Molecular determinants of Ras-mTORC2 signaling.

Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested the involvement of the Switch I (SWI) effector domain of Ras in binding mTORC2 components, the regulation of the Ras-mTORC2 pathway is not entirely understood. In Dictyostelium, mTORC2 is selectively activated by the Ras protein RasC, and the RasC-mTORC2 pathway then mediates chemotaxis to cAMP and cellular aggregation by regulating the actin cytoskeleton and promoting cAMP signal relay. Here, we investigated the role of specific residues in RasC's SWI, C-terminal allosteric domain, and hypervariable region (HVR) related to mTORC2 activation. Interestingly, our results suggest that RasC SWI residue A31, which was previously implicated in RasC-mediated aggregation, regulates RasC's specific activation by the Aimless RasGEF. On the other hand, our investigation identified a crucial role for RasC SWI residue T36, with secondary contributions from E38 and allosteric domain residues. Finally, we found that conserved basic residues and the adjacent prenylation site in the HVR, which are crucial for RasC's membrane localization, are essential for RasC-mTORC2 pathway activation by allowing for both RasC's own cAMP-induced activation and its subsequent activation of mTORC2. Therefore, our findings revealed new determinants of RasC-mTORC2 pathway specificity in Dictyostelium, contributing to a deeper understanding of Ras signaling regulation in eukaryotic cells.

Ras, PI3K and mTORC2 - three's a crowd?

The Ras oncogene is notoriously difficult to target with specific therapeutics. Consequently, there is interest to better understand the Ras signaling pathways to identify potential targetable effectors. Recently, the mechanistic target of rapamycin complex 2 (mTORC2) was identified as an evolutionarily conserved Ras effector. mTORC2 regulates essential cellular processes, including metabolism, survival, growth, proliferation and migration. Moreover, increasing evidence implicate mTORC2 in oncogenesis. Little is known about the regulation of mTORC2 activity, but proposed mechanisms include a role for phosphatidylinositol (3,4,5)-trisphosphate - which is produced by class I phosphatidylinositol 3-kinases (PI3Ks), well-characterized Ras effectors. Therefore, the relationship between Ras, PI3K and mTORC2, in both normal physiology and cancer is unclear; moreover, seemingly conflicting observations have been reported. Here, we review the evidence on potential links between Ras, PI3K and mTORC2. Interestingly, data suggest that Ras and PI3K are both direct regulators of mTORC2 but that they act on distinct pools of mTORC2: Ras activates mTORC2 at the plasma membrane, whereas PI3K activates mTORC2 at intracellular compartments. Consequently, we propose a model to explain how Ras and PI3K can differentially regulate mTORC2, and highlight the diversity in the mechanisms of mTORC2 regulation, which appear to be determined by the stimulus, cell type, and the molecularly and spatially distinct mTORC2 pools.

Protein kinase A regulates the Ras, Rap1 and TORC2 pathways in response to the chemoattractant cAMP in Dictyostelium.

Efficient directed migration requires tight regulation of chemoattractant signal transduction pathways in both space and time, but the mechanisms involved in such regulation are not well understood. Here, we investigated the role of protein kinase A (PKA) in controlling signaling of the chemoattractant cAMP in Dictyostelium discoideum We found that cells lacking PKA display severe chemotaxis defects, including impaired directional sensing. Although PKA is an important regulator of developmental gene expression, including the cAMP receptor cAR1, our studies using exogenously expressed cAR1 in cells lacking PKA, cells lacking adenylyl cyclase A (ACA) and cells treated with the PKA-selective pharmacological inhibitor H89, suggest that PKA controls chemoattractant signal transduction, in part, through the regulation of RasG, Rap1 and TORC2. As these pathways control the ACA-mediated production of intracellular cAMP, they lie upstream of PKA in this chemoattractant signaling network. Consequently, we propose that the PKA-mediated regulation of the upstream RasG, Rap1 and TORC2 signaling pathways is part of a negative feedback mechanism controlling chemoattractant signal transduction during Dictyostelium chemotaxis.

Caffeine inhibits PI3K and mTORC2 in Dictyostelium and differentially affects multiple other cAMP chemoattractant signaling effectors.

