Evolution cheats, or how to get an old enzyme to do new tricks

It is of course a cliche to state that eukaryotic cells (i.e., cells that are not bacteria) are complex. In the case of an animal, tens of thousands of proteins engage in fantastically elaborate interactions that somehow coax a single cell into generating a unique and magnificent organism. These interactions are often portrayed as exquisitely precise, using metaphorical images such as 'lock-and-key' and employing diagrams that resemble subway maps.

Many of these interacting proteins are enzymes that modify other proteins, and many of those enzymes are of a particular type called kinases. Kinases do just one thing: they attach phosphate groups to other molecules. This kind of modification is centrally important in cell biology, and one way to tell is to look at how many kinases there are: the human genome contains about 500 kinase genes.

Now, kinases tend to be pretty picky about who they stick phosphate onto, and this specificity is known to involve the business end of the kinase, called the active site. The active site is (generally) the part of the kinase that physically interacts with the target and transfers the phosphate. You might think that this interaction, between kinase and target, through the active site, would be by far the most important factor in determining the specificity of kinase function. But that's probably not the case.

It turns out that many kinases have other ways of choosing mates. Some use 'docking' sites that are outside the active site. And some rely on a different set of proteins called scaffold proteins to fix them up with their partners. And this suggests that a kinase signaling system can be rewired by messing around with these alternative modes of specificity, without changing the active site itself. This raises a very interesting question, one with implications for evolution and for synthetic biology: just how flexible are kinase signaling systems? Can they be rewired at all? Or perhaps at will?

Wendell Lim's lab at UCSF is famous for tackling questions like this. (Their excellent lab website includes most of their papers, including a very recent review article on scaffold proteins.) In previous work, they showed that kinase pathways can in fact be rewired: in a widely-discussed 2003 Science paper, Sang-Hyun Park and colleagues converted one yeast kinase pathway (controlling mating) into another (controlling osmotic stress responses) by altering the scaffolds without changing the enzyme active sites at all. It was a remarkable result, suggesting that one could get completely new kinase signaling systems without any need to make new catalytic machinery; i.e., without any monkeying with the touchy active sites.

But in that experiment, only one kinase was involved. The rewiring involved differential deployment of the same kinase for distinct purposes and, importantly, that kinase does normally control both mating and osmotic stress responses. So the 2003 experiment left open this question: can a kinase be forced to perform a completely new function, without changing its basic catalytic machinery? To address that question, Angela Won and colleagues in the Lim lab turned to an interesting set of kinases in yeast: the MAP kinase kinases, or MAPKKs. (The nomenclature is awful, I know.) Their work was published in an open-access article in PNAS last month, titled "Recruitment interactions can override catalytic interactions in determining the functional identity of a protein kinase." Figure 1A illustrates their experimental system:

All those ovals are kinases. The MAPKKKs act on the MAPKKs, which act on the MAPKs, which further elicit the biological responses listed across the bottom (the triggers for these responses are listed across the top). Won et al. focused on the MAPKKs, in the second row. Note that there are four yeast MAPKKs: Mkk1, Mkk2, Pbs2, and Ste7. The Mkks are both involved in cell wall remodeling, whereas Ste7 controls both mating and filamentation (i.e., growing as a mold). Notice that each stimulus-response pair (e.g., pheromone and mating) is controlled by a cassette of kinases (e.g., Ste11-Ste7-Fus3 in the case of mating). And notice the box thingy on the far right, which enfolds the mating cassette. It represents Ste5, which is a scaffold protein. Ste5 assembles the cassette, bringing the players into proximity, and that scaffolding function is critical: when Park et al. mutated Ste5 to abolish its binding to Ste7, the mating response disappeared. Ste7 also bears two docking sites for the kinase Fus3, and those too are known to be important for mating function.

So, we have Ste7 at the center of a specific kinase signaling cassette. It relies on two docking sites and a scaffold protein to get into an effective signaling complex. How important are those interactions in determining the specific effect of Ste7 on mating? In other words, could we force Pbs2 to control mating, by giving it a Ste5-like scaffold? Or those two docking sites? Or all of the above? Or is Pbs2 catalytically designed to only act on Hog1, and thus to only control osmotic stress responses?

The first experiment showed that Ste7 does need both the docking sites and the scaffold to fully function. The authors showed this by making a mutant of Ste5 that can't stick to Ste7: that mutant kills the pathway. (Second bar in the graph below, which is Figure 2C. The top bar shows normal mating with normal components.) When they took this dead mutant and tethered it to Ste7, poof, mating was fine (third bar), unless they removed the docking sites from the Ste7 beforehand, in which case mating was reduced by 100-fold.

The next experiment was the big one: the authors attempted to force the other MAPKKs to induce mating by giving them the scaffold (by tethering to Ste5) and/or the docking sites (by adding in the relevant piece of Ste7). With just one or the other, the system was still dead. (Check out Figure 3B.) But with both a scaffold and docking sites, two of the three other kinases activated mating. For example, Pbs2, which normally controls osmotic stress responses, induced mating when it was tethered to Ste5 and to the two docking sites in Ste7. And, interestingly, that rewired Pbs2 variant was now incompetent at its normal job.

What this means is that kinases can acquire most or all of their legendary specificity from the company that they keep, such that their functional roles can be completely changed without altering their catalytic machinery. It means that signaling systems in cells are a lot more flexible than we used to think.

For synthetic biologists, this is good news: creating completely new signaling systems need not mean engineering new enzymes, as previous work from the Lim lab and elsewhere had already demonstrated. And the result strengthens the notion of evolvability and concepts central to evo-devo (especially modularity); in fact, Lim and his colleagues have compared modularity of signaling systems with that of gene expression:

The ability to recombine pathways and regulate signaling behaviors with scaffolds can be likened to how the modular architecture of promoters gives rise to the diverse transcriptional responses that differentiate cell and tissue types.

Results like this point to flexibility in system architecture, but also demonstrate how enzyme specificity – something thought to be particularly difficult to evolve – can be altered through changes in recruitment and location. Remarkably, changes in these interactions, known to be labile under normal conditions, can lead to new biochemical functions. It doesn't happen every time; there are no "universal laws of cellular evolution" being proposed here. But such flexible wiring is something to keep in mind every time you read how impossibly hard evolutionary changes must be.

I'll let Won et al. provide the closing summary:

A growing body of work supports the idea that sophisticated cellular signaling networks in complex eukaryotes have arisen through the generation of new circuitry using a limited toolbox of parts rather than the evolution of novel proteins. Here, we examine the specificity of MAPKKs through attempts to use existing connections to convert MAPKK identity. We have successfully converted two alternative MAPKKs to Ste7 functionality, showing that we can use these simple protein interaction elements to redefine kinase behavior. However, we were unable to find an absolutely generic formula for converting kinases, as we find that some intrinsic contributions to specificity cannot be overcome by recruitment interactions.

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Won, A., Garbarino, J., and Lim, W. (2011). Recruitment interactions can override catalytic interactions in determining the functional identity of a protein kinase. Proceedings of the National Academy of Sciences, 108 (24), 9809-9814 DOI: 10.1073/pnas.1016337108