Guanine Exchange Factor Activity Research Articles

Defective regulation of the eIF2-eIF2B translational axis underlies depressive-like behavior in mice and correlates with major depressive disorder in humans

Major depressive disorder (MDD) is a significant cause of disability in adults worldwide. However, the underlying causes and mechanisms of MDD are not fully understood, and many patients are refractory to available therapeutic options. Impaired control of brain mRNA translation underlies several neurodevelopmental and neurodegenerative conditions, including autism spectrum disorders and Alzheimer’s disease (AD). Nonetheless, a potential role for mechanisms associated with impaired translational control in depressive-like behavior remains elusive. A key pathway controlling translation initiation relies on the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α-P) which, in turn, blocks the guanine exchange factor activity of eIF2B, thereby reducing global translation rates. Here we report that the expression of EIF2B5 (which codes for eIF2Bε, the catalytic subunit of eIF2B) is reduced in postmortem MDD prefrontal cortex from two distinct human cohorts and in the frontal cortex of social isolation-induced depressive-like behavior model mice. Further, pharmacological treatment with anisomycin or with salubrinal, an inhibitor of the eIF2α phosphatase GADD34, induces depressive-like behavior in adult C57BL/6J mice. Salubrinal-induced depressive-like behavior is blocked by ISRIB, a compound that directly activates eIF2B regardless of the phosphorylation status of eIF2α, suggesting that increased eIF2α-P promotes depressive-like states. Taken together, our results suggest that impaired eIF2-associated translational control may participate in the pathophysiology of MDD, and underscore eIF2-eIF2B translational axis as a potential target for the development of novel approaches for MDD and related mood disorders.

Tumor-derived hypoxic small extracellular vesicles promote endothelial cell migration and tube formation via ALS2/Rab5/β-catenin signaling.

Tumor hypoxia has been associated with cancer progression, angiogenesis, and metastasis via modifications in the release and cargo composition of extracellular vesicles secreted by tumor cells. Indeed, hypoxic extracellular vesicles are known to trigger a variety of angiogenic responses via different mechanisms. We recently showed that hypoxia promotes endosomal signaling in tumor cells via HIF-1α-dependent induction of the guanine exchange factor ALS2, which activates Rab5, leading to downstream events involved in cell migration and invasion. Since Rab5-dependent signaling is required for endothelial cell migration and angiogenesis, we explored the possibility that hypoxia promotes the release of small extracellular vesicles containing ALS2, which in turn activate Rab5 in recipient endothelial cells leading to pro-angiogenic properties. In doing so, we found that hypoxia promoted ALS2 expression and incorporation as cargo within small extracellular vesicles, leading to subsequent transfer to recipient endothelial cells and promoting cell migration, tube formation, and downstream Rab5 activation. Consequently, ALS2-containing small extracellular vesicles increased early endosome size and number in recipient endothelial cells, which was followed by subsequent sequestration of components of the β-catenin destruction complex within endosomal compartments, leading to stabilization and nuclear localization of β-catenin. These events converged in the expression of β-catenin target genes involved in angiogenesis. Knockdown of ALS2 in donor tumor cells precluded its incorporation into small extracellular vesicles, preventing Rab5-downstream events and endothelial cell responses, which depended on Rab5 activity and guanine exchange factor activity of ALS2. These findings indicate that vesicular ALS2, secreted in hypoxia, promotes endothelial cell events leading to angiogenesis. Finally, these events might explain how tumor angiogenesis proceeds in hypoxic conditions.

Autoinhibition of the GEF activity of cytoskeletal regulatory protein Trio is disrupted in neurodevelopmental disorder-related genetic variants

TRIO encodes a cytoskeletal regulatory protein with three catalytic domains—two guanine exchange factor (GEF) domains, GEF1 and GEF2, and a kinase domain—as well as several accessory domains that have not been extensively studied. Function-damaging variants in the TRIO gene are known to be enriched in individuals with neurodevelopmental disorders (NDDs). Disease variants in the GEF1 domain or the nine adjacent spectrin repeats (SRs) are enriched in NDDs, suggesting that dysregulated GEF1 activity is linked to these disorders. We provide evidence here that the Trio SRs interact intramolecularly with the GEF1 domain to inhibit its enzymatic activity. We demonstrate that SRs 6-9 decrease GEF1 catalytic activity both in vitro and in cells and show that NDD-associated variants in the SR8 and GEF1 domains relieve this autoinhibitory constraint. Our results from chemical cross-linking and bio-layer interferometry indicate that the SRs primarily contact the pleckstrin homology region of the GEF1 domain, reducing GEF1 binding to the small GTPase Rac1. Together, our findings reveal a key regulatory mechanism that is commonly disrupted in multiple NDDs and may offer a new target for therapeutic intervention for TRIO-associated NDDs.

