Quantitative studies of RET activation, deactivation and trafficking kinetics upon stimulation by its natural ligand Artemin

Abstract

Receptor tyrosine kinases (RTKs) are key regulators of critical cellular processes, such as cell cycle, differentiation, proliferation, apoptosis and survival. Mutations, hyperactivity and loss of function of RTKs are responsible for numerous diseases. Because of the therapeutic importance of RTK signaling, intensive studies have been devoted to understanding the signaling mechanisms of RTKs, and the key components in their signaling networks. However, studying the cellular responses to RTK stimulation in a native cellular context is technically challenging. Consequently, many details of RTK signaling kinetics, and the underlying molecular mechanisms of action, remain unclear. The RET receptor tyrosine kinase is important for neuronal cell survival and function, and for the development of the kidneys and nervous system. Gain of function of RET leads to tumor formation, while loss of function in RET’s kinase activity is associated with the developmental kidney defect Hirschsprung’s disease. RET is activated by ligands of glial cell line-derived neurotrophic factor (GDNF) family, which consist of four homologs—GDNF, Neuturin, Artemin (ART) and Persephin. GDNF family ligands activate RET only in the presence of GPI-linked co-receptors (GFRα1–4). Formation of the pentameric ligand/co-receptor2/RET2 complex leads to dimerization of RET and autophosphrylation of its cytoplasmic kinase domain. RET phosphorylation results in the activation of multiple downstream signaling pathways, including the Ras-Raf-MEK-ERK and PI3K-Akt pathways. The ERK and Akt signaling pathways participate in a variety of cellular activities, including cell survival, proliferation, and differentiation. My project addresses the following questions: (1) What are the kinetics of RET activation and deactivation processes after ART stimulation? (2) How is RET activation coupled to the phosphorylation of ERK1/2 and Akt? (3) How does ligand-induced internalization of RET affect RET signaling? (4) How does each step in the RET-Ras-Raf-MEK-ERK cascade quantitatively regulate ERK phosphorylation levels? The results will elucidate the spatial and temporal dynamics of RET signaling upon stimulation by ART, and to determine how downstream signaling is regulated by the amplitude and timing of RET activation. Overall, the thesis aims to advance our understanding of RTK signaling, by establishing methods and principles that can potentially be applied to other RTK systems.