How Signaling Evolved

Your genome holds genes for 518 protein kinases, and 58 of them are receptor tyrosine kinases (RTKs). Baker’s yeast runs on roughly 120 to 130 kinases and has no receptor tyrosine kinase at all. Chris Deppmann asks his class: “How did we get such rapid expansion of RTKs and signaling pathways??” His second question holds most of the answer: “How are new pathways coupled to receptors??”

The idea: a few old pathways, copied many times

You have now met the core animal pathways in this course, and the list is short. Comparisons of animal genomes keep returning the same core set. A widely cited review of animal development (Pires-daSilva and Sommer, 2003) names seven, and a 2007 review of receptor tyrosine kinase networks (Amit, Wides and Yarden) adds G-protein-coupled receptors to make eight. This lesson uses the eight:

• G-protein-coupled receptors (lesson)
• nuclear receptors (lesson)
• TGF-beta (lesson)
• Notch (lesson)
• JAK and STAT (lesson)
• Hedgehog (lesson)
• Wnt, called Wingless in flies (lesson)
• receptor tyrosine kinases (lesson)

A fruit fly has all eight, and so do you. What differs is the number of parts. Humans have four EGF receptors where the fly and the nematode worm have one each. A 2007 review of sequenced genomes (Amit, Wides and Yarden, Molecular Systems Biology 3:151) compared the human genome with four invertebrate animals, a sea squirt, a sea urchin, the fruit fly and a nematode worm, after adjusting for the number of genes in each genome. By that count humans have about 2.9 times as many RTKs, 2.5 times as many EGF receptors and 1.8 times as many MAP kinases.

Receptors, at the top of a pathway, multiplied more than the kinases in its core. The record does not show animals inventing one new pathway after another. It shows old pathways copied, mostly at the receptor end, and the copies changed. Start with the simplest relay known.

The simplest relay: two proteins and one phosphate

A bacterium senses most of its surroundings with two-component systems. Component one is a sensor kinase, usually set in the membrane. When it detects its signal, it takes the end phosphate from ATP and puts it on one of its own histidines. Component two is the responder (its formal name is response regulator). The responder moves that phosphate onto one of its own aspartates, and the change in shape that follows switches it on. Most responders are transcription factors, so the whole pathway is a sensor and a gene switch with one phosphate passed between them.

ATP → histidine on the sensor → aspartate on the responder → output

E. coli has about thirty of these pairs, each for a different condition such as osmotic strength or nitrogen supply. Its swimming uses a variant: the kinase, CheA, is a separate protein that sits beneath the receptors, and the responder, CheY, binds the flagellar motor instead of DNA (the circuit built on it is in signaling circuits). In 1985 Ann Stock, with Daniel Koshland and Jeffry Stock, reported that the sequence of CheY resembled regulators of sporulation and of membrane protein synthesis, so unrelated behaviours ran on one family of proteins. In 1988 Melvin Simon’s laboratory mixed purified chemotaxis proteins and followed the phosphate from CheA to CheY, and Jeffry Stock’s group at Princeton reported the same transfer that year.

Set this beside an animal pathway and you can list what the animal pathway adds.

A mark that stays. A phosphate on aspartate is labile, and how long it lasts differs from one responder to another, from seconds for CheY to minutes or more for others. CheY sheds its phosphate in well under a minute unaided, and a helper protein, CheZ, makes it faster still. A phosphate on serine, threonine or tyrosine stays until a phosphatase removes it (the phosphorylation switch), so the cell controls “off” separately from “on”.

A reader. Because the mark stays, another protein can bind it. An SH2 domain reads a phosphotyrosine. In the bacterial relay the phosphate changes the responder that carries it, and no separate domain reads it.

Branches and layers. Most sensor kinases pass their phosphate to one partner responder and largely ignore the rest. An animal receptor recruits several pathways at once, and each runs through tiers of kinases.

The enzymes differ too. The catalytic domain of a sensor kinase has a different fold from the serine, threonine and tyrosine kinases you know. Animals have no two-component systems, though plants and fungi do.

Copy, then change: three fates for a spare gene

Mistakes in copying DNA sometimes duplicate a single gene, and now and then a whole genome. Susumu Ohno argued in his 1970 book Evolution by Gene Duplication that this is the main source of new genes, because a spare copy can change while the original keeps doing the job. The genomes of jawed vertebrates, ours included, carry the traces of two whole-genome doublings early in their history, the likely route from one EGF receptor to four.

A spare copy has three possible fates.

