Imaging EphA3 receptor tyrosine kinase substrate interaction

Abstract

Receptor tyrosine kinases (RTKs) function as key signaling molecules to influence several physiological processes such as cell growth, survival and migration. Crystal structure and biochemical evidence suggests that the kinase activity of some RTKs, like the Eph RTK family, is affected by the phosphorylation of several key tyrosine residues in the activation loop (AL) of the tyrosine kinase domain and in the conserved juxtamembrane segment (JMS). However, in living cells, little is known about this regulatory mechanism in which tyrosine phosphorylation affects the catalytic activity and substrate accessibility of RTKs in both space and time. In this thesis, we used the EphA3 RTK as an example to investigate the JMS auto- inhibition involved in the Eph RTK activity regulatory mechanism. To achieve this, a generic cellular EphA3 RTK substrate interaction-imaging assay was developed that is based on Forster Resonance Energy Transfer as measured by Fluorescence Lifetime Imaging Microscopy (FRET-FLIM). A high affinity EphA3 peptide substrate was applied to increase the probability of kinase-substrate complexes forming. Using this assay, it was possible to quantitatively investigate the formation of steady-state enzyme- substrate (ES) complexes between the purified EphA3 catalytic domain fused to mCitrine and the Cy3.5 labeled peptide substrate. A kinase/phosphatase reaction cycle could be reconstituted that gave rise to stable ES-intermediates as measured by FRET- FLIM. By using this ES imaging approach, EphA3 RTK (WT)-substrate interactions could be observed in EphA3-mCitrine expressing quiescent Cos-7 cells that had been microinjected with the Cy3.5-substrate. This suggests that a population of EphA3 receptors exists with a temporarily relieved JMS auto-repression in the absence of ephrin-A5 ligands. Stimulation with soluble ephrin-A5 ligands led to a detectable increase in ES-intermediates, that pointed to an autocatalytic phosphorylation mechanism of JMS auto-repression relieve. To further study the role of JMS tyrosine phosphorylation on active site accessibility, the ES imaging approach was applied to analyze the active site accessibility of JMS regulation deficient mutants (MTs). Different substrate accessibilities were observed for these JMS regulation-deficient mutants (MTs), which revealed a possible function of JMS tyrosine phosphorylation in the regulation of the EphA3 RTK active site accessibility in living cells. In this work, a generic living cell compatible FRET-FLIM approach has been developed that allows direct observation of kinase-substrate interactions and helps to elucidate conformational dynamics of EphA3 RTK in living cells. In principle, this assay could also be used to quantitatively address conformational dynamics of other RTKs in living cells.%%%%Rezeptortyrosinkinasen (RTKs) fungieren als Schlusselmolekule in der Signalubertragung und beeinflussen dadurch mehrere physiologische Prozesse der Zelle, wie beispielsweise Wachstum, Uberleben und Migration. Kristallstrukturen wie auch biochemische…