Our work is designed to provide fundamental understanding of RASopathies, a large class of developmental abnormalities caused by the germline mutations in components of the RAS/MAPK pathway. Patients with these conditions display a broad spectrum of phenotypes, including heart defects, short stature, facial dysmorphisms, and neurocognitive delays. Whole genome sequencing of RASopathies provides new sequence variants in the well characterized components of the RAS pathway, but the functional consequences of these sequence variations is poorly understood. We will quantitatively characterize the functional and phenotypic consequences of the activating mutations in MEK, a core component of the RAS pathway. Focusing on the same group of mutations, we will first determine their effects on the regulation and enzymatic activity of MEK (Aim 1). This will be done using mass spectrometry-based kinetic assays with purified proteins. Next, we will quantify the consequences of activating mutations for the kinetics of RAS signaling in vivo (Aim 2). This will be done using a quantitative imaging approach, with which we will monitor RAS signaling in Drosophila embryos. Finally, we will determine how the same mutations influence RAS-dependent tissue morphogenesis (Aim 3). This will be done using live imaging experiments in zebrafish, focusing on heart development as a model of a morphogenetic event that is commonly affected in RASopathies. Our studies should provide mechanistic insights into the biochemical and morphogenetic effects of mutations identified in a large group of human developmental abnormalities and highlight the importance of using multiple models and quantitative approaches for answering a specific biological question.

Public Health Relevance

Our work will contribute to fundamental understanding of developmental abnormalities caused by the germline mutations within the highly conserved RAS/MAPK signaling pathway. By combining quantitative studies with purified components and developing embryos, we will establish how the same set of mutations affect the biochemical activity and tissue-level functions of MEK, a core component of the RAS/MAPK pathway.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM086537-07
Application #
9333420
Study Section
Modeling and Analysis of Biological Systems Study Section (MABS)
Program Officer
Hoodbhoy, Tanya
Project Start
2011-09-15
Project End
2019-08-31
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
7
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Princeton University
Department
Engineering (All Types)
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08543
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Grossman, Rona; Paroush, Ze'ev (2017) High-Throughput In Vitro Identification of Direct MAPK/Erk Substrates. Methods Mol Biol 1487:127-135
Jindal, Granton A; Goyal, Yogesh; Humphreys, John M et al. (2017) How activating mutations affect MEK1 regulation and function. J Biol Chem 292:18814-18820
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Jindal, Granton A; Goyal, Yogesh; Yamaya, Kei et al. (2017) In vivo severity ranking of Ras pathway mutations associated with developmental disorders. Proc Natl Acad Sci U S A 114:510-515
Song, Yonghyun; Marmion, Robert A; Park, Junyoung O et al. (2017) Dynamic Control of dNTP Synthesis in Early Embryos. Dev Cell 42:301-308.e3
Goyal, Yogesh; Jindal, Granton A; Pelliccia, José L et al. (2017) Divergent effects of intrinsically active MEK variants on developmental Ras signaling. Nat Genet 49:465-469
Johnson, Heath E; Goyal, Yogesh; Pannucci, Nicole L et al. (2017) The Spatiotemporal Limits of Developmental Erk Signaling. Dev Cell 40:185-192
Rogers, William A; Goyal, Yogesh; Yamaya, Kei et al. (2017) Uncoupling neurogenic gene networks in the Drosophila embryo. Genes Dev 31:634-638
Lim, Bomyi; Dsilva, Carmeline J; Kevrekidis, Ioannis G et al. (2017) Reconstructing ERK Signaling in the Drosophila Embryo from Fixed Images. Methods Mol Biol 1487:337-351

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