Identification of Aurora kinases as RasGAP Src homology 3 domain-binding proteins

The GTPase-activating protein RasGAP functions as both a negative regulator and an effector of Ras proteins. In tumor cells, RasGAP is no longer able to deactivate oncogenic Ras proteins, and its effector function becomes predominant. As RasGAP itself has no obvious enzymatic function that may explain this effector function, we looked for downstream RasGAP effectors that could fulfill this role. We looked for the existence of RasGAP Src homology 3 (SH3) domain partners as this domain is involved in the regulation of cell proliferation and has an anti-apoptotic effect. We report here the identification of a new RasGAP SH3 domain-binding protein, named Aurora. This Drosophila melanogaster Ser/Thr kinase has three human orthologs called Aurora/Ipl1-related kinase or HsAIRK-1, -2, and -3. Coimmunoprecipitation experiments in COS cells confirmed that HsAIRK-1 and HsAIRK-2 both interact with RasGAP. RasGAP pull-down experiments showed that it interacts with HsAIRK-1 in G(2)/M HeLa cells. We also demonstrated that RasGAP binds to the kinase domain of Aurora and that this interaction inhibits the kinase activity of HsAIRK-1 and HsAIRK-2. Finally we showed that RasGAP forms a ternary complex with HsAIRK and survivin. This complex may be involved in the regulation of the balance between cell division and apoptosis.     

Nectin-like molecule-5/Tage4 enhances cell migration in an integrin-dependent, Nectin-3-independent manner

Cell migration plays roles in invasion of transformed cells and scattering of embryonic mesenchymal cells into surrounding tissues. We have found that Ig-like Necl-5/Tage4 is up-regulated in NIH3T3 cells transformed by an oncogenic Ras (V12Ras-NIH3T3 cells) and heterophilically trans-interacts with a Ca(2+)-independent Ig-like cell adhesion molecule nectin-3, eventually enhancing their intercellular motility. We show here that Necl-5 furthermore enhances cell migration in a nectin-3-independent manner. Studies using L fibroblasts expressing various mutants of Necl-5, NIH3T3 cells, and V12Ras-NIH3T3 cells have revealed that Necl-5 enhances serum- and platelet-derived growth factor-induced cell migration. The extracellular region of Necl-5 is necessary for directional cell migration, but not for random cell motility. The cytoplasmic region of Necl-5 is necessary for both directional and random cell movement. Necl-5 colocalizes with integrin alpha(V)beta(3) at leading edges of migrating cells. Analyses using an inhibitor or an activator of integrin alpha(V)beta(3) or a dominant negative mutant of Necl-5 have shown the functional association of Necl-5 with integrin alpha(V)beta(3) in cell motility. Cdc42 and Rac small G proteins are activated by the action of Necl-5 and required for the serum-induced, Necl-5-enhanced cell motility. These results indicate that Necl-5 regulates serum- and platelet-derived growth factor-induced cell migration in an integrin-dependent, nectin-3-independent manner, when cells do not contact other cells. We furthermore show here that enhanced motility and metastasis of V12Ras-NIH3T3 cells are at least partly the result of up-regulated Necl-5.     

p200 RhoGAP promotes cell proliferation by mediating cross-talk between Ras and Rho signaling pathways

