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Title: Solution structure properties and (dys)function of phospholipase Cγ
Author: Koss, H.
ISNI:       0000 0004 7964 7789
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2016
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Phosphatidylinositol phospholipase Cγ (PLCγ) is an intracellular membrane-associated second messenger signalling protein, which is activated by tyrosine kinases such as fibroblast growth factor receptor 1 (FGFR1). PLCγ contains the pseudocyclic γ-specific array (γSA), in which a SH2 domain pair (tandem-SH2), a SH3 domain, and a split-PH domain are connected by linker segments, effectively within a loop that is an insert in the catalytic domain. Activated FGFR1 binding to the nSH2 domain leads to phosphorylation of Y783, a residue in the γSA cSH2-SH3 linker. This modification terminates PLCγ autoinhibition by the cSH2 domain and reduces nSH2 affinity to FGFR1. This work has focused on the effect of Y783 phosphorylation on the tandem-SH2 solution structure and on PLCγ-FGFR dissociation. This work also aims to shed light on the mechanistic basis of activating mutations in PLCγ relevant for cancer and autoimmune disease. Molecular dynamics simulations followed by principal component analysis and contact analysis suggest a substantial solution structure difference between phospho- and non-phospho-tandem-SH2, the crystal structures of which are similar. After NMR backbone resonance assignment for both forms of tandem-SH2, chemical shift perturbation analyses, relaxation data analysis, residual dipolar coupling and SAXS experiments suggest that phosphorylation reduces the inter-SH2-domain flexibility, but does not lead to a change of relative SH2 domain orientation. The cSH2-Y783 linker is mechanistically important for allosterically communicating Y783 phosphorylation to the nSH2-cSH2 junction, which had been predicted by MD simulations. The C-terminus, along with the cSH2-Y783 linker, might thereby play an important role in an allosteric mechanism leading to FGFR1 kinase release. Activating PLCγ mutations were characterized in terms of their effects on autoinhibition, on the ability of Y783 to be phosphorylated, on and protein stability and on relative SH2 domain orientation. The PLCγ1 mutation R707Q was found to act by destabilizing the cSH2 domain and thereby promote release of PLCγ autoinhibition. The PLCγ1 mutation R687W leads to a complex modulation of the allosteric pathways in cSH2; it was also observed to be phosphorylated faster in a custom-made real-time in-NMR-tube FGFR kinase assay. An NMR analysis in the context of the γSA became feasible after transfer of backbone resonance assignments from the individual domains.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available