Caffeine is commonly used in Dictyostelium to inhibit the synthesis of the chemoattractant cAMP and, therefore, its secretion and the autocrine stimulation of cells, in order to prevent its interference with the study of chemoattractant-induced responses. However, the mechanism through which caffeine inhibits cAMP synthesis in Dictyostelium has not been characterized. Here, we report the effects of caffeine on the cAMP chemoattractant signaling network. We found that caffeine inhibits phosphatidylinositol 3-kinase (PI3K) and mechanistic target of rapamycin complex 2 (mTORC2). Both PI3K and mTORC2 are essential for the chemoattractant-stimulated cAMP production, thereby providing a mechanism for the caffeine-mediated inhibition of cAMP synthesis. Our results also reveal that caffeine treatment of cells leads to an increase in cAMP-induced RasG and Rap1 activation, and inhibition of the PKA, cGMP, MyoII, and ERK1 responses. Finally, we observed that caffeine has opposite effects on F-actin and ERK2 depending on the assay and Dictyostelium strain used, respectively. Altogether, our findings reveal that caffeine considerably affects the cAMP-induced chemotactic signaling pathways in Dictyostelium, most likely acting through multiple targets that include PI3K and mTORC2.

Degradation of activated K-Ras orthologue via K-Ras-specific lysine residues is required for cytokinesis.

Mammalian cells encode three closely related Ras proteins, H-Ras, N-Ras, and K-Ras. Oncogenic K-Ras mutations frequently occur in human cancers, which lead to dysregulated cell proliferation and genomic instability. However, mechanistic role of the Ras isoform regulation have remained largely unknown. Furthermore, the dynamics and function of negative regulation of GTP-loaded K-Ras have not been fully investigated. Here, we demonstrate RasG, the Dictyostelium orthologue of K-Ras, is targeted for degradation by polyubiquitination. Both ubiquitination and degradation of RasG were strictly associated with RasG activity. High resolution tandem mass spectrometry (LC-MS/MS) analysis indicated that RasG ubiquitination occurs at C-terminal lysines equivalent to lysines found in human K-Ras but not in H-Ras and N-Ras homologues. Substitution of these lysine residues with arginines (4KR-RasG) diminished RasG ubiquitination and increased RasG protein stability. Cells expressing 4KR-RasG failed to undergo proper cytokinesis and resulted in multinucleated cells. Ectopically expressed human K-Ras undergoes polyubiquitin-mediated degradation in Dictyostelium, whereas human H-Ras and a Dictyostelium H-Ras homologue (RasC) are refractory to ubiquitination. Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium, and further identification of the responsible E3-ligase may provide a novel therapeutic approach against K-Ras-mutated cancers.

Evaluating the Activity of Ras/Rap GTPases in Dictyostelium.

Ras and Rap small GTPases of the Ras superfamily act as molecular switches to control diverse cellular processes as part of different signaling pathways. Dictyostelium expresses several Ras and Rap proteins, and their study has and continues to greatly contribute to our understanding of their role in eukaryote biology. To study the activity of Ras and Rap proteins in Dictyostelium, several assays based on their interaction with the Ras binding domain of known eukaryotic Ras/Rap effectors have been developed and proved extremely useful to study their regulation and cellular roles. Here, we describe methods to assess Ras/Rap activity biochemically using a pull-down assay and through live-cell imaging using fluorescent reporters.

APEX2-Mediated Proximity Protein Labeling in Dictyostelium.

Largely due to its simplicity, while being more like human cells compared to other experimental models, Dictyostelium continues to be of great use to discover basic molecular mechanisms and signaling pathways underlying evolutionarily conserved biological processes. However, the identification of new protein interactions implicated in signaling pathways can be particularly challenging in Dictyostelium due to its extremely fast signaling kinetics coupled with the dynamic nature of signaling protein interactions. Recently, the proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells was shown to allow the detection of weak and/or transient protein interactions and also to obtain spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium. Coupled with the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium's proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium.

Extracellular signals induce dynamic ER remodeling through αTAT1-dependent microtubule acetylation.

