Calmodulin binds RalA and RalB and is required for the thrombin-induced activation of Ral in human platelets

Ral GTPases may be involved in calcium/calmodulin-mediated intracellular signaling pathways. RalA and RalB are activated by calcium, and RalA binds calmodulin in vitro. It was examined whether RalA can bind calmodulin in vivo, whether RalB can bind calmodulin, and whether calmodulin is functionally involved in Ral activation. Yeast two-hybrid analyses demonstrated both Rals interact directly but differentially with calmodulin. Coimmunoprecipitation experiments determined that calmodulin and RalB form complexes in human platelets. In vitro pull-down experiments in platelets and in vitro binding assays showed endogenous Ral and calmodulin interact in a calcium-dependent manner. Truncated Ral constructs determined in vitro and in vivo that RalA has an additional calmodulin binding domain to that previously described, that although RalB binds calmodulin, its C-terminal region is involved in partially inhibiting this interaction, and that in vitro RalA and RalB have an N-terminal calcium-independent and a C-terminal calcium-dependent calmodulin binding domain. Functionally, in vitro Ral-GTP pull-down experiments determined that calmodulin is required for the thrombin-induced activation of Ral in human platelets. We propose that differential binding of calmodulin by RalA and RalB underlies possible functional differences between the two proteins and that calmodulin is involved in the regulation of the activation of Ral-GTPases.     

Involvement of Ras and Ral in chemotactic migration of skeletal myoblasts

In skeletal myoblasts, Ras has been considered to be a strong inhibitor of myogenesis. Here, we demonstrate that Ras is involved also in the chemotactic response of skeletal myoblasts. Expression of a dominant-negative mutant of Ras inhibited chemotaxis of C2C12 myoblasts in response to basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1), key regulators of limb muscle development and skeletal muscle regeneration. A dominant-negative Ral also decreased chemotactic migration by these growth factors, while inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase (MEK) showed no effect. Activation of the Ras-Ral pathway by expression of an activated mutant of either Ras, the guanine-nucleotide dissociation stimulator for Ral, or Ral resulted in increased motility of myoblasts. The ability of Ral to stimulate motility was reduced by introduction of a mutation which prevents binding to Ral-binding protein 1 or phospholipase D. These results suggest that the Ras-Ral pathway is essential for the migration of myoblasts. Furthermore, we found that Ras and Ral are activated in C2C12 cells by bFGF, HGF and IGF-1 and that the Ral activation is regulated by the Ras- and the intracellular Ca(2+)-mediated pathways. Taken together, our data indicate that Ras and Ral regulate the chemotactic migration of skeletal muscle progenitors.     

Ral-GTPase interacts with the beta1 subunit of Na+/K+-ATPase and is activated upon inhibition of the Na+/K+ pump

Na+/K+-ATPase functions as both an ion pump and a signal transducer. Cardiac glycosides partially inhibit Na+/K+-ATPase, causing activation of multiple interrelated growth pathways via the Na+/K+-ATPase/c-Src/epidermal growth factor receptor complex. Such pathways include Ras/MEK/ERK and Ral/RalGDS cascades, which can lead to cardiac hypertrophy. In search of novel Ral-GTPase binding proteins, we used RalB as the bait to screen a human testes cDNA expression library using the yeast 2-hybrid system. The results demonstrated that 1 of the RalB interacting clones represented the C-terminal region of the beta1 subunit of Na+/K+-ATPase. Further analysis using the yeast 2-hybrid system and full-length beta1 subunit of Na+/K+-ATPase confirmed the interaction with RalA and RalB. In vitro binding and pull-down assays demonstrated that the beta1 subunit of Na+/K+-ATPase interacts directly with RalA and RalB. Ral-GTP pull-down assays demonstrated that short-term ouabain treatment of A7r5 cells, a rat aorta smooth muscle cell line, caused activation of Ral GTPase. Maximal activation was observed 10 min after ouabain treatment. Ouabain-mediated Ral activation was inhibited upon the stimulation of Na+/K+-ATPase activity by Ang II. We propose that Ral GTPase is involved in the signal transducing function of Na+/K+-ATPase and provides a possible molecular mechanism connecting Ral to cardiac hypertrophy during diseased conditions.     

