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Dissecting the functional importance of eukaryotic protein phosphorylation

Periodic Reporting for period 3 - PhosFunc (Dissecting the functional importance of eukaryotic protein phosphorylation)

Reporting period: 2018-04-01 to 2019-09-30

Living cells have to adapt to the changes in their environmental conditions. Such changes are very often first sensed at the membrane of cells and communicated internally by reversible post-translational modification (PTM) of proteins in complex and dynamic signaling networks. The reversible post-translational modification of proteins can modify how these behave in different ways by changing protein structural conformations, interactions or enzymatic activities. Protein phosphorylation is one of the most common and well studied PTMs that is catalyzed by specialized enzymes called protein kinases. Kinases can also regulate each other in complex regulatory networks that resemble logic circuits. Signaling networks not only need to be efficient in mounting an appropriate response but must also be evolutionarily adaptable. When species diverge to occupy new niches their regulatory networks need to evolve to cope with these changes. As François Jacob wrote in 1977, unlike engineering, evolution reuses or “tinkers” with pre-existing parts to find solutions. In contrast to this view, signaling networks are often presented as “electronic-circuits” of information cascades, a paradigm that although useful might hinder our progress in the study of cell signaling. Studying the underlying evolutionary process that gives rise to PTM signaling systems will allow us to better understand the functional relevance of PTM regulation in extant species.
Divergence of expression patterns is often asserted as the main driving force in generating phenotypic diversity. However, several studies have challenged this view. It has been noted that protein-protein interactions can diverge quickly, in particular those of lower binding specificity, such as domain-peptide interactions. The specificity of peptide binding domains and post-translational regulatory interactions are determined by a few key amino-acid residues in the target peptide (referred to as linear motifs). It is therefore plausible that these interactions can diverge quickly as they can be easily created and destroyed by a few point mutations. Recent advances in mass spectrometry (MS) now allow for the very large scale identification of protein phosphorylation sties. These advances lead to an increase in throughput with thousands of phosphosites discovered for some model organisms allowing for the first time to study the evolution of PTMs. The first evolutionary studies have shown that there is only weak evolutionary constraint imposed by the modifications which might be explained by the existence of a significant number of sites that serve no biological role in present day species but are the by-product of the high evolutionary rate of creation and destruction of phosphorylation sites.
The increased throughput in identification of protein phosphorylation sites along with the lack of sequence conservation at phosphosites and potential existence of non-functional sites has resulted in a tremendous challenge of identifying functional PTM sites among the many thousands of phosphosites identified to date. For example, there are over 200,000 phosphosites that have been experimentally determined in human proteins of which only 3% have a curated described function. Knowing the extent of non-functional phosphorylation as well as developing methods to rank sites according to functional importance is a major bottleneck in current studies of cell signaling. Tackling these issues will have an impact on many areas of fundamental cell biology (e.g. cell-cycle, DNA damage, response to stress, etc). Protein kinases and phosphorylation signaling networks are very often mutated in cancer and hijacked during infection. Understanding the function of protein phosphorylation will facilitate our understanding of how cancer mutations or some pathogens change these regulatory networks in disease.

In order to study the contribution to fitness of protein phosphorylation we are developing a combined computat
In the first half of the project period we have developed a computational approach to estimate the age of phosphorylation sites using a collection of protein phosphorylation data collected for 18 different fungal species. The selected fungal species span a range of different evolutionary relationships with some species being closely related and others being very distantly related. By mapping the origin of each phosphorylation site to the phylogenetic three of these species we could pinpoint their approximately age. Based on this analysis we have determined that most phosphosites are of very young origin. The speed by which phosphosites are created and destroyed during evolution is similar to the rate of changes in gene expression. It has been long though that changes in gene expression where the most important source of evolutionary changes. Our work has conclusively shown that changes in protein post-translational regulation can also be a strong contributor to evolutionary differences. This work has been made public in a publication (Studer et al. Science 2016).

In addition to the work described above we have made additional progress in several of the other objectives which we have not yet made public through scientific publications.
We expect to conclude all of the major objectives of this grant. We expect to be able to describe the extent by which protein phosphorylation contributes to fitness and to be prioritize which phosphosites are of high importance for living cells.