Pascale G Charest

Pascale G Charest

Associate Professor
Associate Professor, BIO5 Institute
Associate Professor, Cancer Biology - GIDP
Associate Professor, Chemistry and Biochemistry-Sci
Member of the Graduate Faculty
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

Sumita, K., Yoshino, H., Sasaki, M., Majd, N., Kahoud, E. R., Takahashi, H., Takeuchi, K., Kuroda, T., Lee, S., Charest, P. G., Takeda, K., Asara, J. M., Firtel, R. A., Anastasiou, D., & Sasaki, A. T. (2014). Degradation of activated K-ras orthologue via K-ras-specific lysine residues is required for cytokinesis. Journal of Biological Chemistry, 289(7), 3950-3959.

Abstract:

Background: Targeting oncogenic K-Ras for cancer therapy has remained challenging. Results: Ubiquitination specifically occurs on the activated K-Ras orthologue in Dictyostelium via evolutionary conserved K-Ras lysines, which promotes K-Ras protein degradation. Conclusion: Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium. Significance: This work reveals a novel negative feedback regulation for the K-Ras isoform, which is critical for cytokinesis in Dictyostelium. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc..

Charest, P. G., Shen, Z., Lakoduk, A., Sasaki, A. T., Briggs, S. P., & Firtel, R. A. (2010). A ras signaling complex controls the RasC-TORC2 pathway and directed cell migration. Developmental Cell, 18(5), 737-749.

PMID: 20493808;PMCID: PMC2893887;Abstract:

Ras was found to regulate Dictyostelium chemotaxis, but the mechanisms that spatially and temporally control Ras activity during chemotaxis remain largely unknown. We report the discovery of a Ras signaling complex that includes the Ras guanine exchange factor (RasGEF) Aimless, RasGEFH, protein phosphatase 2A (PP2A), and a scaffold designated Sca1. The Sca1/RasGEF/PP2A complex is recruited to the plasma membrane in a chemoattractant- and F-actin-dependent manner and is enriched at the leading edge of chemotaxing cells where it regulates F-actin dynamics and signal relay by controlling the activation of RasC and the downstream target of rapamycin complex 2 (TORC2)-Akt/protein kinase B (PKB) pathway. In addition, PKB and PKB-related PKBR1 phosphorylate Sca1 and regulate the membrane localization of the Sca1/RasGEF/PP2A complex, and thereby RasC activity, in a negative feedback fashion. Thus, our study uncovered a molecular mechanism whereby RasC activity and the spatiotemporal activation of TORC2 are tightly controlled at the leading edge of chemotaxing cells. © 2010 Elsevier Inc.

Islam, T. A., Stepanski, B. M., & Charest, P. G. (2016). Studying chemoattractant signal transduction dynamics in Dictyostelium using BRET. Methods in Molecular Biology, 1407, 63-77. doi:10.1007/978-1-4939-3480-5_5
Perroy, J., Pontier, S., Charest, P. G., Aubry, M., & Bouvier, M. (2004). Real-time monitoring of ubiquitination in living cells by BRET.. Nat Methods, 1(3), 203-208.

PMID: 15782195;Abstract:

Ubiquitin has emerged as an important regulator of protein stability and function in organisms ranging from yeast to mammals. The ability to detect in situ changes in protein ubiquitination without perturbing the physiological environment of cells would be a major step forward in understanding the ubiquitination process and its consequences. Here, we describe a new method to study this dynamic post-translational modification in intact human embryonic kidney cells. Using bioluminescence resonance energy transfer (BRET), we measured the ubiquitination of beta-arrestin 2, a regulatory protein implicated in the modulation of G protein-coupled receptors. In addition to allowing the detection of basal and GPCR-regulated ubiquitination of beta-arrestin 2 in living cells, real-time BRET measurements permitted the recording of distinct ubiquitination kinetics that are dictated by the identity of the activated receptor. The ubiquitination BRET assay should prove to be a useful tool for studying the dynamic ubiquitination of proteins and for understanding which cellular functions are regulated by this post-translational event.

Khanna, A., Lotfi, P., Chavan, A. J., Montano, N. M., Bolourani, P., Weeks, G., Shen, Z., Briggs, S. P., Pots, H., Van Haastert, P. J., Kortholt, A., & Charest, P. G. (2016). The small GTPases Ras and Rap1 bind to and control TORC2 activity. Scientific Reports, 6, 25823. doi:10.1038/srep25823