RLIP mediates downstream signalling from RalB to the actin cytoskeleton during Xenopus early development

The Ras protein activates at least three different pathways during early development. Two of them regulate mesodermal gene expression and the third is thought to participate in the control of actin cytoskeleton dynamics via the Ral protein. From a yeast two-hybrid screen of a Xenopus maternal cDNA library, we identified the Xenopus orthologue of the Ral interacting protein (RLIP, RIP1 or RalBP1), a putative effector of small G protein Ral. Previously, we observed that a constitutively activated form of Ral GTPase (XralB G23V) induced bleaching of the animal hemisphere and disruption of the cortical actin cytoskeleton. To demonstrate that RLIP is the effector of RalB in early development, we show that the artificial targeting of RLIP to the membrane induces a similar phenotype to that of activated RalB. We show that overexpression of the Ral binding domain (RalBD) of XRLIP, which binds to the effector site of Ral, acts in competition with the endogenous effector of Ral and protects against the destructive effect of XralB G23V on the actin cytoskeleton. In contrast, the XRLIP has a synergistic effect on the activated form of XralB, which is dependent on the RalBD of RLIP. We provide evidence for the involvement of RLIP by way of its RalBD on the dynamics of the actin cytoskeleton and propose that signalling from Ral to RLIP is required for gastrulation.     

Activation of the Small GTPase Ral in Platelets

Ral is a ubiquitously expressed Ras-like small GTPase which is abundantly present in human platelets. The biological function of Ral and the signaling pathway in which Ral is involved are largely unknown. Here we describe a novel method to measure Ral activation utilizing the Ral binding domain of the putative Ral effector RLIP76 as an activation-specific probe. With this assay we investigated the signaling pathway that leads to Ral activation in human platelets. We found that Ral is rapidly activated after stimulation with various platelet agonists, including α-thrombin. In contrast, the platelet antagonist prostaglandin I₂ inhibited α-thrombin-induced Ral activation. Activation of Ral by α-thrombin could be inhibited by depletion of intracellular Ca²⁺, whereas the induction of intracellular Ca²⁺ resulted in the activation of Ral. Our results show that Ral can be activated by extracellular stimuli. Furthermore, we show that increased levels of intracellular Ca²⁺ are sufficient for Ral activation in platelets. This activation mechanism correlates with the activation mechanism of the small GTPase Rap1, a putative upstream regulator of Ral guanine nucleotide exchange factors.     

Novel Binding Partners and Differentially Regulated Phosphorylation Sites Clarify Eps8 as a Multi-Functional Adaptor

Eps8 is involved in both cell signalling and receptor trafficking. It is a known phosphorylation substrate for two proteins involved in the fibroblast growth factor receptor (FGFR) signalling pathway: the receptor itself and Src. Here we report a differential proteomic analysis of Eps8 aimed to identify specific FGFR and Src family kinase dependent phosphosites and co-associated phosphodependent binding partners. This study reveals a total of 22 Eps8 pTyr and pSer/Thr phosphorylation sites, including those that are dependent on Src family and FGFR kinase activity. Peptide affinity purification of proteins that bind to a selection of the pTyr phosphosites has identified a range of novel Eps8 binding partners including members of the intracellular vesicle trafficking machinery (clathrin and AP-2), proteins which have been shown to regulate activated receptor trafficking (NBR1 and Vav2), and proteins involved in receptor signalling (IRS4 and Shp2). Collectively this study significantly extends the understanding of Eps8 post-translational modification by regulated phosphorylation, identifies novel Eps8 binding partners implicated in receptor trafficking and signalling, and confirms the functions of Eps8 at the nexus of receptor signalling and vesicular trafficking.     

