Pascale G Charest

Pascale G Charest

Associate Professor
Associate Professor, Chemistry and Biochemistry-Sci
Associate Professor, Cancer Biology - GIDP
Member of the Graduate Faculty
Associate Professor, BIO5 Institute
Primary Department
Contact
(520) 626-2916

Research Interest

Research Interest

Our research focuses on the signal transduction pathways and molecular mechanisms controlling directed cell migration, or chemotaxis, in eukaryotic cells. Chemotaxis is central to many biological processes, including the embryonic development, wound healing, the migration of white blood cells (leukocytes) to sites of inflammation or bacterial infection, as well as the metastasis of cancer cells. Cells can sense chemical gradients that are as shallow as a 2% difference in concentration across the cell, and migrate towards the source of the signal, the chemoattractant. This is achieved through an intricate network of intracellular signaling pathways that are triggered by the chemoattractant signal. These pathways ultimately translate the detected chemoattractant gradient into changes in the cytoskeleton that lead to cell polarization and forward movement. In addition, many cells such as leukocytes and Dictyostelium, transmit the chemoattractant signal to other cells by themselves secreting chemoattractants, which increases the number of cells reaching the chemoattractant source.To investigate key mechanisms of signal transduction underlying chemotaxis, we are using the social amoeba Dictyostelium discoideum as well as human cancer cell models. Cell motility and chemotaxis of Dictyostelium cells is very similar to that of leukocytes and cancer cells, using the same underlying cellular processes as these higher eukaryotic cells. Dictyostelium is amenable to cell biological, biochemical, and genetic approaches that are unavailable in more complex systems. The discoveries we make using Dictyostelium are then confirmed in human cells and, in particular, in the context of directed cancer cell migration and metastasis. Our aim is to understand the molecular foundation of directed cell migration, which is expected to guide the design of efficient anti-metastatic treatments.Our approach is interdisciplinary, in which we combine molecular genetics and proteomics to identify new signaling proteins and pathways involved in the control of chemotaxis, with live cell imaging using fluorescent reporters to understand the spatiotemporal dynamics of the signaling events, as well as biochemical analyses and proximity assays [including Bioluminescence Resonance Energy Transfer (BRET) and FRET] to understand how proteins interact and function within the signaling network. In addition, in collaboration with Dr. Wouter-Jan Rappel at UC San Diego, we generate quantitative models of the chemotactic signaling networks to help identify key regulatory mechanisms and link them to whole cell behavior

Publications

Charest, P. G., Oligny-Longpré, G., Bonin, H., Azzi, M., & Bouvier, M. (2007). The V2 vasopressin receptor stimulates ERK1/2 activity independently of heterotrimeric G protein signalling. Cellular Signalling, 19(1), 32-41.

PMID: 16857342;Abstract:

The V2 vasopressin receptor (V2R) activates the mitogen activated protein kinases (MAPK) ERK1/2 through a mechanism involving the scaffolding protein βarrestin. Here we report that this activating pathway is independent of Gαs, Gαi, Gαq or Gβγ and that the V2R-mediated activation of Gαs inhibits ERK1/2 activity in a cAMP/PKA-dependent manner. In the HEK293 cells studied, the βarrestin-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 βarrestin prevents signal propagation to the nucleus. βarrestin 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 βarrestin-dependent activation of ERK1/2 by the V2R involves c-Src and a metalloproteinase-dependent trans-activation event. These findings demonstrate that βarrestin is a genuine signalling initiator that can, on its own, engage a MAPK activation machinery upon stimulation of a GPCR by its natural ligand. © 2006 Elsevier Inc. All rights reserved.

Kölsch, V., Charest, P. G., & Firtel, R. A. (2008). The regulation of cell motility and chemotaxis by phospholipid signaling. Journal of Cell Science, 121(5), 551-559.