p200 RhoGAP, a member of the Rho GTPase-activating protein (RhoGAP) family, was previously implicated in the regulation of neurite outgrowth through its RhoGAP activity. Here we show that ectopic expression of p200 RhoGAP stimulates fibroblast cell proliferation and cell cycle progression, leading to transformation. The morphology of the foci induced by p200 RhoGAP is distinct from that formed by Rac or Rho activation but similar to that induced by oncogenic Ras, raising the possibility that p200 RhoGAP may engage Ras signaling. Expression of p200 RhoGAP results in a significant increase of Ras-GTP and the activation of two downstream signaling pathways of Ras, ERK1/2 and phosphatidylinositol 3-kinase. Inhibition of Ras or ERK1/2, but not phosphatidylinositol 3-kinase, effectively suppresses the foci formation induced by p200 RhoGAP, suggesting that the Ras-ERK pathway is required for p200 RhoGAP-mediated cell transformation. p200 RhoGAP co-localizes with p120 RasGAP in cells and forms a complex with p120 RasGAP, and this interaction is mediated by the C-terminal region and the Src homology 3 domain of p200 RhoGAP and p120 RasGAP, respectively. Mutations of p200 RhoGAP that disrupt interaction with p120 RasGAP abolish its Ras activation and cell transforming activities. Interestingly, the RhoGAP activity of the N-terminal RhoGAP domain in p200 RhoGAP is also required for its full transforming activity, and expression of a dominant negative RhoA mutant that blocks RhoA cycling between the GDP- and GTP-bound states suppresses p200 RhoGAP transformation. These results suggest that a Rho GTPase-activating protein may have a positive input to cell proliferation and provide evidence that p200 RhoGAP can mediate cross-talks between Ras- and Rho-regulated signaling pathways in cell growth regulation.     

A Cell Motility Screen Reveals Role for MARCKS-Related Protein in Adherens Junction Formation and Tumorigenesis

Invasion through the extracellular matrix (ECM) is important for wound healing, immunological responses and metastasis. We established an invasion-based cell motility screen using Boyden chambers overlaid with Matrigel to select for pro-invasive genes. By this method we identified antisense to MARCKS related protein (MRP), whose family member MARCKS is a target of miR-21, a microRNA involved in tumor growth, invasion and metastasis in multiple human cancers. We confirmed that targeted knockdown of MRP, in both EpRas mammary epithelial cells and PC3 prostate cancer cells, promoted in vitro cell migration that was blocked by trifluoperazine. Additionally, we observed increased immunofluoresence of E-cadherin, β-catenin and APC at sites of cell-cell contact in EpRas cells with MRP knockdown suggesting formation of adherens junctions. By wound healing assay we observed that reduced MRP supported collective cell migration, a type of cell movement where adherens junctions are maintained. However, destabilized adherens junctions, like those seen in EpRas cells, are frequently important for oncogenic signaling. Consequently, knockdown of MRP in EpRas caused loss of tumorigenesis in vivo, and reduced Wnt3a induced TCF reporter signaling in vitro. Together our data suggest that reducing MRP expression promotes formation of adherens junctions in EpRas cells, allowing collective cell migration, but interferes with oncogenic β-catenin signaling and tumorigenesis.     

Tumor suppressor p53 restricts Ras stimulation of RhoA and cancer cell motility

Many features of the cancer cell phenotype emerge as a result of cooperation between multiple oncogenic mutations. Here we show that activated Ras(V12) and loss of p53 function can cooperate to promote cell motility, a feature closely associated with cancer progression to malignancy. Our analysis indicates that Ras(V12) and loss of p53 synergistically induce RhoA activity, revealing a previously unknown role for p53 in tumor suppression. p53 prevents activation of RhoA and thus induction of cell motility by Ras(V12) through a simple signaling circuit, which integrates multiple inputs that converge on RhoA. Our data suggest that p53 suppresses cancer progression to malignancy by modulating the quality of Ras signaling.     

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.     

Differential interference of chlorpromazine with the membrane interactions of oncogenic K-Ras and its effects on cell growth

