Abstract
The African clawed frog Xenopus laevis is an important model organism for studies in developmental and cell biology, including cell-signaling. However, our knowledge of X. laevis protein post-translational modifications remains scarce. Here, we used a mass spectrometry-based approach to survey the phosphoproteome of this species, compiling a list of 2636 phosphosites. We used structural information and phosphoproteomic data for 13 other species in order to predict functionally important phospho-regulatory events. We found that the degree of conservation of phosphosites across species is predictive of sites with known molecular function. In addition, we predicted kinase-protein interactions for a set of cell-cycle kinases across all species. The degree of conservation of kinase-protein interactions was found to be predictive of functionally relevant regulatory interactions. Finally, using comparative protein structure models, we find that phosphosites within structured domains tend to be located at positions with high conformational flexibility. Our analysis suggests that a small class of phosphosites occurs in positions that have the potential to regulate protein conformation.
Highlights
Protein function can be regulated by post-translational modifications (PTMs) by altering diverse protein properties such as their localization, activity or interactions
In addition we have used 3D structural models to identify a set of phosphorylation sites that might regulate protein conformations
Recent advances in phosphoenrichment procedures and mass spectrometry (MS) technologies have resulted in a tremendous increase in the capacity to identify phosphorylation sites on a large scale [3] and over the past few years over 200.000 phosphorylation sites have been identified across a varied number of species
Summary
Protein function can be regulated by post-translational modifications (PTMs) by altering diverse protein properties such as their localization, activity or interactions. Recent advances in phosphoenrichment procedures and mass spectrometry (MS) technologies have resulted in a tremendous increase in the capacity to identify phosphorylation sites on a large scale [3] and over the past few years over 200.000 phosphorylation sites have been identified across a varied number of species (ptmfunc.com) These studies have highlighted the true extent and complexity of PTM regulation and underscored the need to develop large-scale approaches to study PTM function. Different kinases have specific preferences for the amino acids in the vicinity of the target phosphorylated residue [15,16,17] This local sequence context is often referred to as the kinase target consensus sequence or motif and the conservation of these kinase motifs across orthologous proteins can be used to improve the predictions of kinase regulated sites [18,19]. In parallel to conservation based approaches, computational and experimental methods have been developed to identify phosphosites that are more likely to be functionally important by regulating protein interactions [13,20], protein activities [13], metabolic enzymes [21], or cross-regulate other types of modifications [22,23,24]
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