Chemiotics: The vanishing simplicity of chemical pathways in the cell

Posted on behalf of Retread

So nat’ralists observe, a flea

Hath smaller fleas that on him prey,

And these have smaller fleas that bite ’em,

And so proceed ad infinitum.

– Jonathan Swift

Is anything like this going on in the cell? Consider mitogen activated protein kinase kinase kinase (abbreviated MAPKKK) — shades of Major Major Major in Catch-22. Recall that a kinase is an enzyme which attaches a phosphate group to (phosphorylates) one of the 3 amino acids with hydroxyls on their side chains — serine, threonine and tyrosine. A phosphate ester is formed in the process adding a significant amount of negative charge and some local bulk to the protein (and if the protein is an enzyme often significantly altering its activity).

And what is the target that MAPKKK phosphorylates? Why MAPKK, another kinase which itself phosphorylates MAPK (yet another kinase — I’m not making this up). MAPK phosphorylates a variety of proteins, among them transcription factors which turn on various genes.

All quite linear (sequential) and comprehensible. There is a nice chain of causality from the agent outside the cell (the mitogen) to the receptor for it, to MAPKKK and so on to a particular set of genes whose level of expression is altered with the net result being cellular proliferation (e.g., mitosis).

Discovering this pathway took a lot of hard work on the ras protein, which is mutated in 30% of all cancers. Just the steps from the mitogen binding to its receptor to ras and thence to MAPKKK are quite complex. It was a hard slog, one (linear) step at a time. But what if all this work was like the drunk looking under the street light for his key because that’s where the light was. Suppose far more than that is going on.

Instead of teasing out pathways one protein at a time, suppose you just threw a mitogen (in this case epidermal growth factor — EGF ) at a cell (OK, a cancer cell — the Hela cell — the workhorse of cancer research) and looked at every protein to see what was phosphorylated and what was not. Using advanced mass spectroscopy and some other cutting edge techniques [Cell vol. 127 pp. 635–648, 2006] did just that. Some 6,600 distinct phosphorylation sites on 2,244 different proteins were found. 924/6,600 sites showed more than a twofold change in the phosphorylated to unphosphorylated ratio.

In addition, the work was repeated at several time points within 30 minutes of EGF application, allowing the time course of phosphorylation at each site to be determined. The time courses of phosphorylation varied from site to site. Many proteins had more than one site phosphorylation. Even on the same protein the time course of phosphorylation depended on the site studied. At least 46 distinct regulators of gene transcription showed a greater than twofold variation in phosphorylation. It doesn’t take much imagination to see that adding a lot of negative charge would alter the ability of a transcription factor to approach DNA (which has one phosphate per nucleotide).

Where this leaves our notion of causality (which really is quite linear) and whether our minds are strong enough to comprehend these events is the subject of the next post.