Membrane anchorage of Ras proteins is important for their signaling and oncogenic potential. K-Ras4B (K-Ras), the Ras isoform most often mutated in human cancers, is the only Ras isoform where a polybasic motif contributes essential electrostatic interactions with the negatively charged cytoplasmic leaflet. Here we studied the effects of the cationic amphiphilic drug chlorpromazine (CPZ) on the membrane association of oncogenic K-Ras(G12V), cell proliferation, and apoptosis. Combining live cell microscopy, FRAP beam size analysis, and cell fractionation studies, we show that CPZ reduces the association of GFP-K-Ras(G12V) with the plasma membrane and increases its exchange between plasma membrane and cytoplasmic pools. These effects appear to depend on electrostatic interactions because the membrane association of another related protein that has a membrane-interacting polybasic cluster (Rac1(G12V)) was also affected, whereas that of H-Ras was not. The weakened association with the plasma membrane led to a higher fraction of GFP-K-Ras(G12V) in the cytoplasm and in internal membranes, accompanied by either cell cycle arrest (PANC-1 cells) or apoptosis (Rat-1 fibroblasts), the latter being in correlation with the targeting of K-Ras(G12V) to mitochondria. In accord with these results, CPZ compromised the transformed phenotype of PANC-1 cells, as indicated by inhibition of cell migration and growth in soft agar.     

Novel role of Giα2 in cell migration: Downstream of PI3-kinase–AKT and Rac1 in prostate cancer cells

Tumor cell motility is the essential step in cancer metastasis. Previously, we showed that oxytocin and epidermal growth factor (EGF) effects on cell migration in prostate cancer cells require Giα2 protein. In the current study, we investigated the interactions among G-protein coupled receptor (GPCR), Giα2, PI3-kinase, and Rac1 activation in the induction of migratory and invasive behavior by diverse stimuli. Knockdown and knockout of endogenous Giα2 in PC3 cells resulted in attenuation of transforming growth factor β1 (TGFβ1), oxytocin, SDF-1α, and EGF effects on cell migration and invasion. In addition, knockdown of Giα2 in E006AA cells attenuated cell migration and overexpression of Giα2 in LNCaP cells caused significant increase in basal and EGF-stimulated cell migration. Pretreatment of PC3 cells with Pertussis toxin resulted in attenuation of TGFβ1- and oxytocin-induced migratory behavior and PI3-kinase activation without affecting EGF-induced PI3-kinase activation and cell migration. Basal- and EGF-induced activation of Rac1 in PC3 and DU145 cells were not affected in cells after Giα2 knockdown. On the other hand, Giα2 knockdown abolished the migratory capability of PC3 cells overexpressing constitutively active Rac1. The knockdown or knockout of Giα2 resulted in impaired formation of lamellipodia at the leading edge of the migrating cells. We conclude that Giα2 protein acts at two different levels which are both dependent and independent of GPCR signaling to induce cell migration and invasion in prostate cancer cells and its action is downstream of PI3-kinase–AKT–Rac1 axis.     

Oncogenic Ras-transformed human fibroblasts exhibit differential changes in contraction and migration in 3D collagen matrices

Tractional force exerted by tissue cells in 3D collagen matrices can be utilized for matrix remodeling or cell migration. The interrelationship between these motile processes is not well understood. The current studies were carried out to test the consequences of oncogenic Ras (H-Ras^V12) transformation on human fibroblast contraction and migration in 3D collagen matrices. Beginning with hTERT-immortalized cells, we prepared fibroblasts stably transformed with E6/E7 and with the combination HPV16 E6/E7 and H-Ras^V12. Oncogenic Ras-transformed cells lost contact inhibition of cell growth, formed colonies in soft agar and were unable to make adherens junctions. We observed no changes in the extent or growth factor dependence of collagen matrix contraction (floating or stress-relaxation) by oncogenic Ras-transformed cells. On the other hand, transformed cells in nested collagen matrices lost not only growth factor selectivity, but also cell-matrix density-dependent inhibition of migration. These findings demonstrate differential regulation of collagen matrix contraction and cell migration in 3D collagen matrices.     