1. It is lost. A mutation disables it, nothing selects against the loss, and it decays into a pseudogene (a gene-like sequence that no longer makes a working protein). This is the common outcome.
2. The copies divide the old job. Each loses a different part of what the ancestor did, so both must be kept. This is sub-functionalization. Two members of the EGF receptor family look like a case: ErbB2 has no known ligand, ErbB3 has a feeble kinase, and the two signal strongly as a pair.
3. One copy takes a new job, such as answering a new ligand. This is neo-functionalization.

A related word is co-option, the use of an existing gene or pathway for a new purpose without copying it: Toll patterns the fly embryo and also fights infection (lesson).

Design problem

The gene for a receptor tyrosine kinase is copied by accident, so the animal now has two. Describe three different fates for the extra copy. For the fate in which the copy comes to carry a new message, say what must change in the copy, and what must change so that the new message does not leak into the old pathway.

Now carry the duplication through a whole pathway. Begin with one ancestral cascade of ligand, receptor and kinase chain, and duplicate the receptor twice. In one outcome everything downstream is duplicated as well and the copies stay apart, which gives four vertical cascades like four separate wires. In the other, the four receptors go on sharing downstream parts and gain cross-connections. That gives a network in which many inputs funnel into a few heavily connected proteins called hubs. Ras is one. The ratios in the first section point to the second outcome, because receptors multiplied faster than the core they feed.

”The virtue of modularity”: new wiring from old parts

Now the second question: “How are new pathways coupled to receptors??” If every new connection needed a new enzyme, expansion would be slow. Chris’s phrase for the way out is “The virtue of modularity”.

Signalling proteins are assembled from domains that fold and work on their own (interaction modules). That gives evolution cheap ways to make a connection.

Move a domain. A DNA rearrangement that joins the coding segment for an SH2 domain to another gene makes a protein with a new partner in one step. Adaptors such as Grb2 are nothing but binding domains (adaptors and scaffolds).

Add a docking site. A new docking site is a few amino acids. The SH2 domain of Grb2 reads a phosphotyrosine followed two positions later by asparagine. The SH2 domains of PI 3-kinase read a phosphotyrosine followed three positions later by methionine. One or two point mutations in a receptor’s tail can make or remove such a site, and with it a line to Ras or to PI 3-kinase.

Change where and when a gene is on. Short stretches of regulatory DNA beside a gene decide which cells make it and when. A receptor that turns up in a new tissue meets different partners and different target genes, so an unchanged pathway gives a new result.

The logic is the same at both levels. A gene gains a new expression pattern through a short DNA motif, and a protein gains a new partner through a short peptide motif.

Genome counts agree. Interaction domains grow in number from yeast to worm and fly to mouse and human, and the parts for tyrosine phosphorylation show it plainly. Budding yeast has one protein with an SH2 domain. Humans have more than a hundred, alongside 90 tyrosine kinases.

Where did that tyrosine kit come from? Choanoflagellates are the closest living single-celled relatives of animals. Nicole King and Sean Carroll reported a receptor tyrosine kinase gene in one of them, Monosiga brevicollis, in 2001. Its genome, published in 2008, encodes tyrosine kinases, tyrosine phosphatases and SH2 domains in numbers that match or exceed ours, yet it lacks recognisable Wnt and TGF-beta genes. So the kit of a kinase to write the mark, an SH2 domain to read it and a phosphatase to erase it predates animals. What it does in a single cell is still largely unknown.

Why a network and not a set of wires

A network gives up insulation, so what does it buy? Robustness: the output holds when a part fails or an input is noisy (signaling circuits). Remove one receptor and its sisters, which feed the same core, cover part of the loss. Feedback loops in the shared core steady the output for every receptor at once.

The same design has costs.

A hub is a single point of failure. Lock Ras on (GTPase switches) and every receptor above it is overruled.

A shared core allows bypass. A lung tumour held in check by an inhibitor of the EGF receptor can escape by amplifying the gene for MET, a different receptor that feeds the same core.

And relatives resemble each other. Nearly all human kinases descend from one ancestral catalytic domain and keep a similar ATP pocket, so a drug shaped for one pocket often fits a relative. Imatinib, the drug for the BCR-ABL kinase of chronic myeloid leukaemia, also inhibits KIT and the PDGF receptors. KIT and one of the PDGF receptors are sister genes that sit side by side on chromosome 4. That cross-reaction is why imatinib treats a gut tumour driven by KIT, and it is why new kinase drugs are profiled against hundreds of kinases. The lesson on drugs in this unit starts from there.