A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. October 2014. / The c-Jun N-terminal kinases (JNKs) are mitogen-activated protein kinases (MAPKs) that are
activated by the dual phosphorylation of a canonical threonine and tyrosine residue. While it
is well known that the activation of JNK mediates many important cellular processes such as
differentiation, proliferation, and apoptosis, the mechanisms by which phosphorylation
induces its activation are not known. An understanding of the structural and biophysical basis
for the activation of JNK is highly desirable however, as dysregulation of the kinase has been
implicated in numerous prominent diseases. Aiming first to improve upon the previously
reported inadequacies in acquiring active JNK, this work describes a novel method for the
purification of large yields of pure and phosphorylated JNK1β1, the most abundant JNK
isoform. Using codon harmonization as a precautionary measure toward increasing the
soluble overexpression of the kinase raised unique questions about the role of translation
kinetics in both the heterologous and natural co-translational modification of kinases. After
purifying the upstream activating kinases of JNK, phosphorylation of JNK1β1 was achieved
by reconstituting the MEKK1 → MKK4 → JNK MAPK activation cascade in vitro.
Activated JNK1β1 was thereafter able to phosphorylate its substrate, ATF2, with high
catalytic efficiency. Characterising the nature of JNK1β1 modification by MKK4, mass
spectrometry revealed that the latter kinase phosphorylates JNK1β1 not only at its activation
residues (T183 and Y185), but also at a recognised yet uncharacterised phospho-site (S377)
as well as two novel phospho-residues (T228 and S284) whose phosphorylation appear to
have functional significance. Unfolding studies and amide hydrogen-deuterium exchange
(HX) mass spectrometry (MS) were then used to investigate the changes to the stability and
structure/conformational dynamics of JNK1β1 induced by phosphorylation and nucleotide
substrate binding. Increased flexibility detected at the hinge between the N- and C-terminal
domains upon phosphorylation suggested that activation may require interdomain closure.
Patterns of solvent protection by the ATP analogue, AMP-PNP, reflected a novel mode of
nucleotide binding to the C-terminal domain of a destabilised and open domain conformation
of inactive JNK1β1. HX protection at both domains following AMP-PNP binding to active
JNK1β1 revealed that the domains close around nucleotide upon phosphorylation,
simultaneously stabilising the kinase. This reveals that phosphorylation activates JNK1β1 in
part by enhancing the flexibility of the hinge to enable interdomain closure and the formation
of a functional active site. This work thus offers novel insight into the unique molecular
mechanisms by which JNK1β1 is regulated by nucleotide binding and phosphorylation by
MKK4, and by the complex interplay that exists between them.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/16804 |
| Date | 29 January 2015 |
| Creators | Owen, Gavin Ray |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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