PMID: 18287584;PMCID: PMC2671295;Abstract:

Phosphoinositide 3-kinase (P13K), PTEN and localized phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] play key roles in chemotaxis, regulating cell motility by controlling the actin cytoskeleton in Dictyostelium and mammalian cells. PtdIns(3,4,5)P3, produced by PI3K, acts via diverse downstream signaling components, including the GTPase Rac, Arf-GTPases and the kinase Akt (PKB). It has become increasingly apparent, however, that chemotaxis results from an interplay between the P13K-PTEN pathway and other parallel pathways in Dictyostelium and mammalian cells. In Dictyostelium, the phospholipase PLA2 acts in concert with P13K to regulate chemotaxis, whereas phospholipase C (PLC) plays a supporting role in modulating P13K activity. In adenocarcinoma cells, PLC and the actin regulator cofilin seem to provide the direction-sensing machinery, whereas P13K might regulate motility.

Charest, P. G., Terrillon, S., & Bouvier, M. (2005). Monitoring agonist-promoted conformational changes of β-arrestin in living cells by intramolecular BRET. EMBO Reports, 6(4), 334-340.

PMID: 15776020;PMCID: PMC1299283;Abstract:

Recruitment of β-arrestin (β-arr) to agonist-stimulated G-protein-coupled receptors (GPCRs) has a crucial role in controlling signalling efficacy and selectivity. When translocated to the receptor, β-arr is believed to undergo important conformational rearrangement necessary for its downstream actions. To probe these changes in living cells, we constructed an intramolecular bioluminescence resonance energy transfer (BRET)-based biosensor, in which β-arr is sandwiched between the Renilla luciferase (Luc) and the yellow fluorescent protein (YFP). We show that the intramolecular BRET between Luc and YFP was significantly increased following GPCR activation, suggesting a conformational rearrangement bringing the amino terminus and carboxyl terminus of β-arr in closer proximity. Kinetic analysis showed that this conformational change follows the initial β-arr/receptor engagement. In addition to providing new insights into the agonist-induced conformational rearrangements of β-arr in living cells, the double-brilliance β-arr offers a universal biosensor for GPCR activation, allowing the study of native receptors in large-scale screening analysis. ©2005 European Molecular Biology Organization.

Charest, P. G., & Firtel, R. A. (2006). Feedback signaling controls leading-edge formation during chemotaxis. Current Opinion in Genetics and Development, 16(4), 339-347.

PMID: 16806895;Abstract:

Chemotactic cells translate shallow chemoattractant gradients into a highly polarized intracellular response that includes the localized production of PI(3,4,5)P3 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)P3 signal amplifies itself independently of the original stimulus, as well as inhibitory signals that restrict PI(3,4,5)P3 to the leading edge, thereby creating a steep intracellular PI(3,4,5)P3 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)P3. © 2006 Elsevier Ltd. All rights reserved.

Kölsch, V., Shen, Z., Lee, S., Plak, K., Lotfi, P., Chang, J., Charest, P. G., Romero, J. L., Jeon, T. J., Kortholt, A., Briggs, S. P., & Firtel, R. A. (2013). Daydreamer, a Ras effector and GSK-3 substrate, is important for directional sensing and cell motility. Molecular Biology of the Cell, 24(2), 100-114.

PMID: 23135995;PMCID: PMC3541958;Abstract:

How independent signaling pathways are integrated to holistically control a biological process is not well understood. We have identified Daydreamer (DydA), a new member of the Mig10/RIAM/lamellipodin (MRL) family of adaptor proteins that localizes to the leading edge of the cell. DydA is a putative Ras effector that is required for cell polarization and directional movement during chemotaxis. dydA- cells exhibit elevated F-actin and assembled myosin II (MyoII), increased and extended phosphoinositide-3-kinase (PI3K) activity, and extended phosphorylation of the activation loop of PKB and PKBR1, suggesting that DydA is involved in the negative regulation of these pathways. DydA is phosphorylated by glycogen synthase kinase-3 (GSK-3), which is required for some, but not all, of DydA's functions, including the proper regulation of PKB and PKBR1 and MyoII assembly. gskA- cells exhibit very strong chemotactic phenotypes, as previously described, but exhibit an increased rate of random motility. gskA- cells have a reduced MyoII response and a reduced level of phosphatidylinositol (3,4,5)-triphosphate production, but a highly extended recruitment of PI3K to the plasma membrane and highly extended kinetics of PKB and PKBR1 activation. Our results demonstrate that GSK-3 function is essential for chemotaxis, regulating multiple substrates, and that one of these effectors, DydA, plays a key function in the dynamic regulation of chemotaxis. © 2013 Kölsch et al.