I have long been interested in studying the rate by which protein interactions change during evolution. A new chapter in this ongoing research agenda has been published this week (article & perspective) in collaboration with the group of Judit Villén in the University of Washington and many contributions from the labs of Maitreya J. Dunham, Eulàlia de Nadal and Francesc Posas. For the first time I tried to engage with the press by putting out a press-release and it was interesting to work with Mary Todd Bergman at EMBL-EBI to digest the work to its core message. However, to atone for my sins of not being able to give sufficient context and credit to the work that has come before this, I decided that I could use this blog to write a sort of anti-press release. Grab some coffee, get confy and don’t expect a punchy fast message here because this manuscript has a long and branched root.
Cue flashback …
For me, this started 15 years ago (gasp, I can't believe it has been this long) when Andreas Wagner published some work trying to measure the conservation of protein interactions after gene duplication. This in turn was made possible by the first protein interaction mapping efforts. In my PhD lab I was using conservation to predict interactions for SH3 domains that bind short linear proline rich peptides. Influenced by Andreas Wagner’s papers, “linear-motif” research at EMBL and the field of evolution of gene expression I hypothesized that domain-peptide interactions could be poorly conserved since they are mediated only by a few residues in a linear unstructured peptide. This idea was first reported in the literature in a perspective by Neduva and Russell also at the EMBL at the time. I tried to generalize the concept that specificity and evolvability could be related such that very unspecific interactions may be more prone to change during evolution (article, blog post). Other groups have also shown that linear motif interactions can be fast evolving (e.g. Chica et al, Edwards et al.,)
Mass spectrometry to the rescue
The problem with trying to compare protein interactions is that you need to measure them first. The domain-peptide interactions mediated by linear motifs are particularly hard to identify because they are usually of low affinity. So, the work described above was based predicted interface sites for linear motifs. At this point, improvements in mass spectrometry and enrichment strategies really made a difference. The identification of protein phosphorylation sites made it possible to find, in large scale, thousands of sites that represent high-confidence interaction sites. The back-story that resulted in these developments in MS is a story our collaborator Judit Villén has been a part of and that I can’t tell as well.
Kinase-target interactions are also linear motif interactions and if the previous linear motif research was correct the phosphosites that represent these interactions should be rapidly evolving. That was exactly what I ended up testing when I started my postdoc. We were just one of several groups working on it and in 2009 several papers got published on the topic including our work (Beltrao et al., blog post) and others (Landry et al., Tan et al., Holt et al, Amoutzias et al. ). All of these together made a really strong case for the fast divergence of protein phosphorylation, although other articles followed to also note the constraints (Nguyen Ba & Moses and Gray & Kumar). At this point the conversation was also shifting to the consequences of these evolutionary changes. Mirroring similar discussions around the consequences of changes in gene-expression there was a sense that some of these phosphosites, and therefore kinase-substrate interactions do not play a functional role (Gustav E. Lienhard 2008 , Landry et al. 2009.). I also tried to contribute to the debate on functional relevance by trying to assign functions to PTM sites computational and extending the conservation analysis to other PTMs (Beltrao et al. 2013, blog post).
What was left to find then?
Most of the studies mentioned so far have relied on pairwise species comparisons. What we tried to do in this more recent study was to obtain a phylogenetic history of protein phosphorylation across a very broad phylogeny. For this, Judit’s lab obtained phosphorylation data for 18 fungal species that shared a common ancestor hundreds of millions of years ago. Romain Studer in our group then tried to combine the phosphorylation observations, which are known to be incomplete, with sequence based predictions of phosphorylation potential and the species phylogenetic tree. This allowed us to predict a likely evolutionary history for thousands of phosphosites.
If you happen to have kept up with the literature that I mentioned above then you might expect some of the findings we observed next - most phosphosites are recent acquisitions and the small fraction of ancient phosphosites is enriched in functionally relevant sites. From the ancient sites we tested a few cases for fitness and functional consequences and we think these serve as great resource for future cell signaling studies (and yes we are chasing that). Given the breath of species we studied we could also measure the changes in phosphorylation “motifs” that are found across species. Kinases recognize their target sites, in part, by the sequences around the phospho-acceptor residue, so-called kinase target motifs. We could observe that the types of target motifs used across species showed changes that we think relates to changes in the types of kinases or their activities. We are now interested in better understanding what determines kinase specificity so that we can study their evolution - what did the first protein kinase look like ?
So who the hell cares?
Many of the methods we are working on are useful to better understand the impact of mutations related to these signaling circuits in cancer or other diseases. We are working on this too but I care about this because I want to know how nature comes up with all these beautiful diverse mechanisms and forms. Coming up with a history of how these phosphosites have been changing across species is really just the first step. We have almost no clue as to what the thousands of observed phosphosites are doing, if anything. Are the signaling pathways changing in a neutral way that conserves the functional outcomes?
From a personal note it is fantastic to be able to connect this work to things that I did all the way back to my first PhD paper and that I can connect this blog post to a chain of several other blog posts covering the research I have done and that our research group is doing now.