Ras-PI3K信号负性调节的H3K56乙酰化促进乳腺癌细胞MCF-7的增殖和迁移能力

Ras蛋白常见的第12、13氨基酸残基突变引起的Ras信号通路异常与人类恶性肿瘤发生相关。然而,Ras致瘤信号通路是否涉及表观遗传学因素尚不明了。本研究旨在阐明人乳腺癌MCF-7细胞中组蛋白H3第56位赖氨酸残基乙酰化修饰（H3K56ac）水平是否受Ras信号通路调控,以及H3K56ac水平对MCF-7细胞增殖和迁移能力的影响。点突变结合基因转染揭示,与野生型比较,第12位氨基酸突变的Ras质粒（pEGFP-H-RasG12V）转染导致MCF-7细胞内H3K56ac水平明显降低。采用可特异激活Ras下游3条通路（Ras-Raf、Ras-RalGEF和Ras-PI3K）的3种质粒（pEGFP-H-RasG12V T35S,pEGFP-H-RasG12V E37G和pEGFP-H-RasG12V Y40C）转染证明,只有转染pEGFP-H-RasG12V Y40C的MCF-7细胞内不仅有Ras-PI3K-AKT通路被激活,且与H3K56ac水平下调相伴;而其他两条通路的激活不影响H3K56ac水平。MTT法结合Transwell、软琼脂克隆形成能力实验证明,RasG12V Y40C转染增强细胞增殖、迁移和克隆形成能力。上述结果表明,MCF-7细胞中H3K56ac水平受Ras-PI3K通路的负性调控,但不受Raf和RalGEF通路影响。Ras-PI3K激活导致的H3K56ac水平降低可增强乳腺癌MCF-7细胞的增殖和迁移能力。总之,这些结果提示,组蛋白H3K56ac是Ras-PI3K致瘤信号通路中的重要成员。Ras信号通路与组蛋白修饰相结合研究将会加深对乳腺癌细胞增殖和迁移调控机制的认识。     

Activation of the Ral and phosphatidylinositol 3' kinase signaling pathways by the ras-related protein TC21

TC21 is a member of the Ras superfamily of small GTP-binding proteins that, like Ras, has been implicated in the regulation of growth-stimulating pathways. We have previously identified the Raf/mitogen-activated protein kinase pathway as a direct TC21 effector pathway required for TC21-induced transformation (M. Rosário, H. F. Paterson, and C. J. Marshall, EMBO J. 18:1270-1279, 1999). In this study we have identified two further effector pathways for TC21, which contribute to TC21-stimulated transformation: the phosphatidylinositol 3' kinase (PI-3K) and Ral signaling pathways. Expression of constitutively active TC21 leads to the activation of Ral A and the PI-3K-dependent activation of Akt/protein kinase B. Strong activation of the PI-3K/Akt pathway is seen even with very low levels of TC21 expression, suggesting that TC21 may be a key small GTPase-regulator of PI-3K. TC21-induced alterations in cellular morphology in NIH 3T3 and PC12 cells are also PI-3K dependent. On the other hand, activation of the Ral pathway by TC21 is required for TC21-stimulated DNA synthesis but not transformed morphology. We show that inhibition of Ral signaling blocks DNA synthesis in human tumor cell lines containing activating mutations in TC21, demonstrating for the first time that this pathway is required for the proliferation of human tumor cells. Finally, we provide mechanisms for the activation of these pathways, namely, the direct in vivo interaction of TC21 with guanine nucleotide exchange factors for Ral, resulting in their translocation to the plasma membrane, and the direct interaction of TC21 with PI-3K. In both cases, the effector domain region of TC21 is required since point mutations in this region can interfere with activation of downstream signaling.     

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.     

Genetic analysis of Ras signalling pathways in cell proliferation,migration and survival

We have used mouse embryonic fibroblasts (MEFs) devoid of Ras proteins to illustrate that they are essential for proliferation and migration, but not for survival, at least in these cells. These properties are unique to the Ras subfamily of proteins because ectopic expression of other Ras‐like small GTPases, even when constitutively active, could not compensate for the absence of Ras proteins. Only constitutive activation of components of the Raf/Mek/Erk pathway was sufficient to sustain normal proliferation and migration of MEFs devoid of Ras proteins. Activation of the phosphatidylinositol 3‐kinase (PI3K)/PTEN/Akt and Ral guanine exchange factor (RalGEF)/Ral pathways, either alone or in combination, failed to induce proliferation or migration of Rasless cells, although they cooperated with Raf/Mek/Erk signalling to reproduce the full response mediated by Ras signalling. In contrast to current hypotheses, Ras signalling did not induce proliferation by inducing expression of D‐type Cyclins. Rasless MEFs had normal levels of Cyclin D1/Cdk4 and Cyclin E/Cdk2. However, these complexes were inactive. Inactivation of the pocket proteins or knock down of pRb relieved MEFs from their dependence on Ras signalling to proliferate.     