GmGPA3 is involved in post-Golgi trafficking of storage proteins and cell growth in soybean cotyledons

As the major nutritional component in soybean seeds storage proteins are initially synthesized on the endoplasmic reticulum as precursors and subsequently delivered to protein storage vacuoles (PSVs) via the Golgi-mediated pathway where they are converted into mature subunits and accumulated. However, the molecular machinery required for storage protein trafficking in soybean remains largely unknown. In this study, we cloned the sole soybean homolog of OsGPA3 that encodes a plant-unique kelch-repeat regulator of post-Golgi vesicular traffic for rice storage protein sorting. A complementation test showed that GmGPA3 could rescue the rice gpa3 mutant. Biochemical assays verified that GmGPA3 physically interacts with GmRab5 and its guanine exchange factor (GEF) GmVPS9. Expression of GmGPA3 had no obvious effect on the GEF activity of GmVPS9 toward GmRab5a. Notably, knock-down of GmGPA3 disrupted the trafficking of mmRFP-CT10 (an artificial cargo destined for PSVs) in developing soybean cotyledons. We identified two putative GmGPA3 interacting partners (GmGMG3 and GmGMG11) by screening a yeast cDNA library. Overexpression of GmGPA3 or GmGMG3 caused shrunken cotyledon cells. Our overall results suggested that GmGPA3 plays an important role in cell growth and development, in addition to its conserved role in mediating storage protein trafficking in soybean cotyledons.

Probing thermostability of detergent-solubilized CB2 receptor by parallel G protein–activation and ligand-binding assays

G protein-coupled receptors (GPCRs) comprise a large class of integral membrane proteins involved in the regulation of a broad spectrum of physiological processes and are a major target for pharmaceutical drug development. Structural studies can help advance the rational design of novel specific pharmaceuticals that target GPCRs, but such studies require expression of significant quantities of these proteins in pure, homogenous, and sufficiently stable form. An essential precursor for these structural studies is an assessment of protein stability under experimental conditions. Here we report that solubilization of a GPCR, type II cannabinoid receptor CB2, in a Façade detergent enables radioligand thermostability assessments of this receptor with low background from nonspecific interactions with lipophilic cannabinoid ligand. Furthermore, this detergent is compatible with a [35S]GTPγS radionucleotide exchange assay measuring guanine exchange factor activity that can be applied after heat treatment to further assess receptor thermostability. We demonstrate that both assays can be utilized to determine differences in CB2 thermostability caused by mutations, detergent composition, and the presence of stabilizing ligands. We report that a constitutively active CB2 variant has higher thermostability than the WT receptor, a result that differs from a previous thermostability assessment of the analogous CB1 mutation. We conclude that both ligand-binding and activity-based assays under optimized detergent conditions can support selection of thermostable variants of experimentally demanding GPCRs.

Auto-regulation of Rab5 GEF activity in Rabex5 by allosteric structural changes, catalytic core dynamics and ubiquitin binding.

Intracellular trafficking depends on the function of Rab GTPases, whose activation is regulated by guanine exchange factors (GEFs). The Rab5 GEF, Rabex5, was previously proposed to be auto-inhibited by its C-terminus. Here, we studied full-length Rabex5 and Rabaptin5 proteins as well as domain deletion Rabex5 mutants using hydrogen deuterium exchange mass spectrometry. We generated a structural model of Rabex5, using chemical cross-linking mass spectrometry and integrative modeling techniques. By correlating structural changes with nucleotide exchange activity for each construct, we uncovered new auto-regulatory roles for the ubiquitin binding domains and the Linker connecting those domains to the catalytic core of Rabex5. We further provide evidence that enhanced dynamics in the catalytic core are linked to catalysis. Our results suggest a more complex auto-regulation mechanism than previously thought and imply that ubiquitin binding serves not only to position Rabex5 but to also control its Rab5 GEF activity through allosteric structural alterations.

Adaptor Protein p66Shc: A Link Between Cytosolic and Mitochondrial Dysfunction in the Development of Diabetic Retinopathy.

