Site-selective chemical phosphorylation and activation of kinases

Thesis

Phosphorylation and dephosphorylation of proteins play an essential role in their cellular function. However, the study of protein phosphoforms in a molecular level is challenging, as the isolation and characterisation of homogeneous phosphoforms is not always possible. We propose to synthesise and study these phosphoforms using a tag-and-modify approach. This methodology allows the selective chemical phosphorylation of protein via the introduction of a phosphocysteine residue (pC).

The effect of individual phosphorylation of MEK1 kinase, which plays a central role in cell differentiation and proliferation, was examined. The selective formation of homogeneous MEK1pC218, MEK1pC222, as well as the doubly phosphorylated MEK1pC218pC222 mimics of the corresponding activate MEK1 phosphoforms, was successfully achieved. The engineering of new reactants and masking techniques was critical to the task. The development of a LCMS based procedure for kinetic studies unravelled the effect of phosphorylation on MEK1 binding to its substrates, ATP and ERK1. While initial crystallisation attempts showed that optimisation was still needed. Future work should stress the utility of such methodology towards the understanding of the structural variations created by phosphorylation.

The chemical mechanism of formation of dehydroalanine (Dha) was also investigated through the design and use of deuterated probes. The selective formation of α-deuterated cysteine residues in both amino-acid and protein models allowed the elucidation of an E1cB mechanism. Moreover, this study highlighted the particularity of 2,5-dibromohexane diamide (DBHDA) as a mild, water soluble and almost universal reagent for the modification of cysteine to Dha residues in protein.

Authors: Galan, S

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Kinases; Phosphorylation; Mass spectrometry; Site-selective protein modification