Tage4/Nectin-like molecule-5 heterophilically trans-interacts with cell adhesion molecule Nectin-3 and enhances cell migration

Malignant transformation of cells causes disruption of cell-cell adhesion, enhancement of cell motility, and invasion into surrounding tissues. Nectins have both homophilic and heterophilic cell-cell adhesion activities and organize adherens junctions in cooperation with cadherins. We examined here whether Tage4, which was originally identified to be a gene overexpressed in colon carcinoma and has a domain structure similar to those of nectins, is involved in cell adhesion and/or migration. Tage4 heterophilically trans-interacted with nectin-3, but not homophilically with Tage4. Expression of Tage4 was markedly elevated in NIH3T3 cells transformed by an oncogenic Ki-Ras (V12Ras-NIH3T3 cells) as compared with that of wild-type NIH3T3 cells. trans-Interaction of Tage4 with nectin-3 enhanced motility of V12Ras-NIH3T3 cells. Tage4 did not bind afadin, a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton and cadherins through catenins. Thus, Tage4 heterophilically trans-interacts with nectin-3 and regulates cell migration. Tage4 is tentatively re-named here nectin-like molecule-5 (necl-5) on the basis of its function and domain structure similar to those of nectins.     

Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration

RhoA activity is transiently inhibited at the initial phase of integrin engagement, when Cdc42- and/or Rac1-mediated membrane spreading and ruffling predominantly occur. Paxillin, an integrin-assembly protein, has four major tyrosine phosphorylation sites, and the phosphorylation of Tyr31 and Tyr118 correlates with cell adhesion and migration. We found that mutation of Tyr31/118 caused enhanced activation of RhoA and premature formation of stress fibers with substantial loss of efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells. These phenotypes were similar to those induced by RhoA(G14V) in parental cells, and could be abolished by expression of RhoA(T19N), Rac1(G12V), or p190RhoGAP in the mutant-expressing cells. Phosphorylated Tyr31/118 was found to bind to two src homology (SH)2 domains of p120RasGAP, with coprecipitation of endogenous paxillin with p120RasGAP. p190RhoGAP is known to be a major intracellular binding partner for the p120RasGAP SH2 domains. We found that Tyr31/118-phosphorylated paxillin competes with p190RhoGAP for binding to p120RasGAP, and provides evidence that p190RhoGAP freed from p120RasGAP efficiently suppresses RhoA activity during cell adhesion. We conclude that Tyr31/118-phosphorylated paxillin serves as a template for the localized suppression of RhoA activity and is necessary for efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells.     

Adaptor protein Nck1 interacts with p120 Ras GTPase-activating protein and regulates its activity

Adaptor protein Nck1 binds a number of intracellular proteins and influences various signaling pathways. Here we show that Nck1 directly binds and activates the GTPase-activating protein of Ras (RasGAP), which is responsible for the down-regulation of Ras. The first and the third SH3 domains of Nck1 and the NH₂-terminal proline-rich sequence of RasGAP contribute most to the complex formation causing direct molecular interaction between the two proteins. Cell adhesion to the substrate is obligatory for the Nck1 and RasGAP association, as cell detachment makes RasGAP incapable of associating with Nck1. This leads to the complex dissipation, decrease of RasGAP activity and the increase of H-Ras-GTP level in the detached cells. Our findings reveal unexpected feature of adaptor protein Nck1 as the regulator of RasGAP activity.     

The Ras-GTPase-activating protein SH3 domain is required for Cdc2 activation and mos induction by oncogenic Ras in Xenopus oocytes independently of mitogen-activated protein kinase activation.

