When doing science, there’s generally one totally optimal way of performing an experiment. But, there may also be several other less optimal means of gathering similar data, and one of those may be much more feasible than the totally optimal method. As a scientist, you have to determine whether this other method is sufficient, or whether it’s necessary to expend the extra effort and/or resources on the more difficult method. Sometimes it’s totally fine to take the simpler approach (and this will spare some of your precious time and your lab’s precious funding), but this post is about a case where it’s not.
My colleagues and I have a new paper in JBC (the Journal of Biological Chemistry) that went online late last month. Although I think that the science is pretty interesting, I’m not going to write at length about it here. Instead, you should check out my post about an earlier paper on this subject (integrin phosphorylation) here. Superficially, the two are reasonably similar (at least on the level that I would discuss things on the blog). In fact, much of the newly published work was was actually conducted around the same time as the earlier work (a few years ago), but for a variety of reasons we didn’t publish it then. The new paper marks a major advancement of that work, and the new biological data from Jake Haling (in Mark Ginsberg’s lab at UCSD) also increases its depth considerably.
However, what I want to write about here is how one goes about studying tyrosine phosphorylation. Tyrosine is one of the 20 amino acids that form the building blocks of the proteins in our bodies. Proteins are often modified after they are produced in the body, and one type of modification is phosphorylation (the addition of a phosphate group to the -OH group of serine, threonine, or tyrosine, i.e. changing -OH to -PO42-). This modification is catalyzed by a type of enzyme called a kinase, and phosphorylation is a type of “posttranslational modification” because it occurs after the mRNA has been translated into a chain of amino acids (i.e. a protein).
Producing proteins in the lab (often in bacteria) is pretty routine work. However, producing a posttranslationally modified protein can be much more difficult, because you generally have to have access to an enzyme that will perform the desired modification. On the other hand, it is generally much simpler to mutate the source DNA, and then use that DNA to produce a modified protein (generally with one amino acid substituted for another), and this strategy can be used to study tyrosine phosphorylation indirectly. For example, look at panel A in the following figure:
This panel shows the chemical structure of tyrosine (on the left), and the structures of two other amino acids that tyrosine is often mutated to in order to study tyrosine phosphorylation. Phenylalanine is identical to tyrosine, except it is missing the -OH group. Therefore, it is basically a “non-phosphorylatable” tyrosine. Alanine, on the other hand, totally lacks the aromatic ring (and -OH) of tyrosine, so mutating tyrosine to alanine effectively removes the tyrosine side chain from a protein.
But, what if you want to study the effect of tyrosine phosphorylation more directly? Are there any “phosphomimetic” mutations that you could make? Well, for serine and threonine, this generally works quite well. Panel B above shows the chemical structures of glutamate and phosphorylated serine and threonine. As you can see, glutamate looks fairly similar to these two phosphorylated amino acids. Thus, it is generally an effective phosphomimetic substitution for serine or threonine. (Aspartate is also often used as a phosphomimetic, but it is one chemical bond shorter than glutamate, so it seems to me that glutamate would always be a better substitution.)
Panel B also shows the chemical structure of phosphorylated tyrosine. Even the non-expert should be able to see that phosphotyrosine looks nothing like glutamate. Sure, they both have a negative charge, but that’s about the only similarity. Glutamate is much shorter than phosphotyrosine, and it totally lacks the important aromatic ring. However, this hasn’t stopped some scientists from using glutamate (or, even worse, aspartate) as a phosphotyrosine mimic. The scientific literature is full of examples.
For my studies, I decided to give it a shot, since it would have saved a great deal of time, effort, and resources. Not surprisingly, though, it didn’t work. In all cases I studied, mutating tyrosine to glutamate had the same effect as mutating tyrosine to alanine. In some cases, this was the exact opposite effect of phosphorylating tyrosine. So, I instead had to develop a system to phosphorylate my protein, and that’s what enabled the work published in the new paper.
I’m not saying that glutamate can never be used to mimic phosphotyrosine. But, if it is, it needs to be extensively validated. Optimally, the protein with the glutamate substitution would be compared to the phosphorylated protein. At the very least, the glutamate mutant should be compared to an alanine mutant. Of the first couple of examples I found in the literature where this was done, all showed that these two mutations had the same effect. In these cases, at least, one can’t say that the effects of the mutation were due to mimicking phosphotyrosine (as opposed to just removing the aromatic ring).
So if you come across an example in the literature of glutamate being used to mimic phosphotyrosine, I’d encourage you to be extra skeptical–and make sure that the mutant is validated in some way. And, if you’re a scientist doing these experiments, you had better do the proper controls. Just know that if I end up reviewing your manuscript, I’ll definitely ask for them!
Anthis, N., Haling, J., Oxley, C., Memo, M., Wegener, K., Lim, C., Ginsberg, M., & Campbell, I. (2009). integrin tyrosine phosphorylation is a conserved mechanism for regulating talin-induced integrin activation Journal of Biological Chemistry DOI: 10.1074/jbc.M109.061275