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.