Dynamic changes in the endoplasmic reticulum (ER) morphology are central to maintaining cellular homeostasis. Microtubules (MT) facilitate the continuous remodeling of the ER network into sheets and tubules by coordinating with many ER-shaping protein complexes, although how this process is controlled by extracellular signals remains unknown. Here we report that TAK1, a kinase responsive to various growth factors and cytokines including TGF-ß and TNF-α, triggers ER tubulation by activating αTAT1, an MT-acetylating enzyme that enhances ER-sliding. We show that this TAK1/αTAT1-dependent ER remodeling promotes cell survival by actively downregulating BOK, an ER membrane-associated proapoptotic effector. While BOK is normally protected from degradation when complexed with IP3R, it is rapidly degraded upon their dissociation during the ER sheets-to-tubules conversion. These findings demonstrate a distinct mechanism of ligand-induced ER remodeling and suggest that the TAK1/αTAT1 pathway may be a key target in ER stress and dysfunction.

CRISPR-mediated reversion of oncogenic KRAS mutation results in increased proliferation and reveals independent roles of Ras and mTORC2 in the migration of A549 lung cancer cells.

Although the RAS oncogene has been extensively studied, new aspects concerning its role and regulation in normal biology and cancer continue to be discovered. Recently, others and we have shown that the mechanistic Target of Rapamycin Complex 2 (mTORC2) is a Ras effector in Dictyostelium and mammalian cells. mTORC2 plays evolutionarily conserved roles in cell survival and migration and has been linked to tumorigenesis. Because RAS is often mutated in lung cancer, we investigated whether a Ras-mTORC2 pathway contributes to enhancing the migration of lung cancer cells expressing oncogenic Ras. We used A549 cells and CRISPR/Cas9 to revert the cells' KRAS G12S mutation to wild-type and establish A549 revertant (REV) cell lines, which we then used to evaluate the Ras-mediated regulation of mTORC2 and cell migration. Interestingly, our results suggest that K-Ras and mTORC2 promote A549 cell migration but as part of different pathways and independently of Ras's mutational status. Moreover, further characterization of the A549REV cells revealed that loss of mutant K-Ras expression for the wild-type protein leads to an increase in cell growth and proliferation, suggesting that the A549 cells have low KRAS-mutant dependency and that recovering expression of wild-type K-Ras protein increases these cells tumorigenic potential.

Proximity Protein Labeling In Dictyostelium With Engineered Ascorbic Acid Peroxidase 2.

To fully understand any cellular process, we not only need to identify the proteins implicated, but also how the protein network is structurally and spatially organized and changes over time. However, the dynamic nature of many protein interactions involved in cellular signaling pathways continues to be the bottleneck in mapping and studying protein networks. Fortunately, a recently developed proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells allows the identification of weak and/or transient protein interactions with spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium, using the cAMP receptor cAR1 as example. Coupled to the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium's proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium.

Ras-mediated activation of mTORC2 promotes breast epithelial cell migration and invasion.

We previously identified the mechanistic target of rapamycin complex 2 (mTORC2) as an effector of Ras for the control of directed cell migration in Dictyostelium. Recently, the Ras-mediated regulation of mTORC2 was found to be conserved in mammalian cells, and mTORC2 was shown to be an effector of oncogenic Ras. Interestingly, mTORC2 has been linked to cancer cell migration, and particularly in breast cancer. Here, we investigated the role of Ras in promoting the migration and invasion of breast cancer cells through mTORC2. We observed that both Ras and mTORC2 promote the migration of different breast cancer cells and breast cancer cell models. Using HER2 and oncogenic Ras-transformed breast epithelial MCF10A cells, we found that both wild-type Ras and oncogenic Ras promote mTORC2 activation and an mTORC2-dependent migration and invasion in these breast cancer models. We further observed that, whereas oncogenic Ras-transformed MCF10A cells display uncontrolled cell proliferation and invasion, disruption of mTORC2 leads to loss of invasiveness only. Together, our findings suggest that, whereas the Ras-mediated activation of mTORC2 is expected to play a minor role in breast tumor formation, the Ras-mTORC2 pathway plays an important role in promoting the migration and invasion of breast cancer cells.

