PTPS · Spatio-temporal regulation of growth factor signaling by PTPs in living cells

Receptor tyrosine kinases (RTK) are involved in the regulation of cell proliferation, survival, differentiation and motility upon binding of growth factors. Disturbance of the normal balance between tyrosine kinase and Protein Tyrosine Phosphatase (PTP) activity results in aberrant tyrosine phosphorylation and has been implicated in the etiology of several human diseases, including cancer, diabetes and inflammation. Thus, it is important to understand how phosphorylation is regulated by members of both enzyme families. The objective of this proposal is to analyze the spatial regulation of Epidermal Growth Factor (EGF) signaling by PTPs in intact living cells. For this we propose to use quantitative fluorescence lifetime imaging microscopy (FLIM) in combination with functional genomic approaches. We will obtain information about which PTPs regulate the kinase activity of the EGF receptor, and the biochemical conectivity among PTP activities in response to EGF and other growth factors or hormones. Finally we will also investigate how PTP activities are regulated in response to EGF and where this happens in the cell. This spatio-temporal regulation of PTP activities will be studied by a novel approach in which enzyme-substrate complexes are directly imaged in living cells. Observation of `live´ biochemistry on a microscopic level has the advantage of preserving the network interconnectivity and spatial organization, allowing the investigation of the molecular dynamics that gives rise to the specificity in intracellular signal transduction. Since cellular responses are generated by the action of different gene products, cellular biology demands the regulation of signaling pathways at a proteomic scale to be studied to understand how signal specificity is achieved in cells and how stimuli are integrated to develop specific responses. Thus, our goal will be to obtain new information about dynamic properties of signaling networks at the proteomic scale.