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Structural analysis of phosphorylation hotspots and kinase target preferences. 10.17863/CAM.46589 Strumillo, Marta Julia 0000000315272482 Beltrao, Pedro Cells are constantly sensing and adapting to changes in conditions. Protein post-translational regulation is one of the fastest mechanism used by cells to relay signals from sensors to effectors during such adaptations. Mass spectrometry allows for the study of posttranslational modifications on a very large scale and has been extensively applied to study protein phospho- rylation. On the order of 75% of human proteins have been estimated to be phosphorylated and approximately 160,000 human phosphosites are listed in public repositories. This wealth of knowledge, remains mostly uncharacterized with around 5% of human phosphosites having an annotated regulatory role or known regulatory kinase. Devising ways to study the functional importance of phosphosites is therefore a crucial research question. The recognition of target sites by a kinase is thought to be determined by a short contiguous sequence motif around the target phosphosite. It has been reported that kinases can, in some cases, recognize a 3D epitope instead of a linear peptide sequence. However, the extent by which 3D epitopes are important for kinase recognition is unknown. To study the usage of 3D kinase recognition motifs, I firstly examined if known in vitro and in vivo human kinase targets can be explained by 3D epitopes. For this I devised a computational pipeline mapping known kinase target phosphosites to structural models. Using these I identified potential cases where the important specificity determinant residues are not observed in contiguous sequences in the targets but may exist as a 3D epitope, and performed docking simulations to examine the possible kinase interactions. The 3D epitope examples were found to be rather exceptions than a rule, and the analysis confirms the general rule of linear motif recognition by kinases. To better predict phosphorylation of high functional relevance I analysed phosphosites that are highly conserved across species within protein domains families. These regions of conserved phosphorylation, defined as phosphorylation hotspots, were determined us- ing phosphosite data for a total of 40 eukaryotic species. A total of 241 domain regions were identified as hotspots within 162 domain families that were then mapped to proteins structures. These regions were shown to predict known regulatory sites and overlap with important structural features (i.e. protein interfaces and residues near or at catalytic sites). To further study the regulatory regions of protein domains I searched for regions of conserved ubiquitination and/or a high degree of recurrent mutations found in cancer. Of 68 domains that had enough data for analysis of all 3 types of hotspots I present the analysis of interesting cases and domains containing overlapping PTM and/or mutational hotspots. University of Cambridge 2019-12-11 Thesis Doctoral Doctor of Philosophy (PhD) en https://www.repository.cam.ac.uk/handle/1810/299516 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a2602c3a-b5e3-4655-bedd-c3d02bccf0ce/download 87eda9de84448d1f82354d60eee3eb5f https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2dad1cc9-e4d4-496e-bee9-aba2f688170d/download b28500a86f82ea9f77b8a01531807a94 https://www.rioxx.net/licenses/all-rights-reserved/ phosphorylation hotspots hotspot kinase PTMs Protein kinase Ubiquitination