Imaging EphA3 receptor tyrosine kinase substrate interaction
2012
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…
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