Compartment-specific feedback loop and regulated trafficking can result in sustained activation of Ras at the Golgi

Imaging experiments have shown that cell signaling components such as Ras can be activated by growth factors at distinct subcellular locations. Trafficking between these subcellular locations is a regulated dynamic process. The effects of trafficking and the molecular mechanisms underlying compartment-specific Ras activation were studied using numerical simulations of an ordinary differential equation-based multi-compartment model. The simulations show that interplay between two distinct mechanisms, a palmitoylation cycle that controls Ras trafficking and a phospholipase C-epsilon (PLC-epsilon) driven feedback loop, can convert a transient calcium signal into prolonged Ras activation at the Golgi. Detailed analysis of the network identified PLC-epsilon as a key determinant of "compartment switching". Modulation of PLC-epsilon activity switches the location of activated Ras between the plasma membrane and Golgi through a new mechanism termed "kinetic scaffolding". These simulations indicate that multiple biochemical mechanisms, when appropriately coupled, can give rise to an intracellular compartment-specific sustained Ras activation in response to stimulation of growth factor receptors at the plasma membrane.     

A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes

Insulin-stimulated glucose uptake in fat and muscle is mediated by the major facilitative glucose transporter Glut4. Insulin controls the trafficking of Glut4 to the plasma membrane via regulation of a series of small G proteins, including RalA and Rab10. We demonstrate here that Rab10 is a bona fide target of the GTPase-activating protein AS160, which is inhibited after phosphorylation by the protein kinase Akt. Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2. Rab10 and RalA reside in the same pool of Glut4-storage vesicles in untreated cells, and, together with Rlf, they ensure maximal glucose transport. Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10. Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.     

The Microtubule-Associated Rho Activating Factor GEF-H1 Interacts with Exocyst Complex to Regulate Vesicle Traffic

The exocyst complex plays a critical role in targeting and tethering vesicles to specific sites of the plasma membrane. These events are crucial for polarized delivery of membrane components to the cell surface, which is critical for cell motility and division. Though Rho GTPases are involved in regulating actin dynamics and membrane trafficking, their role in exocyst-mediated vesicle targeting is not very clear. Herein, we present evidence that depletion of GEF-H1, a guanine nucleotide exchange factor for Rho proteins, affects vesicle trafficking. Interestingly, we found that GEF-H1 directly binds to exocyst component Sec5 in a Ral GTPase-dependent manner. This interaction promotes RhoA activation, which then regulates exocyst assembly/localization and exocytosis. Taken together, our work defines a mechanism for RhoA activation in response to RalA-Sec5 signaling and involvement of GEF-H1/RhoA pathway in the regulation of vesicle trafficking.     

Ral家族与肿瘤

Ras相关蛋白Ral家族包括RalA和RalB,参与介导了多种细胞内外信号转导、跨膜运输、细胞形态的维持、转录因子的调节、细胞分化和肿瘤发生与转移等重要的生命活动。Ral家族上游信号通路以及广泛分布的效应器网络对其各种功能起着重要的调控作用,对Ral家族的深入研究将为预防和治疗肿瘤提供了一条新的途径。     

Dual effects of Ral-activated pathways on p27 localization and TGF-β signaling

Constitutive activation or overactivation of Ras signaling pathways contributes to epithelial tumorigenesis in several ways, one of which is cytoplasmic mislocalization of the cyclin-dependent kinase inhibitor p27^Kip1 (p27). We previously showed that such an effect can be mediated by activation of the Ral-GEF pathway by oncogenic N-Ras. However, the mechanism(s) leading to p27 cytoplasmic accumulation downstream of activated Ral remained unknown. Here, we report a dual regulation of p27 cellular localization by Ral downstream pathways, based on opposing effects via the Ral effectors RalBP1 and phospholipase D1 (PLD1). Because RalA and RalB are equally effective in mislocalizing both murine and human p27, we focus on RalA and murine p27, which lacks the Thr-157 phosphorylation site of human p27. In experiments based on specific RalA and p27 mutants, complemented with short hairpin RNA–mediated knockdown of Ral downstream signaling components, we show that activation of RalBP1 induces cytoplasmic accumulation of p27 and that this event requires p27 Ser-10 phosphorylation by protein kinase B/Akt. Of note, activation of PLD1 counteracts this effect in a Ser-10–independent manner. The physiological relevance of the modulation of p27 localization by Ral is demonstrated by the ability of Ral-mediated activation of the RalBP1 pathway to abrogate transforming growth factor-β–mediated growth arrest in epithelial cells.     