Diabetes increases oxidative stress in the retina and dysfunctions their mitochondria, accelerating capillary cell apoptosis. A 66 kDa adaptor protein, p66Shc, is considered as a sensor of oxidative stress-induced apoptosis. In the pathogenesis of diabetic retinopathy, a progressive disease, reactive oxygen species (ROS) production by activation of a small molecular weight G-protein (Ras-related C3 botulinum toxin substrate 1 [Rac1])-Nox2 signaling precedes mitochondrial damage. Rac1 activation is facilitated by guanine exchange factors (GEFs), and p66Shc increases Rac1-specific GEF activity of Son of Sevenless 1 (Sos1). p66Shc also possesses oxidoreductase activity and can directly stimulate mitochondrial ROS generation. Our aim was to investigate the role of p66Shc in the development of diabetic retinopathy and mechanism of its transcription. High glucose increased p66Shc expression in human retinal endothelial cells, and elevated acetylated histone 3 lysine 9 (H3K9) levels and transcriptional factor p53 binding at its promoter. Glucose also augmented interactions between Rac1 and Sos1 and activated Rac1-Nox2. Phosphorylation of p66Shc was increased, allowing it to interact with peptidyl prolyl isomerase to facilitate its localization inside the mitochondria, culminating in mitochondrial damage. P66shc-small interfering RNA (siRNA) inhibited glucose-induced Rac1 activation and mitochondrial damage. Similar results are observed in retinal microvessels from diabetic rats. This is the first report identifying the role of p66Shc in the development of diabetic retinopathy and implicating increased histone acetylation in its transcriptional regulation. Thus, p66Shc has dual role in the development of diabetic retinopathy; its regulation in the early stages of the disease should impede Rac1-ROS production and, in the later stages, prevent mitochondrial damage and initiation of a futile cycle of free radicals.

C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking.

Intronic expansion of a hexanucleotide GGGGCC repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene is the major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. However, the cellular function of the C9ORF72 protein remains unknown. Here, we demonstrate that C9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictions that C9ORF72 bears Rab guanine exchange factor activity. Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin from the plasma membrane to Golgi apparatus, internalization of TrkB receptor and altered the ratio of autophagosome marker light chain 3 (LC3) II:LC3I, indicating that C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence that C9ORF72 regulates autophagy. Investigation of proteins interacting with C9ORF72 using mass spectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.

Scaffold-mediated gating of Cdc42 signalling flux.

Scaffold proteins modulate signalling pathway activity spatially and temporally. In budding yeast, the scaffold Bem1 contributes to polarity axis establishment by regulating the GTPase Cdc42. Although different models have been proposed for Bem1 function, there is little direct evidence for an underlying mechanism. Here, we find that Bem1 directly augments the guanine exchange factor (GEF) activity of Cdc24. Bem1 also increases GEF phosphorylation by the p21-activated kinase (PAK), Cla4. Phosphorylation abrogates the scaffold-dependent stimulation of GEF activity, rendering Cdc24 insensitive to additional Bem1. Thus, Bem1 stimulates GEF activity in a reversible fashion, contributing to signalling flux through Cdc42. The contribution of Bem1 to GTPase dynamics was borne-out by in vivo imaging: active Cdc42 was enriched at the cell pole in hypophosphorylated cdc24 mutants, while hyperphosphorylated cdc24 mutants that were resistant to scaffold stimulation displayed a deficit in active Cdc42 at the pole. These findings illustrate the self-regulatory properties that scaffold proteins confer on signalling pathways.

Structure-Activity Studies of Bis-O-Arylglycolamides: Inhibitors of the Integrated Stress Response.

The integrated stress response comprises multiple signaling pathways for detecting and responding to cellular stress that converge at a single event-the phosphorylation of Ser51 on the α-subunit of eukaryotic translation initiation factor 2 (eIF2α). Phosphorylation of eIF2α (eIF2α-P) results in attenuation of global protein synthesis via the inhibitory effects of eIF2α-P on eIF2B, the guanine exchange factor (GEF) for eIF2. Herein we describe structure-activity relationship (SAR) studies of bis-O-arylglycolamides, first-in-class integrated stress response inhibitors (ISRIB). ISRIB analogues make cells insensitive to the effects of eIF2α-P by activating the GEF activity of eIF2B and allowing global protein synthesis to proceed with residual unphosphorylated eIF2α. The SAR studies described herein support the proposed pharmacology of ISRIB analogues as binding across a symmetrical protein-protein interface formed between protein subunits of the dimeric eIF2B heteropentamer.