The Ras-GTPase-activating protein (RasGAP) is an important modulator of p21ras - dependent signal transduction in Xenopus oocytes and in mammalian cells. We investigated the role of the RasGAP SH3 domain in signal transduction with a monoclonal antibody against the SH3 domain of RasGaP. This antibody prevented the activation of the maturation-promoting factor complex (cyclin B-p34cdc2) by oncogenic Ras. The antibody appears to be specific because as little as 5 ng injected per oocyte reduced the level of Cdc2 activation by 50% whereas 100 ng of nonspecific immunoglobulin G did not affect Cdc2 activation. The antibody blocked the Cdc2 activation induced by oncogenic Ras but not that induced by progesterone, which acts independently of Ras. A peptide corresponding to positions 317 to 326 of a sequence in the SH3 domain of human RasGAP blocked Cdc2 activation, whereas a peptide corresponding to positions 273 to 305 of a sequence in the N-terminal moiety of the SH3 domain of RasGAP had no effect. The antibody did not block the mitogen-activated protein (MAP) kinase cascade (activation of MAPK/ERK kinase [MEK], MAP kinase, and S6 kinase p90rsk). Surprisingly, injection of the negative MAP kinase mutant protein ERK2 K52R (containing a K-to-R mutation at position 52) blocked the Cdc2 activation induced by oncogenic Ras as well as blocking the activation of MAP kinase. Thus, MAP kinase is also implicated in the regulation of Cdc2 activity. In this study, we further investigated the regulation of the synthesis of the c-mos oncogene product, which is necessary for the activation of Cdc2. We report that the synthesis of the c-mos oncogene product, which is necessary for the activation antibody to the SH3 domain of RasGAP and by injecting the negative MAP kinase mutant protein ERK2 K52R. These results suggest that oncogenic Ras activates two signaling mechanisms: the MAP kinase cascade and a signaling pathway implicating the SH3 domain of RasGAP. These mechanisms might control Mos protein expression implicated in Cdc2 activation.     

A direct interaction between oncogenic Ha-Ras and phosphatidylinositol 3-kinase is not required for Ha-Ras-dependent transformation of epithelial cells

Cells expressing oncogenic Ras proteins transmit a complex set of signals that ultimately result in constitutive activation of signaling molecules, culminating in unregulated cellular function. Although the role of oncogenic Ras in a variety of cellular responses including transformation, cell survival, differentiation, and migration is well documented, the direct Ras/effector interactions that contribute to the different Ras biological end points have not been as clearly defined. Observations by other groups in which Ras-dependent transformation can be blocked by expression of either dominant negative forms of Phosphatidylinositol (PI) 3-kinase or PTEN, a 3-phosphoinositide-specific phosphatase, support an essential role for PI 3-kinase and its lipid products in the transformation process. These observations coupled with the in vitro observations that the catalytic subunits of PI 3-kinase, the p110 isoforms, bind directly to Ras-GTP foster the implication that a direct interaction between an oncogenic Ras protein and PI 3-kinase are causal in the oncogenicity of mutant Ras proteins. Using an activated Ha-Ras protein (Y64G/Y71G/F156L) that fails to interact with PI 3-kinase, we demonstrate that oncogenic Ha-Ras does not require a direct interaction with PI 3-kinase to support anchorage-independent growth of IEC-6 epithelial cells. We do find, however, that IEC-6 cells expressing an oncogenic Ha-Ras protein that no longer binds PI 3-kinase are greatly impaired in their ability to migrate toward fibronectin.     

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.     

Activation of c-Ha-Ras by nitric oxide modulates survival responsiveness in neuronal PC12 cells

p21(c-Ha-Ras) (Ras) can be activated by the guanine nucleotide exchange factor mSOS1 or by S-nitrosylation of cysteine 118 via nitric oxide (NO). To determine whether these two Ras-activating mechanisms modulate distinct biological effects, a NO-nonresponsive Ras mutant (Ras(C118S)) was stably expressed in the PC12 cells, a cell line that generates NO upon nerve growth factor treatment. We report here that Ras(C118S) functions indistinguishably from wild type Ras in activating and maintaining the mSOS1- and Raf-1-dependent mitogen-activated protein kinase cascade necessary for neuronal differentiation. However, continuous (>5 days) exposure to nerve growth factor reveals that, in contrast to parental or wild-type Ras-overexpressing PC12 cells, Ras(C118S)-expressing PC12 cells cannot sustain the basal interaction between Ras and phosphatidylinositol 3-kinase. This results in spontaneous apoptosis of these cells despite the presence of nerve growth factor and serum. Thus unique downstream effector interactions and biological outcomes can be differentially modulated by distinct modes of Ras activation.