A novel RalGEF-like protein, RGL3, as a candidate effector for rit and Ras

The small GTPase Rit is a close relative of Ras, and constitutively active Rit can induce oncogenic transformation. Although the effector loops of Rit and Ras are highly related, Rit fails to interact with the majority of the known Ras candidate effector proteins, suggesting that novel cellular targets may be responsible for Rit transforming activity. To gain insight into the cellular function of Rit, we searched for Rit-binding proteins by yeast two-hybrid screening. We identified the C-terminal Rit/Ras interaction domain of a protein we have designated RGL3 (Ral GEF-like 3) that shares 35% sequence identity with the known Ral guanine nucleotide exchange factors (RalGEFs). RGL3, through a C-terminal 99-amino acid domain, interacted in a GTP- and effector loop-dependent manner with Rit and Ras. Importantly, RGL3 exhibited guanine nucleotide exchange activity toward the small GTPase Ral that was stimulated in vivo by the expression of either activated Rit or Ras. These data suggest that RGL3 functions as an exchange factor for Ral and may serve as a downstream effector for both Rit and Ras.     

Activation of the RalGEF/Ral pathway promotes prostate cancer metastasis to bone

A hallmark of metastasis is organ specificity; however, little is known about the underlying signaling pathways responsible for the colonization and growth of tumor cells in target organs. Since tyrosine kinase receptor activation is frequently associated with prostate cancer progression, we have investigated the role of a common signaling intermediary, activated Ras, in prostate cancer metastasis. Three effector pathways downstream of Ras, Raf/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase, and Ral guanine nucleotide exchange factors (RalGEFs), were assayed for their ability to promote the metastasis of a tumorigenic, nonmetastatic human prostate cancer cell line, DU145. Oncogenic Ras promoted the metastasis of DU145 to multiple organs, including bone and brain. Activation of the Raf/ERK pathway stimulated metastatic colonization of the brain, while activation of the RalGEF pathway led to bone metastases, the most common organ site for prostate cancer metastasis. In addition, loss of RalA in the metastatic PC3 cell line inhibited bone metastasis but did not affect subcutaneous tumor growth. Loss of Ral appeared to suppress expansive growth of prostate cancer cells in bone, whereas homing and initial colonization were less affected. These data extend our understanding of the functional roles of the Ral pathway and begin to identify signaling pathways relevant for organ-specific metastasis.     

Phylogeny of the CDC25 homology domain reveals rapid differentiation of Ras pathways between early animals and fungi

The members of the Ras-like superfamily of small GTP-binding proteins are molecular switches that are in general regulated in time and space by guanine nucleotide exchange factors and GTPase activating proteins. The Ras-like G-proteins Ras, Rap and Ral are regulated by a variety of guanine nucleotide exchange factors that are characterized by a CDC25 homology domain. Here we study the evolution of the Ras pathway by determining the evolutionary history of CDC25 homology domain coding sequences. We identified CDC25 homology domain coding sequences in animals, fungi and a wide range of protists, but not in plants. This suggests that the CDC25 homology domain originated in or before the last eukaryotic ancestor but was subsequently lost in plant. We provide evidence that at least seven different ancestral Ras guanine nucleotide exchange factors were present in the ancestor of fungi and animals. Differences between present day fungi and animals are the result of loss of ancestral Ras guanine nucleotide exchange factors early in fungal and animal evolution combined with lineage specific duplications and domain acquisitions. In addition, we identify Ral guanine exchange factors and Ral in early diverged fungi, dating the origin of Ral signaling back to before the divergence of animals and fungi. We conclude that the Ras signaling pathway evolved by gradual change as well as through differential sampling of the ancestral CDC25 homology domain repertoire by both fungi and animals. Finally, a comparison of the domain composition of the Ras guanine nucleotide exchange factors shows that domain addition and diversification occurred both prior to and after the fungal–animal split.     

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.     

Pseudomonas aeruginosa exoenzyme S disrupts Ras-mediated signal transduction by inhibiting guanine nucleotide exchange factor-catalyzed nucleotide exchange.