Modulation of B-cell receptor and microenvironment signaling by a guanine exchange factor in B-cell malignancies.

Objective: Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cells over-express a guanine exchange factor (GEF), Rasgrf-1. This GEF increases active Ras as it catalyzes the removal of GDP from Ras so that GTP can bind and activate Ras. This study aims to study the mechanism of action of Rasgrf-1 in B-cell malignancies. Methods: N-terminus truncated Rasgrf-1 variants have a higher GEF activity as compared to the full-length transcript therefore a MCL cell line with stable over-expression of truncated Rasgrf-1 was established. The B-cell receptor (BCR) and chemokine signaling pathways were compared in the Rasgrf-1 over-expressing and a control transfected cell line. Results: Cells over-expressing truncated form of Rasgrf-1 have a higher proliferative rate as compared to control transfected cells. BCR was activated by lower concentrations of anti-IgM antibody in Rasgrf-1 over-expressing cells as compared to control cells indicating that these cells are more sensitive to BCR signaling. BCR signaling also phosphorylates Rasgrf-1 that further increases its GEF function and amplifies BCR signaling. This activation of Rasgrf-1 in over-expressing cells resulted in a higher expression of phospho-ERK, AKT, BTK and PKC-alpha as compared to control cells. Besides BCR, Rasgrf-1 over-expressing cells were also more sensitive to microenvironment stimuli as determined by resistance to apoptosis, chemotaxis and ERK pathway activation. Conclusions: This GEF protein sensitizes B-cells to BCR and chemokine mediated signaling and also upregulates a number of other signaling pathways which promotes growth and survival of these cells.

Plasma membrane restricted RhoGEF activity is sufficient for RhoA-mediated actin polymerization.

The small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regulated in time and space and depends on guanine exchange factors (GEFs). However, the kinetics and subcellular localization of GEF activity towards RhoA are poorly defined. To study the mechanism underlying the spatiotemporal control of RhoA activity by GEFs, we performed single cell imaging with an improved FRET sensor reporting on the nucleotide loading state of RhoA. By employing the FRET sensor we show that a plasma membrane located RhoGEF, p63RhoGEF, can rapidly activate RhoA through endogenous GPCRs and that localized RhoA activity at the cell periphery correlates with actin polymerization. Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA. The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellular morphology. Together, our data demonstrate that GEF activity at the plasma membrane is sufficient for actin polymerization via local RhoA signaling.

TD-60 links RalA GTPase function to the CPC in mitosis.

TD-60 (also known as RCC2) is a highly conserved protein that structurally resembles the Ran guanine exchange factor (GEF) RCC1, but has not previously been shown to have GEF activity. TD-60 has a typical chromosomal passenger complex (CPC) distribution in mitotic cells, but associates with integrin complexes and is involved in cell motility during interphase. Here we show that TD-60 exhibits GEF activity, in vitro and in cells, for the small GTPase RalA. TD-60 or RalA depletion causes spindle abnormalities in prometaphase associated with abnormal centromeric accumulation of CPC components. TD-60 and RalA apparently work together to contribute to the regulation of kinetochore–microtubule interactions in early mitosis. Importantly, several mitotic phenotypes caused by TD-60 depletion are reverted by the expression of a GTP-locked mutant, RalA (Q72L). The demonstration that a small GTPase participates in the regulation of the CPC reveals a level of mitotic regulation not suspected in previous studies.

Angiotensin II Activates the RhoA Exchange Factor Arhgef1 in Humans

Although a causative role for RhoA-Rho kinase has been recognized in the development of human hypertension, the molecular mechanism(s) and the RhoA guanine exchange factor(s) responsible for the overactivation of RhoA remain unknown. Arhgef1 was identified as a RhoA guanine exchange factor involved in angiotensin II (Ang II)-mediated regulation of vascular tone and hypertension in mice. The aim of this study was to determine whether Arhgef1 is activated and involved in the activation of RhoA-Rho kinase signaling by Ang II in humans. In vitro stimulation of human coronary artery smooth muscle cells and human peripheral blood mononuclear cells by Ang II (0.1 μmol/L) induced activation of Arhgef1 attested by its increased tyrosine phosphorylation. Silencing of Arhgef1 expression by siRNA inhibited Ang II-induced activation of RhoA-Rho kinase signaling. In normotensive subjects, activation of the renin-angiotensin system by a low-salt diet for 7 days increased RhoA-Rho kinase signaling and stimulated Arhgef1 activity in peripheral blood mononuclear cells. In conclusion, our results strongly suggest that Arhgef1 mediates Ang II-induced RhoA activation in humans. Moreover, they show that measurement of RhoA guanine exchange factor activity in peripheral blood mononuclear cells might be a useful method to evaluate RhoA guanine exchange factor activity in humans.