G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility.

Phosphoinositide 3-kinase (PI3K)gamma and Dictyostelium PI3K are activated via G protein-coupled receptors through binding to the Gbetagamma subunit and Ras. However, the mechanistic role(s) of Gbetagamma and Ras in PI3K activation remains elusive. Furthermore, the dynamics and function of PI3K activation in the absence of extracellular stimuli have not been fully investigated. We report that gbeta null cells display PI3K and Ras activation, as well as the reciprocal localization of PI3K and PTEN, which lead to local accumulation of PI(3,4,5)P(3). Simultaneous imaging analysis reveals that in the absence of extracellular stimuli, autonomous PI3K and Ras activation occur, concurrently, at the same sites where F-actin projection emerges. The loss of PI3K binding to Ras-guanosine triphosphate abolishes this PI3K activation, whereas prevention of PI3K activity suppresses autonomous Ras activation, suggesting that PI3K and Ras form a positive feedback circuit. This circuit is associated with both random cell migration and cytokinesis and may have initially evolved to control stochastic changes in the cytoskeleton.

Activated membrane patches guide chemotactic cell motility.

Many eukaryotic cells are able to crawl on surfaces and guide their motility based on environmental cues. These cues are interpreted by signaling systems which couple to cell mechanics; indeed membrane protrusions in crawling cells are often accompanied by activated membrane patches, which are localized areas of increased concentration of one or more signaling components. To determine how these patches are related to cell motion, we examine the spatial localization of RasGTP in chemotaxing Dictyostelium discoideum cells under conditions where the vertical extent of the cell was restricted. Quantitative analyses of the data reveal a high degree of spatial correlation between patches of activated Ras and membrane protrusions. Based on these findings, we formulate a model for amoeboid cell motion that consists of two coupled modules. The first module utilizes a recently developed two-component reaction diffusion model that generates transient and localized areas of elevated concentration of one of the components along the membrane. The activated patches determine the location of membrane protrusions (and overall cell motion) that are computed in the second module, which also takes into account the cortical tension and the availability of protrusion resources. We show that our model is able to produce realistic amoeboid-like motion and that our numerical results are consistent with experimentally observed pseudopod dynamics. Specifically, we show that the commonly observed splitting of pseudopods can result directly from the dynamics of the signaling patches.

Spatiotemporal regulation of Ras activity provides directional sensing.

Cells' ability to detect and orient themselves in chemoattractant gradients has been the subject of numerous studies, but the underlying molecular mechanisms remain largely unknown [1]. Ras activation is the earliest polarized response to chemoattractant gradients downstream from heterotrimeric G proteins in Dictyostelium, and inhibition of Ras signaling results in directional migration defects [2]. Activated Ras is enriched at the leading edge, promoting the localized activation of key chemotactic effectors, such as PI3K and TORC2 [2-5]. To investigate the role of Ras in directional sensing, we studied the effect of its misregulation by using cells with disrupted RasGAP activity. We identified an ortholog of mammalian NF1, DdNF1, as a major regulator of Ras activity in Dictyostelium. We show that disruption of nfaA leads to spatially and temporally unregulated Ras activity, causing cytokinesis and chemotaxis defects. By using unpolarized, latrunculin-treated cells, we show that tight regulation of Ras is important for gradient sensing. Together, our findings suggest that Ras is part of the cell's compass and that the RasGAP-mediated regulation of Ras activity affects directional sensing.

Feedback signaling controls leading-edge formation during chemotaxis.

Chemotactic cells translate shallow chemoattractant gradients into a highly polarized intracellular response that includes the localized production of PI(3,4,5)P(3) on the side of the cell facing the highest chemoattractant concentration. Research over the past decade began to uncover the molecular mechanisms involved in this localized signal amplification controlling the leading edge of chemotaxing cells. These mechanisms have been shown to involve multiple positive feedback loops, in which the PI(3,4,5)P(3) signal amplifies itself independently of the original stimulus, as well as inhibitory signals that restrict PI(3,4,5)P(3) to the leading edge, thereby creating a steep intracellular PI(3,4,5)P(3) gradient. Molecules involved in positive feedback signaling at the leading edge include the small G-proteins Rac and Ras, phosphatidylinositol-3 kinase and F-actin, as part of interlinked feedback loops that lead to a robust production of PI(3,4,5)P(3).

