Cell cycle: Cells divide and multiply over time, and the rate at which they do so is tightly controlled by a network of proteins, the core group of which participate directly in regulating the cell cycle. This cycle describes a cell as it goes from resting to preparing itself for division (primarily by duplicating all of its DNA for splitting) to finally dividing into two daughter cells. Cdk1 is one of a family of kinases that positively regulate the cell cycle. In cancer, overactivation of Cdk2, Cdk4, Cdk6, and/or Cdk8 have been implicated in a good percentage of cases: they cause the cells to multiply too often. However, the Cdk1 this paper is concerned with is yeast Cdk1. It phosphorylates serines (S) and threonines (T) on hundreds of proteins. Part of how Cdk1 knows which S/T to phosphorylate is whether it is followed by a proline (P), and sometimes also by a lysine (K) or arginine (R) two amino acids further down from the proline. Biologists write this "consensus phosphorylation sequence" as S/T*-P-x-K/R, where * is the phosphorylation site and x is any amino acid. Different kinases have different consensus sequences.
The basic results: What they did here is first to determine all of the proteins that yeast Cdk1 phosphorylates and where. They did this by comparing normal-growing yeast to yeast that had Cdk1's normal control of the cell cycle interrupted by chemicals. They then used an insanely expensive machine called a tandem mass spectrometer to figure out which serines and threonines were differentially phosphorylated (if you really want to get technical, they made this comparison possible by growing one set of yeast normally and the other set using "heavy" carbon and nitrogen; this allows the spectrometer to distinguish a mixture of the two by mass which makes everything a lot simpler). In total, they found 547 serines and threonines that were phosphorylated by Cdk1.
Ok, now the evolution part. They took the yeast amino acid sequence of all 547 sites and went to the web to download the amino acid sequences of the same 547 sites from 27 different types of fungal organisms, including 7 other kinds of yeast. Then, for each protein, they aligned the sequences for the 28 total organisms. Now, normally, if the amino acid sequence for a particular stretch lines up across all these species, it means that that stretch was specially preserved through natural selection because it's important. They found that about 60 of the phosphorylation sites retained an almost-perfect sequence alignment across the 28 organisms: since the cell cycle is so fundamental to growth, it should have a strong hand in guiding phosphorylation site evolution.
What's surprising is how low that number is. To understand why this happened a little better, the researchers used a different way of looking at phosphorylation site conservation. Instead of looking for precise alignment of the sites, they instead looked to see if the protein had some other way of conserving these crucial serines and threonines. What they found is that over 300 of the sites had phosphorylatable serines and threonines (remember, this is defined by the consensus Cdk1 sequence S/T*-P-x-K/R) at least somewhere in the very close vicinity of the original yeast site. In other words, for most of the sites, it doesn't matter what the specific larger sequence context is, as long as there's a Cdk1 consensus phosphorylation sequence in about the right region. This kind of mushiness might help to integrate new and evolving redundant kinase signals without upsetting the overall balance.
What this means for evolution: The high flexibility of Cdk1 phosphorylation site evolution within just the relatively small cluster of fungal organisms strongly suggests that evolution can be a highly adaptable process. This might be particularly true of such fundamental processes as the cell cycle, which likely faces multiple sources of constant environmental selective pressure. It will be interesting to see if similar experiments are performed on higher organisms, which may provide further insight into whether the rates of phosphorylation site evolution correlates with kinase family complexity, to tie this into the last post (it's no coincidence that these papers were published back-to-back in the same issue). Once again, we get to see the power that kinases - and in this case one particular kinase - can have on protein sequence evolution: significant swaths of the protein pantheon conform to the mercurial nature of the kinase.
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