0077 : DOCK1 a new candidate gene in inherited form of mitral valve prolapse

Mitral valve prolapse (MVP) affects 2-4% of the general population and remains one of the most frequent indications for valvular surgery. So far, the only gene described in MVP, with an X-linked form of inheritance, is FLNA but only represents a small part of MVP. To this respect, we investigated a french MVP pedigree to uncover new molecular insights associated to the disease. We focus on a family of 5 affected patients characterized by a dysmorphic myxomatous phenotype with 2 operated patients. From 4 MVP patients, we performed a Whole Exome Sequencing screening. From bioinformatics analysis, we focused on rare (MAF<0.1%) functional variants shared by the 4 affected patients. Among 25 variants of interest, 11 were novel. One of them, found in a highly conserved residue (GERP=4.88), in DOCK1 (c.4646G>A; p. R1549Q) co-segregates in all affected members in the family. DOCK1 is highly expressed in the mitral valve as evidenced by RNA sequencing experiments using human mitral valve tissue. DOCK1 , encodes an atypical Rac exchange factor, Dock180, which acts as a guanine exchange factors (GEF) for small Rho family G proteins. Interestingly, Sanematsu et al. (2010) described a major role of Dock180 during cardiovascular development. Mitral valves of Dock1 depleted mice are thickened and lead to blood retention in left atrium. We currently investigate repercussions of p. R1549Q (located in GEF activity domain of Dock180) on Dock180 activity in the cellular adhesion phenotype (XCELLigence; IF assays) and GEF activity (Pull-Down assays) using a heterologous transfection system. Finally, a cohort of 285 MVP affected patients is screened to evaluate the prevalence of DOCK1 in the disease. This study reports a familial approach, coupled to an exome sequencing strategy which identifies a novel DOCK1 missense mutation associated with MVP phenotype. In vivo previous studies and cellular mechanisms learning will allow us to better understand mechanisms involved in the MVP pathogenesis.

The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif

Eukaryotic EnguLfment and cell MOtility (ELMO) proteins form an evolutionary conserved family of regulators involved in small GTPase dependent actin remodeling processes that regulates the guanine exchange factor activity of some of the Downstream Of CrK (DOCK) family members. Gathered data strongly suggest that DOCK activation by ELMO and the subsequent signaling result from a subtle balance in the binding of partners to ELMO. Among its putative upward modulators, the Hematopoietic cell kinase (Hck), a member of the Src kinase superfamily, has been identified as a binding partner and a specific tyrosine kinase for ELMO1. Indeed, Hck is implicated in distinct molecular signaling pathways governing phagocytosis, cell adhesion, and migration of hematopoietic cells. Although ELMO1 has been shown to interact with the regulatory Src Homology 3 (SH3) domain of Hck, no direct evidence indicating the mode of interaction between Hck and ELMO1 have been provided in the literature. In the present study, we report convergent pieces of evidence that demonstrate the specific interaction between the SH3 domain of Hck and the polyproline motif of ELMO1. Our results also suggest that the tyrosine-phosphorylation state of ELMO1 tail might act as a putative modulator of Hck kinase activity towards ELMO1 that in turn participates in DOCK180 activation and further triggers subsequent signaling towards actin remodeling.

