To hear Tony Hunter tell the story, his discovery of tyrosine phosphorylation was nothing more than a happy accident. It was 1979, and researchers had known for decades that protein kinases were involved in regulating cell growth, proliferation, and metabolism. But, as far as everyone thought, kinases phosphorylated their target proteins only on serine or threonine residues, the only modified amino acids that had been detected.
Enter Tony Hunter. An assistant professor at the Salk Institute for Biological Studies, Hunter was studying how certain tumor viruses - in particular, polyomavirus and the Rous sarcoma virus - transform human cells. Although researchers had identified the key proteins involved, that is, middle T antigen for polyomavirus and Src for Rous, they weren't sure what those proteins did. "Two groups had reported a kinase activity associated with the viral Src protein," recalls Hunter: Marc Collett...
The next step was to determine the identity of the amino acid it modified. The experiment was fairly routine. Hunter ran the P32-labeled phosphorylated protein on a gel. He excised the middle T band, eluted the protein, and performed a partial acid hydrolysis to release the phosphorylated amino acids. He then spotted this hydrolysate onto a thin-layer plate along with markers for phosphoserine and phosphothreonine and ran the electrophoresis. If the phosphorylated amino acid released from the protein comigrated with phosphoserine, then middle T must phosphorylate serine. If it comigrated with phosphothreonine, then middle T would have threonine kinase activity.
"Much to my amazement, the P32 ran between phosphoserine and phosphothreonine," says Hunter. He repeated the experiment and got the same strange result. Then Hunter remembered a third amino acid with a free hydroxyl group, tyrosine, which could potentially be phosphorylated. "I called around and no one had ever heard of a tyrosine kinase," says Hunter, but he thought he should check it out. So he synthesized some phosphotyrosine and spotted that onto a plate along with his other markers and sample. "Sure enough, phosphotyrosine ran in this space between phosphothreonine and phosphoserine," he says. "But more importantly, it comigrated with the labeled compound I was generating from middle T."
The reason that Hunter saw phosphotyrosine where no one else had, he says, was because he was "too lazy to make up fresh buffer" before doing the experiment. To electrophoretically separate phosphothreonine from phosphoserine, Hunter, like everyone else in the field, had been using a buffer of pH 1.9. At that pH, "phosphotyrosine and phosphothreonine run on top of one another," he says. But Hunter's buffer wasn't actually pH 1.9. "Because the buffer was old and had been used many times," he says, its pH had dropped to 1.7, allowing Hunter to resolve, for the first time, phosphothreonine and phosphotyrosine. Indeed, he then demonstrated that the viral Src protein was also a tyrosine kinase, and not a threonine kinase as had previously been reported.
Crediting the discovery entirely to happenstance might be erring on the side of modesty, says Tomas Mustelin of the Burnham Institute in La Jolla, Calif. "The real genius lies in the fact that Tony realized that an accident was as significant as an intentionally discovered result," he says. "Most people would have said, 'I screwed up, let me fix the problem.' But Tony was smart enough to say, 'If the standard and the label did not comigrate, maybe they aren't the same.'"
Tyrosine Targets Galore
Hunter's discovery was "the spark that set off a forest fire" of interest in tyrosine phosphorylation, says Jack Dixon of UC-San Diego. "Phosphotyrosine became the thing to look for in your system because it was playing a role in cancer." And not only cancer: "Tyrosine phosphorylation is now known to be involved in a large number of absolutely crucial cellular functions from normal cell growth to intracellular vesicle trafficking to immune responses. It's everywhere," says Mustelin. In addition to being integral to cell biology, tyrosine kinases also present targets for new anticancer therapies: One of the most highly touted, rationally designed anticancer compounds, Gleevec, inhibits an oncogenic tyrosine kinase whose aberrant activity fuels the rampant growth of cells in patients with chronic myeloid leukemia.
Perhaps Hunter's background (both his undergraduate and graduate training at the University of Cambridge revolved around biochemistry) helped prepare him to make that chemical distinction. "He's a very good biochemist," notes Tony Pawson of the Mt. Sinai Hospital in Toronto. "It was a popular subject at the time, and I think I was quite good at it," says Hunter, who in 1975 accepted a faculty position at the Salk, where he has remained ever since.