Pseudomonas aeruginosa exoenzyme S double ADP-ribosylates Ras at Arg(41) and Arg(128). Since Arg(41) is adjacent to the switch 1 region of Ras, ADP-ribosylation could interfere with Ras-mediated signal transduction via several mechanisms, including interaction with Raf, or guanine nucleotide exchange factor-stimulated or intrinsic nucleotide exchange. Initial experiments showed that ADP-ribosylated Ras (ADP-r-Ras) and unmodified Ras (Ras) interacted with Raf with equal efficiencies, indicating that ADP-ribosylation did not interfere with Ras-Raf interactions. While ADP-r-Ras and Ras possessed equivalent intrinsic nucleotide exchange rates, guanine nucleotide exchange factor (Cdc25) stimulated the nucleotide exchange of ADP-r-Ras at a 3-fold slower rate than Ras. ADP-r-Ras did not affect the nucleotide exchange of Ras, indicating that the ADP-ribosylation of Ras was not a dominant negative phenotype. Ras-R41K and ADP-r-Ras R41K possessed similar exchange rates as Ras, indicating that ADP-ribosylation at Arg(128) did not inhibit Cdc25-stimulated nucleotide exchange. Consistent with the slower nucleotide exchange rate of ADP-r-Ras as compared with Ras, ADP-r-Ras bound its guanine nucleotide exchange factor (Cdc25) less efficiently than Ras in direct binding experiments. Together, these data indicate that ADP-ribosylation of Ras at Arg(41) disrupts Ras-Cdc25 interactions, which inhibits the rate-limiting step in Ras signal transduction, the activation of Ras by its guanine nucleotide exchange factor.     

Ral small GTPase signaling and oncogenesis: More than just 15 minutes of fame

Since their discovery in 1986, Ral (Ras-like) GTPases have emerged as critical regulators of diverse cellular functions. Ral-selective guanine nucleotide exchange factors (RalGEFs) function as downstream effectors of the Ras oncoprotein, and the RalGEF–Ral signaling network comprises the third best characterized effector of Ras-dependent human oncogenesis. Because of this, Ral GTPases as well as their effectors are being explored as possible therapeutic targets in the treatment of RAS mutant cancer. The two Ral isoforms, RalA and RalB, interact with a variety of downstream effectors and have been found to play key and distinct roles in both normal and neoplastic cell physiology including regulation of vesicular trafficking, migration and invasion, tumor formation, metastasis, and gene expression. In this review we provide an overview of Ral biochemistry and biology, and we highlight recent discoveries.     

Ral is both necessary and sufficient for the inhibition of myeloid differentiation mediated by Ras

Hyperactivation of Ras is one of the most common abnormalities in acute myeloid leukemia. In experimental models, Ras inhibits myeloid differentiation, which is characteristic of leukemia; however, the mechanism through which it disrupts hematopoiesis is poorly understood. In multipotent FDCP-mix cells, Ras inhibits terminal neutrophil differentiation, thereby indefinitely extending their proliferative potential. Ras also strongly promotes the sensitivity of these cells to granulocyte-macrophage colony-stimulating factor (GM-CSF). Using this model, we have dissected the signaling elements downstream of Ras to determine their relative contribution to the dysregulation of hematopoiesis. Cells expressing Ras mutants selectively activating Raf (Ras*T35S) or phosphatidylinositol 3-kinase (Ras*Y40C) did not significantly affect differentiation or proliferative capacity, whereas Ras*E37G (which selectively activates RalGEFs) perpetuated proliferation and blocked neutrophil development in a manner similar to that of Ras. Correspondingly, expression of constitutively active versions of these effectors confirmed the overriding importance of Ral guanine nucleotide exchange factors. Cells expressing Ras demonstrated hyperactivation of Ral, which itself was able to exactly mimic the phenotype of Ras, including hypersensitivity to GM-CSF. Conversely, dominant negative Ral promoted spontaneous neutrophil development. Ral, in turn, appears to influence differentiation through multiple effectors. These data show, for the first time, the importance of Ral in regulating differentiation and self-renewal in hematopoietic cells.     

Thermodynamics of Ras/effector and Cdc42/effector interactions probed by isothermal titration calorimetry

Proliferation, differentiation, and morphology of eucaryotic cells is regulated by a large network of signaling molecules. Among the major players are members of the Ras and Rho/Rac subfamilies of small GTPases that bind to different sets of effector proteins. Recognition of multiple effectors is important for communicating signals into different pathways, leading to the question of how an individual GTPase achieves tight binding to diverse targets. To understand the observed specificity, detailed information about binding energetics is expected to complement the information gained from the three-dimensional structures of GTPase/effector protein complexes. Here, the thermodynamics of the interaction of four closely related members of the Ras subfamily with four different effectors and, additionally, the more distantly related Cdc42/WASP couple were quantified by means of isothermal titration calorimetry. The heat capacity changes upon complex formation were rationalized in light of the GTPase/effector complex structures. Changes in enthalpy, entropy, and heat capacity of association with various Ras proteins are similar for the same effector. In contrast, although the structures of the Ras-binding domains are similar, the thermodynamics of the Ras/Raf and Ras/Ral guanine nucleotide dissociation stimulator interactions are quite different. The energy profile of the Cdc42/WASP interaction is similar to Ras/Ral guanine nucleotide dissociation stimulator, despite largely different structures and interface areas of the complexes. Water molecules in the interface cannot fully account for the observed discrepancy but may explain the large range of Ras/effector binding specificity. The differences in the thermodynamic parameters, particularly the entropy changes, could help in the design of effector-specific inhibitors that selectively block a single pathway.