Amplification of B cell receptor–Erk signaling by Rasgrf-1 overexpression in chronic lymphocytic leukemia

Rasgrf-1 is a guanine exchange factor (GEF) that catalyzes the exchange of GDP for GTP. In a RNA microarray analysis of chronic lymphocytic leukemia (CLL) specimens (n = 5), this gene was found to be overexpressed in CLL as compared to normal peripheral blood mononuclear cell (PBMC) CD19 + B cells (n = 3). CLL specimens (n = 29) expressed Rasgrf-1 RNA at levels 5–300-fold higher as compared to normal B cells. CLL specimens expressed a 75 kDa isoform that was smaller than the expected full-length protein (140 kDa) and the truncated variant had higher GEF activity. Knockdown of Rasgrf-1 in CLL specimens inhibited active GTP-bound Ras and the Ras/Erk/mitogen-activated protein kinase (MAPK) pathway. Rasgrf-1 was phosphorylated and activated by B cell receptor (BCR) signaling that increased its GEF function, and this phosphorylation was blocked by Src and Bruton's tyrosine kinase (BTK) inhibitors. Rasgrf-1 is a novel GEF protein that has a role in BCR signaling and its overexpression further activates the Ras/Erk/MAPK pathway in CLL specimens.

C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking.

Intronic expansion of a hexanucleotide GGGGCC repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene is the major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. However, the cellular function of the C9ORF72 protein remains unknown. Here, we demonstrate that C9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictions that C9ORF72 bears Rab guanine exchange factor activity. Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin from the plasma membrane to Golgi apparatus, internalization of TrkB receptor and altered the ratio of autophagosome marker light chain 3 (LC3) II:LC3I, indicating that C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence that C9ORF72 regulates autophagy. Investigation of proteins interacting with C9ORF72 using mass spectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.

Abstract PD1-9: P-REX1 employs a positive feedback loop to activate growth factor receptor/PI3K signaling

Abstract A mass spectrometry-based proteomic screen in ER+ breast cancer cells revealed that levels of P-REX1 are decreased upon PTEN loss, and increased upon PI3K inhibition. P-REX1 integrates signaling inputs from receptor tyrosine kinases (RTKs)/PI3K (via the PI3K phospholipid product PIP3) and G protein-coupled receptors (via Gbg subunits) to drive guanine exchange factor (GEF) activity on Rac1. RNAi knockdown of PREX1 and overexpression of exogenous PREX1 in ER+ breast cancer cells respectively decreased and increased activation of insulin-like growth factor receptor-1 (IGF-1R)/insulin receptor (InsR), PI3K/AKT/SGK3, and MEK/Erk under steady-state and growth factor (IGF-1, Heregulin)-stimulated conditions. While the P-REX1 homologue P-REX2a was previously shown to inhibit PTEN phosphatase activity to activate the PI3K/AKT pathway, we did not detect an effect of P-REX1 on PTEN activity. PREX1 knockdown suppressed PI3K/AKT signaling in PTEN-null breast cancer cells; therefore, P-REX1 and P-REX2a may not be functionally redundant. Knockdown and overexpression of PREX1 respectively increased and decreased doxorubicin-induced apoptosis in ER+ breast cancer cells, linking PREX1 with a pro-survival phenotype. Inhibition of signaling nodes downstream of PI3K (AKT, mTOR) derepresses feedback to activate RTKs and PI3K; knockdown of PREX1 abrogated the PI3K activation induced by inhibition of mTORC1/mTORC2. Structural analysis of P-REX1 revealed that the DH domain (which binds Gβγ and is required for GEF activity) is dispensable for P-REX1 effects on PI3K signaling, while the PH domain [which binds PIP3 and PI(3,4)P2] is required. These data place P-REX1 in a positive feedback loop, whereby PI3K generates PIP3 and PI(3,4)P2, and P-REX1 binds these phospholipids at the plasma membrane. In turn, P-REX1 promotes RTK activation, and RTKs activate PI3K/AKT and MEK/Erk signaling. Gene expression profiling of diverse types of solid tumors (n = 2,009) and cancer cell lines (n = 807) revealed that PREX1 mRNA is most abundant in ER+ breast tumors compared to other subtypes. Reverse-phase protein array (RPPA) analysis of lysates from 712 breast tumors showed that P-REX1 levels are inversely correlated with markers of PI3K/AKT/mTOR pathway activation. Further, P-REX1 levels are higher in ER+ tumors than ER- tumors. In another series of 1,293 carcinomas, PREX1 was genetically amplified or mutated in 6.2% of cases, and in 5% of breast cancers. We then tested whether PREX1 lesions co-exist with other PI3K pathway-activating lesions. Among genes encoding proteins implicated in RTK/PI3K signaling and phosphatidylinositol metabolism, we found a significant enrichment for PREX1 mutation/amplification in 54/79 (68%) genes across 1,523 carcinomas. We tested the effects of 7 PREX1 mutants found in breast tumors on PI3K signaling in vitro. A G344R mutation in the PREX1 PH domain conferred increased affinity for PIP3 and PI(3,4)P2, and increased the levels of phospho-AKT. These findings suggest that P-REX1 is an ER+ breast tumor-specific oncogene, and PREX1 mutations increase its oncogenic effects in breast cancer. We propose that neutralizing P-REX1 function is a novel therapeutic approach to selectively abrogate oncogenic signaling in ER+ breast cancers while sparing normal tissues. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD1-9.