His affinity for biochemistry also helped Hunter and his colleagues confirm that tyrosine phosphorylation is a fairly widespread cellular phenomenon, says Jonathan Cooper, a former postdoc and now at the Fred Hutchison Cancer Research Center in Seattle. "They would label up control and retrovirus-infected cultures with large amounts of P32 and then do something which was, I think, really quite bizarre," he says. "They'd perform phenol extractions, like one would do to purify DNA. But they'd take the phenol phase, which had all the protein in it. All screaming hot, of course."
After extracting all the proteins they could get, Hunter and his colleagues would measure the amount of phosphotyrosine. "And they made a rather remarkable discovery," says Cooper. In cells transformed by viral Src, or some other oncoproteins, the amount of phosphotyrosine shot up 10-fold compared to normal cells. "And they realized that oncoproteins were doing a lot inside cells, more than just phosphorylating themselves," he says. "So the hunt was on to try to find out what else was getting phosphorylated."
"Those were exciting times," says Cooper, "although when I first arrived in the lab I didn't necessarily see the significance. But it didn't take long for me to get sucked into trying to identify substrates." Searching for those substrates and trying to unravel how phosphorylation affects their activity has kept Hunter and his lab busy for more than three decades. The work has led him to explore the role of protein phosphorylation in a diverse array of cellular functions, including transformation, cell communication, cell adhesion, cell-cycle regulation, the control of gene expression, and protein degradation.
A Scientific Smorgasbord
At any given time, the projects pursued in the Hunter lab depend largely on the interests of the postdocs in residence. "Tony has quite a hands-off approach," says UCSD's Peter van der Geer, a former graduate student. "You can essentially do anything you want. Actually, you have to. Tony's supportive, but he encourages you to be independent." The variety is one of the strengths of the lab. "Tony sets the dinner table and the postdocs all bring a dish and you have a terrific meal," says Dixon, who two years ago moved from Michigan to UCSD, in part because "Tony was across the street."
The scientific smorgasbord also keeps Hunter intellectually sated. "What makes Tony tick is data, and more data," says David Schlaepfer, another former postdoc who is now at the Scripps Research Institute. "Rumor has it that as a graduate student Tony would eagerly await the publication of the new metabolic charts," adds Pawson. "He has a scholarly attitude towards science, in the sense that his interests are broad and he likes to pick up information of all sorts and synthesize them into a coherent story."
Van der Geer agrees. "Tony is one of those people who is truly interested in trying to understand everything." Thanks to his natural curiosity and his copious note taking at meetings, Hunter's knowledge is notoriously encyclopedic. "Anything we want to know, we basically just go ask Tony. We don't even bother to look it up," says Walter Eckhart, who was Hunter's postdoctoral advisor and has shared a lab with him at the Salk for the past 30 years.
The lust for information has recently driven Hunter to survey the kinome, the full set of human protein kinases. His interest in cataloging these enzymes arose in the mid-1980s when the number of new kinases seemed to grow exponentially. The expansion prompted Hunter to predict, only somewhat tongue-in-cheek, that the number of kinases would hit 1,001. Although that has turned out to be an overestimate (the real number peaked at 518) Mustelin points out that the total number of predicted human genes has also fallen well short of the mark. So, Hunter's original estimate, as a percentage, wasn't too far off the mark.
The kinome project has yielded some interesting observations. Hunter and his colleague Gerard Manning discovered that the human genome contains quite a few dead kinases whose sequences have nonetheless been carefully preserved. Although these otherwise lifeless enzymes don't actively phosphorylate target proteins themselves, they often form complexes with active kinases, perhaps tweaking their structures in a way that allows them to perform their duties.
Determining the kinases expressed in different cell types could help researchers draw up a short list of the genes that might be involved in a variety of human diseases, says Hunter. "You can then look for mutations that change the activity or expression of those particular kinases." In the meantime, Hunter and his colleagues will continue to probe the behavior of a select handful of this cavalcade of kinases one by one. "Until he has 518 postdocs," says Mustelin, "he can't work on them all."