 30 years ago. In the last decade, however, the identification of unexpected in vivo functions of pathway components, as well as the discovery of Raf mutations in human cancer, the ensuing quest for inhibitors, and the efforts to understand their mechanism of action, have boosted interest tremendously. From this large body of work, protein–protein interaction has emerged as a recurrent, crucial theme. This review focuses on the role of protein complexes in the regulation of the Raf/Mek/Erk pathway and in its cross-talk with other signaling cascades. Mapping these interactions and finding a way of exploiting them for therapeutic purposes is one of the challenges of future molecule-targeted therapy.     

Mek1/2 MAPK kinases are essential for Mammalian development, homeostasis, and Raf-induced hyperplasia

The p42/p44 mitogen-activated protein kinase (MAPK) cascade includes Ras, Raf, Mek, and Erk MAPK. To determine the effect of a full knockout at a single level of this signaling pathway in mammals, and to investigate functional redundancy between Mek1 and Mek2, we disrupted these genes in murine and human epidermis. Loss of either protein alone produced no phenotype, whereas combined Mek1/2 deletion in development or adulthood abolished Erk1/2 phosphorylation and led to hypoproliferation, apoptosis, skin barrier defects, and death. Conversely, a single copy of either allele was sufficient for normal development. Combined Mek1/2 loss also abolished Raf-induced hyperproliferation. Human tissue deficient in either Mek isoform was normal, whereas loss of both proteins led to hypoplasia, which was rescued by active Erk2 expression. These data indicate that Mek1/2 are functionally redundant in the epidermis, where they act as a linear relay in the MAPK pathway to mediate development and homeostasis.     

The molecular scaffold kinase suppressor of Ras 1 is a modifier of RasV12-induced and replicative senescence

In primary mouse embryo fibroblasts (MEFs), oncogenic Ras induces growth arrest via Raf/MEK/extracellular signal-regulated kinase (ERK)-mediated activation of the p19ARF/p53 and INK4/Rb tumor suppressor pathways. Ablation of these same pathways causes spontaneous immortalization in MEFs, and oncogenic transformation by Ras requires ablation of one or both of these pathways. We show that Kinase Suppressor of Ras 1 (KSR1), a molecular scaffold for the Raf/MEK/ERK cascade, is necessary for RasV12-induced senescence, and its disruption enhances primary MEF immortalization. RasV12 failed to induce p53, p19ARF, p16INK4a, and p15INK4b expression in KSR1-/- MEFs and increased proliferation instead of causing growth arrest. Reintroduction of wild-type KSR1, but not a mutated KSR1 construct unable to bind activated ERK, rescued RasV12-induced senescence. On continuous culture, deletion of KSR1 accelerated the establishment of spontaneously immortalized cultures and increased the proportion of cultures escaping replicative crisis. Despite enhancing escape from both RasV12-induced and replicative senescence, however, both primary and immortalized KSR1-/- MEFs are completely resistant to RasV12-induced transformation. These data show that escape from senescence is not necessarily a precursor for oncogenic transformation. Furthermore, these data indicate that KSR1 is a member of a unique class of proteins whose deletion blocks both senescence and transformation.     

Raf-Induced Cell Cycle Progression in Human TF-1 Hematopoietic Cells

Ras/Raf/MEK/ERK is a crucial pathway regulating cell cycle progression, apoptosis, and drug resistance. The Ras oncogene is frequently mutated in human cancer, which can result in the activation of the downstream Raf/MEK/ERK cascade leading to cell cycle progression in the absence of a growth stimulus. Raf-induced proliferation has been observed in hematopoietic cells. However, the mechanisms by which Raf affects cell cycle progression are not well described. To investigate the importance of Raf/MEK/ERK signaling in human hematopoietic cell growth, the effects of three different Raf genes, A-Raf, B-Raf and Raf-1, on cell cycle progression and regulatory gene expression were examined in TF-1 cells transformed to grow in response to b-estradiol-regulated DRaf:ER genes. Raf activation increased the expression of cyclin A, cyclin D, cyclin E, and p21Cip1, which are associated with G1 progression. Activated DRaf-1:ER and DA-Raf:ER but not DB-Raf:ER increased Cdk2 and Cdk4 kinase activity. The regulatory role of p16Ink4a, a potent Cdk4 kinase inhibitor, on the kinase activity of Cdk2 and Cdk4 was also examined. Raf induced p16Ink4a suppressor but this did not eliminate Cdk4 kinase activity. These results indicate that human hematopoietic cells transformed to grow in response to activated Raf can be used to elucidate the mechanisms by which various cell cycle regulatory molecules effect cell cycle progression. Furthermore, the differences that the various Raf isoforms have on Cdk4 activity and other cell cycle regulatory molecules can be determined in these cells.