FKBP12: A partner of Snx10 required for vesicular trafficking in osteoclasts

Osteoclasts employ highly specialized intracellular trafficking controls for bone resorption and organelle homeostasis. The sorting nexin Snx10 is a (Phosphatidylinositol 3-phosphate) PI3P-binding protein, which localizes to osteoclast early endosomes. Osteoclasts from humans and mice lacking functional Snx10 are severely dysfunctional. They show marked impairments in endocytosis, extracellular acidification, ruffled border formation, and bone resorption, suggesting that Snx10 regulates membrane trafficking. To better understand how SNx10 regulates vesicular formation and trafficking in osteoclasts, we set out on a search for Snx10 partners. We performed a yeast two-hybrid screening and identified FKBP12. FKBP12 is expressed in receptor activator of nuclear factor kB ligand–stimulated RAW264.7 monocytes, coimmunoprecipitates with Snx10, and colocalizes with Snx10 in osteoclasts. We also found that FKBP12, Snx10, and early endosome antigen 1 (EEA1) are present in the same subcellular fractions obtained by centrifugation in sucrose gradients, which confirms localization of FKBP12 to early endosomes. Taken together, these results indicate that Snx10 and FKBP12 are partners and suggest that Snx10 and FKBP12 are involved in the regulation of endosome/lysosome homeostasis via the synthesis. These findings may suggest novel therapeutic approaches to control bone loss by targeting essential steps in osteoclast membrane trafficking.     

PDK1 mediates growth factor-induced Ral-GEF activation by a kinase-independent mechanism

Ras proteins transduce extracellular signals to intracellular signaling pathways by binding to and promoting the activation of at least three classes of downstream signaling molecules: Raf kinases, phosphoinositide-3-kinase (PI3-K) and Ral guanine nucleotide exchange factors (Ral-GEFs). Previous work has demonstrated that epidermal growth factor (EGF) activates Ral-GEFs, at least in part, by a Ras-mediated redistribution of the GEFs to their target, Ral-GTPases, in the plasma membrane. Here we show that Ral-GEF stimulation by EGF involves an additional mechanism, PI3-K-dependent kinase 1 (PDK1)-induced enhancement of Ral-GEF catalytic activity. Remarkably, this PDK1 function is not dependent upon its kinase activity. Instead, the non-catalytic N-terminus of PDK1 mediates the formation of an EGF-induced complex with the N-terminus of the Ral-GEF, Ral-GDS, thereby relieving its auto-inhibitory effect on the catalytic domain of Ral-GDS. These results elucidate a novel function for PDK1 and demonstrate that two Ras effector pathways cooperate to promote Ral-GTPase activation.     

SNAP-29 is a promiscuous syntaxin-binding SNARE.

SNARE proteins are key regulators of membrane fusion and are proposed to dictate the specificity with which particular vesicles fuse with particular target organelles. On intracellular organelles that serve as targets for transport vesicles, organelle-specific syntaxins form heterodimers with either SNAP-23 or its recently described homolog SNAP-29. We have performed a variety of in vitro and in vivo binding assays in an attempt to determine whether SNAP-23 and SNAP-29 differ in their ability to form binary SNARE complexes with different intracellular syntaxins. While SNAP-23 preferentially binds to plasma membrane-localized syntaxins, SNAP-29 binds to both plasma membrane and intracellular syntaxins equally well. Furthermore, binding to SNAP-29 augments the ability of syntaxin to bind to vesicle-associated SNAREs and the presence of vesicle SNAREs dramatically increases SNAP-29 binding to syntaxin. These data suggest that SNAP-23 preferentially regulates plasma membrane-vesicle fusion events while SNAP-29 plays a role in the maintenance of various intracellular protein trafficking pathways.     

