All research conducted at EMBL-EBI under the supervision of Dr. Pedro Beltrao. Work on the PhD project was paused temporarily in the Spring of 2017 for me to undertake a 3-month internship at EMBO Press (in Heidelberg).Protein phosphorylation represents one of the most important post-translational modifica-
tions (PTMs) for cell signalling, and is is catalysed by a group of enzymes called protein
kinases. Through this activity they serve as key regulators of almost all cellular processes.
This is achieved at any time by a network of different kinases that are transiently active. The
fidelity of cell systems control therefore requires that each kinase targets only a restricted set
of substrates. This specificity is achieved partly by contextual factors that separate kinases
spatially and temporally, but also by sequence features that are encoded in the kinase domain
itself.
For this thesis I focus on elements of kinase specificity that are encoded in the the active
site of the enzyme. During these investigations I have tried to address three main questions:
1) How is specificity for residues surrounding the phosphorylation site determined in the
kinase? 2) How did these specificities evolve? and 3) To what extent does kinase evolution
correlate with the evolution of its substrates?
First, I developed a sequence-based method for the automated detection of kinase speci-
ficity determining residues (SDRs). The putative determinants were then rationalised using
available structural data, and in two specific cases were validated experimentally. I also used
mutation data from The Cancer Genome Atlas (TCGA) to demonstrate that kinase SDRs are
often targeted during cancer.
Second, a global analysis of SDR evolution was performed for kinases following gene
duplication and speciation, revealing that SDRs often diverge between paralogues but not
between orthologues. This global analysis is followed by a detailed case study of G-protein
coupled receptor kinase (GRKs) evolution using ancestral sequence reconstructions.
Third, I inferred global substrate preferences in a taxonomically broad range of species
using phosphoproteome data. I then related the evolution of substrate motif sequences to
that of their cognate effector kinases where possible. The results strongly suggest that many
of the motifs emerged in a universal eukaryotic ancestor.
I finish by summarising the major findings of this doctoral research, which to my knowl-
edge represents the most comprehensive analysis to date of protein kinase specificity and its
evolution.BBSR