The V2 vasopressin receptor stimulates ERK1/2 activity independently of heterotrimeric G protein signalling.

The V2 vasopressin receptor (V2R) activates the mitogen activated protein kinases (MAPK) ERK1/2 through a mechanism involving the scaffolding protein beta arrestin. Here we report that this activating pathway is independent of G alpha s, G alpha i, G alpha q or G betagamma and that the V2R-mediated activation of G alpha s inhibits ERK1/2 activity in a cAMP/PKA-dependent manner. In the HEK293 cells studied, the beta arrestin-promoted activation was found to dominate over the PKA-mediated inhibition of the pathway, leading to a strong vasopressin-stimulated ERK1/2 activation. Despite the strong MAPK activation and in contrast with other GPCR, V2R did not induce any significant increase in DNA synthesis, consistent with the notion that the stable interaction between V2R and beta arrestin prevents signal propagation to the nucleus. Beta arrestin was found to be essential for the ERK1/2 activation, indicating that the recruitment of the scaffolding protein is necessary and sufficient to initiate the signal in the absence of any other stimulatory cues. Based on the use of selective pharmacological inhibitors, dominant negative mutants and siRNA, we conclude that the beta arrestin-dependent activation of ERK1/2 by the V2R involves c-Src and a metalloproteinase-dependent trans-activation event. These findings demonstrate that beta arrestin is a genuine signalling initiator that can, on its own, engage a MAPK activation machinery upon stimulation of a GPCR by its natural ligand.

Big roles for small GTPases in the control of directed cell movement.

Small GTPases are involved in the control of diverse cellular behaviours, including cellular growth, differentiation and motility. In addition, recent studies have revealed new roles for small GTPases in the regulation of eukaryotic chemotaxis. Efficient chemotaxis results from co-ordinated chemoattractant gradient sensing, cell polarization and cellular motility, and accumulating data suggest that small GTPase signalling plays a central role in each of these processes as well as in signal relay. The present review summarizes these recent findings, which shed light on the molecular mechanisms by which small GTPases control directed cell migration.

The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules.

To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2ßγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2ßγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2ßγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2ßγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2ßγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2ßγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing.

Studying Chemoattractant Signal Transduction Dynamics in Dictyostelium by BRET.

Understanding the dynamics of chemoattractant signaling is key to our understanding of the mechanisms underlying the directed migration of cells, including that of neutrophils to sites of infections and of cancer cells during metastasis. A model frequently used for deciphering chemoattractant signal transduction is the social amoeba Dictyostelium discoideum. However, the methods available to quantitatively measure chemotactic signaling are limited. Here, we describe a protocol to quantitatively study chemoattractant signal transduction in Dictyostelium by monitoring protein-protein interactions and conformational changes using Bioluminescence Resonance Energy Transfer (BRET).

The small GTPases Ras and Rap1 bind to and control TORC2 activity.

Target of Rapamycin Complex 2 (TORC2) has conserved roles in regulating cytoskeleton dynamics and cell migration and has been linked to cancer metastasis. However, little is known about the mechanisms regulating TORC2 activity and function in any system. In Dictyostelium, TORC2 functions at the front of migrating cells downstream of the Ras protein RasC, controlling F-actin dynamics and cAMP production. Here, we report the identification of the small GTPase Rap1 as a conserved binding partner of the TORC2 component RIP3/SIN1, and that Rap1 positively regulates the RasC-mediated activation of TORC2 in Dictyostelium. Moreover, we show that active RasC binds to the catalytic domain of TOR, suggesting a mechanism of TORC2 activation that is similar to Rheb activation of TOR complex 1. Dual Ras/Rap1 regulation of TORC2 may allow for integration of Ras and Rap1 signaling pathways in directed cell migration.