An important group of growth factors and hormones acts on liver cells through receptor tyrosine kinases (RTKs) such as the epidermal growth factor (EGF) receptor, hepatocyte growth factor/scatter factor (HGF/SF) receptor and insulin receptor. Despite an explosive growth of our knowledge of molecular mechanisms of tyrosine kinase signaling during the past decade, the regulation of RTK pathways at the cellular level remains poorly understood. RTKs activate multiple signaling pathways that often overlap at the level of early adapter/target protein activation, stimulation of mitogen activated protein kinase (MAPK) cascades, and transcriptional events triggered by their stimuli. Recent literature data, also supported by findings from Dr. Kholodenko's group, suggest that specificity of signaling is generated by the timing, duration and amplitude of activation of the different component processes. However, the lack of a quantitative and integrative description of the regulation of RTK pathways hampers our understanding of the causes of particular alterations in complex signaling responses and their immediate consequences for downstream pathways. This proposal aims to fill this gap by quantitative kinetic monitoring and computational analysis of the levels of phosphorylation and activation of RTK pathway intermediates in hepatocytes stimulated with EGF, HGF/SF and insulin. Dr. Kholodenko will employ kinetic and control analyses of growth factor signaling in intact hepatocytes to identify the principal molecular and kinetic factors controlling RTK signaling pathways.
The Specific Aims are: (1) To analyze the factors controlling transient and sustained response patterns in multiple signaling proteins targeted by EGF stimulation in freshly isolated hepatocytes; (2) To analyze the kinetics and control of early signaling responses of the EGF-stimulated MAPK cascade; (3) To extend the experimental and computational analyses to investigate the responses to other RTK-mediated signals, specifically HGF/SF and insulin, singly or in combination.
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