Abstract PR05: P-REX1 creates a positive feedback loop to activate growth factor receptor/PI3K signaling

Abstract A mass spectrometry-based proteomic screen in ER+ breast cancer cells revealed that levels of P-REX1 are decreased upon loss of PTEN, and increased upon inhibition of PI3K. P-REX1 is a cytoplasmic protein that integrates signaling inputs from receptor tyrosine kinases (RTKs)/PI3K (via the PI3K phospholipid product PIP3) and G protein-coupled receptors (via Gβγ subunits) to drive guanine exchange factor (GEF) activity on Rac1, promoting cytoskeletal remodeling and cell migration. RNAi-mediated knockdown of PREX1 and overexpression of exogenous PREX1 in ER+ breast cancer cells, respectively, decreased and increased activation of insulin-like growth factor receptor-1 (IGF-1R)/insulin receptor (InsR), PI3K/AKT/SGK3, and MEK/Erk under steady-state and growth factor (IGF-1, Heregulin)-stimulated conditions. While the P-REX1 homologue P-REX2a was previously shown to inhibit PTEN phosphatase activity to activate the PI3K/AKT pathway, we did not detect an effect of P-REX1 on PTEN activity. PREX1 knockdown suppressed PI3K/AKT signaling in PTEN-null breast cancer cells; therefore, P-REX1 and P-REX2a may not be functionally redundant. Inhibition of signaling nodes downstream of PI3K (AKT, mTOR) derepresses feedback to activate RTKs and PI3K; knockdown of PREX1 abrogated the PI3K activation induced by inhibition of mTORC1/mTORC2. Structural analysis of P-REX1 revealed that the DH domain (which binds Gβγ and is required for GEF activity) is dispensable for P-REX1 effects on PI3K signaling, while the PH domain [which binds PIP3 and PI(3,4)P2] is required. These data place P-REX1 in a positive feedback loop, whereby PI3K generates PIP3 and PI(3,4)P2, P-REX1 binds these phospholipids at the plasma membrane, P-REX1 promotes RTK activation, and RTKs activate PI3K/AKT and MEK/Erk signaling. Gene expression profiling of diverse types of solid tumors (n = 2,009) and cancer cell lines (n = 807) revealed that PREX1 mRNA is most abundant in ER+ breast tumors compared to other subtypes. Reverse-phase protein array (RPPA) analysis of lysates from 712 breast tumors revealed that P-REX1 levels are inversely correlated with markers of PI3K/AKT/mTOR pathway activation. Furthermore, P-REX1 levels are higher in ER+ tumors than ER- tumors. In another series of 1,293 carcinomas, PREX1 was amplified or mutated in 6.2% of cases, and in 5% of breast cancers. Finally, we tested whether PREX1 lesions co-exist with other PI3K pathway-activating lesions. Among genes encoding proteins implicated in RTK/PI3K signaling and phosphatidylinositol metabolism, we found a significant enrichment for PREX1 mutation/amplification in 54/79 (68%) genes across 1,523 carcinomas. We tested the effects of 7 PREX1 mutants found in breast tumors on PI3K signaling in vitro. A G344R mutation in the PREX1 PH domain conferred increased affinity for PIP3 and PI(3,4)P2, and increased levels of phospho-AKT. These findings suggest that P-REX1 is an ER+ breast tumor-specific oncogene, and PREX1 mutations increase its oncogenic effects in breast cancer. We propose that neutralizing P-REX1 function is a novel therapeutic approach to selectively abrogate oncogenic signaling in ER+ breast cancers while sparing normal tissues. This abstract is also presented as Poster A060. Citation Format: Lloye M. Dillon, Jennifer R. Bean, Wei Yang, Justin M. Balko, W. Hayes McDonald, David B. Friedman, Ana M. Gonzalez-Angulo, Gordon B. Mills, Carlos L. Arteaga, Todd W. Miller. P-REX1 creates a positive feedback loop to activate growth factor receptor/PI3K signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr PR05.