Key Words:

Cell cycle, Raf, p21Cip1, p27Kip1, Cyclins, Cdks, Hematopoietic cells     

Raf-induced cell cycle progression in human TF-1 hematopoietic cells

Ras/Raf/MEK/ERK is a crucial pathway regulating cell cycle progression, apoptosis, and drug resistance. The Ras oncogene is frequently mutated in human cancer, which can result in the activation of the downstream Raf/MEK/ERK cascade leading to cell cycle progression in the absence of a growth stimulus. Raf-induced proliferation has been observed in hematopoietic cells. However, the mechanisms by which Raf affects cell cycle progression are not well described. To investigate the importance of Raf/MEK/ERK signaling in human hematopoietic cell growth, the effects of three different Raf genes, A-Raf, B-Raf and Raf-1, on cell cycle progression and regulatory gene expression were examined in TF-1 cells transformed to grow in response to beta-estradiol-regulated DeltaRaf:ER genes. Raf activation increased the expression of cyclin A, cyclin D, cyclin E, and p21(Cip1), which are associated with G(1) progression. Activated DeltaRaf-1:ER and DeltaA-Raf:ER but not DeltaB-Raf:ER increased Cdk2 and Cdk4 kinase activity. The regulatory role of p16(Ink4a), a potent Cdk4 kinase inhibitor, on the kinase activity of Cdk2 and Cdk4 was also examined. Raf induced p16(Ink4a) suppressor but this did not eliminate Cdk4 kinase activity. These results indicate that human hematopoietic cells transformed to grow in response to activated Raf can be used to elucidate the mechanisms by which various cell cycle regulatory molecules effect cell cycle progression. Furthermore, the differences that the various Raf isoforms have on Cdk4 activity and other cell cycle regulatory molecules can be determined in these cells.     

The role of RBF in developmentally regulated cell proliferation in the eye disc and in Cyclin D/Cdk4 induced cellular growth

During Drosophila eye development, cell proliferation is coordinated with differentiation. Immediately posterior to the morphogenetic furrow, cells enter a synchronous round of S phase called second mitotic wave. We have examined the role of RBF, the Drosophila RB family homolog, in cell cycle progression in the second mitotic wave. RBF-280, a mutant form of RBF that has four putative cdk phosphorylation sites mutated, can no longer be regulated by Cyclin D or Cyclin E. Expression of RBF-280 in the developing eye revealed that RBF-280 does not inhibit G1/S transition in the second mitotic wave, rather it delays the completion of S phase and leads to abnormal eye development. These observations suggest that RB/E2F control the rate of S-phase progression instead of G1/S transition in the second mitotic wave. Characterization of the role of RBF in Cyclin D/Cdk4-mediated cellular growth showed that RBF-280 blocks Cyclin D/Cdk4 induced cellular growth in the proliferating wing disc cells but not in the non-dividing eye disc cells. By contrast, RBF-280 does not block activated Ras-induced cellular growth. These results suggest that the ability of Cyclin D/Cdk4 to drive growth in the proliferating wing cells is distinct from that in the none-dividing eye cells or the ability of activated Ras to induce growth, and that RBF may have a role in regulating growth in the proliferating wing discs.     

Oncogenic Ras in tumour progression and metastasis

The ras genes give rise to a family of related GTP-binding proteins that exhibit potent transforming potential. Mutational activation of Ras proteins promotes oncogenesis by disturbing a multitude of cellular processes, such as gene expression, cell cycle progression and cell proliferation, as well as cell survival, and cell migration. Ras signalling pathways are well known for their involvement in tumour initiation, but less is known about their contribution to invasion and metastasis. This review summarises the role and mechanisms of Ras signalling, especially the role of the Ras effector cascade Raf/MEK/ERK, as well as the phosphatidylinositol 3-kinase/Akt pathway in Ras-mediated transformation and tumour progression. In addition, it discusses the impact of Rho GTPases on Ras-mediated transformation and metastasis.     

RGS14 is a multifunctional scaffold that integrates G protein and Ras/Raf MAPkinase signalling pathways

MAPkinase signalling is essential for cell growth, differentiation and cell physiology. G proteins and tyrosine kinase receptors each modulate MAPkinase signalling through distinct pathways. We report here that RGS14 is an integrator of G protein and MAPKinase signalling pathways. RGS14 contains a GPR/GoLoco (GL) domain that forms a stable complex with inactive Giα1/3–GDP, and a tandem (R1, R2) Ras binding domain (RBD). We find that RGS14 binds and regulates the subcellular localization and activities of H-Ras and Raf kinases in cells. Activated H-Ras binds RGS14 at the R1 RBD to form a stable complex at cell membranes. RGS14 also co-localizes with and forms a complex with Raf kinases in cells. The regulatory region of Raf-1 binds the RBD region of RGS14, and H-Ras and Raf each facilitate one another's binding to RGS14. RGS14 selectively inhibits PDGF-, but not EGF- or serum-stimulated Erk phosphorylation. This inhibition is dependent on H-Ras binding to RGS14 and is reversed by co-expression of Giα1, which binds and recruits RGS14 to the plasma membrane. Giα1 binding to RGS14 inhibits Raf binding, indicating that Giα1 and Raf binding to RGS14 are mutually exclusive. Taken together, these findings indicate that RGS14 is a newly appreciated integrator of G protein and Ras/Raf signalling pathways.     