Ral GTPases regulate exocyst assembly through dual subunit interactions

Ral GTPases have been implicated in the regulation of a variety of dynamic cellular processes including proliferation, oncogenic transformation, actin-cytoskeletal dynamics, endocytosis, and exocytosis. Recently the Sec6/8 complex, or exocyst, a multisubunit complex facilitating post-Golgi targeting of distinct subclasses of secretory vesicles, has been identified as a bona fide Ral effector complex. Ral GTPases regulate exocyst-dependent vesicle trafficking and are required for exocyst complex assembly. Sec5, a membrane-associated exocyst subunit, has been identified as a direct target of activated Ral; however, the mechanism by which Ral can modulate exocyst assembly is unknown. Here we report that an additional component of the exocyst, Exo84, is a direct target of activated Ral. We provide evidence that mammalian exocyst components are present as distinct subcomplexes on vesicles and the plasma membrane and that Ral GTPases regulate the assembly interface of a full octameric exocyst complex through interaction with Sec5 and Exo84.     

Membrane and protein interactions of oxysterols

PURPOSE OF REVIEW: Oxysterols, oxidation products of cholesterol, mediate numerous and diverse biological processes. The objective of this review is to explain some of the biochemical and cell biological properties of oxysterols based on their membrane biophysical properties and their interaction with integral and peripheral membrane proteins. RECENT FINDINGS: According to their biophysical properties, which can be distinct from those of cholesterol, oxysterols can promote or inhibit the formation of membrane microdomains or lipid rafts. Oxysterols that inhibit raft formation are cytotoxic. The stereo-specific binding of cholesterol to sterol-sensing domains in cholesterol homeostatic pathways is not duplicated by oxysterols, and some oxysterols are poor substrates for the pathways that detoxify cells of excess cholesterol. The cytotoxic roles of oxysterols are, at least partly, due to a direct physical effect on membranes involved in cholesterol-induced cell apoptosis and raft mediated cell signaling. Oxysterols regulate cellular functions by binding to oxysterol binding protein and oxysterol binding protein-related proteins. Oxysterol binding protein is a sterol-dependent scaffolding protein that regulates the extracellular signal-regulated kinase signaling pathway. According to a recently solved structure for a yeast oxysterol binding protein-related protein, Osh4, some members of this large family of proteins are likely sterol transporters. SUMMARY: Given the association of some oxysterols with atherosclerosis, it is important to identify the mechanisms by which their association with cell membranes and intracellular proteins controls membrane structure and properties and intracellular signaling and metabolism. Studies on oxysterol binding protein and oxysterol binding protein-related proteins should lead to new understandings about sterol-regulated signal transduction and membrane trafficking pathways in cells.     

Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L

Coxiella burnetii is a gram‐negative intracellular bacterium that forms a large, lysosome‐like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking and signalling are unknown. Oxysterol‐binding protein‐related protein 1 long (ORP1L) is a mammalian lipid‐binding protein that plays a dual role in cholesterol‐dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 h of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signalling pathways. The ORP1L N‐terminal ankyrin repeats were necessary and sufficient for PV localization, indicating that ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co‐localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L‐depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest that ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.     

RalA and RalB Proteins Are Ubiquitinated GTPases, and Ubiquitinated RalA Increases Lipid Raft Exposure at the Plasma Membrane

Ras GTPases signal by orchestrating a balance among several effector pathways, of which those driven by the GTPases RalA and RalB are essential to Ras oncogenic functions. RalA and RalB share the same effectors but support different aspects of oncogenesis. One example is the importance of active RalA in anchorage-independent growth and membrane raft trafficking. This study has shown a new post-translational modification of Ral GTPases: nondegradative ubiquitination. RalA (but not RalB) ubiquitination increases in anchorage-independent conditions in a caveolin-dependent manner and when lipid rafts are endocytosed. Forcing RalA mono-ubiquitination (by expressing a protein fusion consisting of ubiquitin fused N-terminally to RalA) leads to RalA enrichment at the plasma membrane and increases raft exposure. This study suggests the existence of an ubiquitination/de-ubiquitination cycle superimposed on the GDP/GTP cycle of RalA, involved in the regulation of RalA activity as well as in membrane raft trafficking.