Ral-specific guanine nucleotide exchange factor activity opposes other Ras effectors in PC12 cells by inhibiting neurite outgrowth

Ras proteins can activate at least three classes of downstream target proteins: Raf kinases, phosphatidylinositol-3 phosphate (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). In NIH 3T3 cells, activated Ral-GEFs contribute to Ras-induced cell proliferation and oncogenic transformation by complementing the activities of Raf and PI3 kinases. In PC12 cells, activated Raf and PI3 kinases mediate Ras-induced cell cycle arrest and differentiation into a neuronal phenotype. Here, we show that in PC12 cells, Ral-GEF activity acts opposite to other Ras effectors. Elevation of Ral-GEF activity induced by transfection of a mutant Ras protein that preferentially activates Ral-GEFs, or by transfection of the catalytic domain of the Ral-GEF Rgr, suppressed cell cycle arrest and neurite outgrowth induced by nerve growth factor (NGF) treatment. In addition, Rgr reduced neurite outgrowth induced by a mutant Ras protein that preferentially activates Raf kinases. Furthermore, inhibition of Ral-GEF activity by expression of a dominant negative Ral mutant accelerated cell cycle arrest and enhanced neurite outgrowth in response to NGF treatment. Ral-GEF activity may function, at least in part, through inhibition of the Rho family GTPases, CDC42 and Rac. In contrast to Ras, which was activated for hours by NGF treatment, Ral was activated for only approximately 20 min. These findings suggest that one function of Ral-GEF signaling induced by NGF is to delay the onset of cell cycle arrest and neurite outgrowth induced by other Ras effectors. They also demonstrate that Ras has the potential to promote both antidifferentiation and prodifferentiation signaling pathways through activation of distinct effector proteins. Thus, in some cell types the ratio of activities among Ras effectors and their temporal regulation may be important determinants for cell fate decisions between proliferation and differentiation.     

G1/S cell cycle arrest provides anoikis resistance through Erk-mediated Bim suppression

Proper attachment to the extracellular matrix is essential for cell survival. Detachment from the extracellular matrix results in an apoptotic process termed anoikis. Anoikis induction in MCF-10A mammary epithelial cells is due not only to loss of survival signals following integrin disengagement, but also to consequent downregulation of epidermal growth factor (EGFR) and loss of EGFR-induced survival signals. Here we demonstrate that G(1)/S arrest by overexpression of the cyclin-dependent kinase inhibitors p16(INK4a), p21(Cip1), or p27(Kip1) or by treatment with mimosine or aphidicolin confers anoikis resistance in MCF-10A cells. G(1)/S arrest-mediated anoikis resistance involves suppression of the BH3-only protein Bim. Furthermore, in G(1)/S-arrested cells, Erk phosphorylation is maintained in suspension and is necessary for Bim suppression. Following G(1)/S arrest, known proteins upstream of Erk, including Raf and Mek, are not activated. However, retained Erk activation under conditions in which Raf and Mek activation is lost is observed, suggesting that G(1)/S arrest acts at the level of Erk dephosphorylation. Thus, anoikis resistance by G(1)/S arrest is mediated by a mechanism involving Bim suppression through maintenance of Erk activation. These results provide a novel link between cell cycle arrest and survival, and this mechanism could contribute to the survival of nonreplicating, dormant tumor cells that avert apoptosis during early stages of metastasis.     

Membrane localization of Raf assists engagement of downstream effectors

We have previously described a small molecule-directed protein dimerization strategy, using coumermycin to juxtapose Raf fusion proteins containing the coumermycin-binding domain GyrB. Oligomerization of cytoplasmically localized Raf-GyrB fusion proteins leads to an increase in the kinase activity of both Raf and its substrate Mek. Surprisingly, more distal targets, such as Erk1 and Erk2, are not activated using this approach. Here we report that coumermycin-induced oligomerization of a membrane-localized Raf-GyrB fusion protein potently activated Erk1 and Erk2, up-regulated Fos protein levels, and induced expression of many immediate-early response genes. Thus, both membrane localization and oligomerization of Raf-GyrB are required to target Raf signals to downstream effectors. The ability to activate the entire Raf signal transduction cascade conditionally, using coumermycin-induced oligomerization, should prove useful for dissecting Raf-mediated effects on gene expression and cellular differentiation.     

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.     

Involvement of H- and N-Ras isoforms in transforming growth factor-beta1-induced proliferation and in collagen and fibronectin synthesis

Transforming growth factor beta1 (TGF-beta1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-beta and Ras signaling pathways are closely related: TGF-beta1 overcomes Ras mitogenic effects and Ras counteracts TGF-beta signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-beta1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras(-/-)/N-ras(-/-)) isoforms and from heterozygote mice (H-ras(+/-)/N-ras(+/-)). ECM synthesis is increased in basal conditions in H-ras(-/-)/N-ras(-/-) fibroblasts, this increase being higher after stimulation with TGF-beta1. TGF-beta1-induced fibroblast proliferation is smaller in H-ras(-/-)/N-ras(-/-) than in H-ras(+/-)/N-ras(+/-) fibroblasts. Erk activation is decreased in H-ras(-/-)/N-ras(-/-) fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.