Before 1979, kinases were only thought to stick phosphates on two of the twenty amino
acids: serine and threonine. But then Tony Hunter discovered that tyrosine could also
be phosphorylated (1, 2), thereby uncovering an entirely new mechanism of protein
regulation in cells. Since then, Hunter has worked on all sorts of protein phosphorylation
events and the kinases that deliver them. Indeed, he has been instrumental in deducing
the human kinome (3, 4).
Figure 1
Tony Hunter
Protein phosphorylation events have an impact on practically every cellular pathway
to some degree, but the main focus of Hunter's work has been their particular relevance
in cancer (5). Hunter, who is a fellow of the Royal Society and member of the National
Academy of Sciences, is director of the Cancer Center and American Cancer Society
professor at the Salk Institute in San Diego.
In a recent interview, Hunter recounted the tale of his tyrosine phosphorylation discovery.
And it's a tale with an important lesson: never dismiss an anomalous result—even if
you're using out-of-date reagents!
EARLY FOCUS
How did you get started in science?
My father was a surgeon in the UK National Health Service. He got me interested in
biology fairly early. Then, when I went to public school at the age of 13, I was pushed
up a class, and within the first week a decision had to be made whether I should take
classics or science as my major subject.
My father and the headmaster had a conversation, the result of which was that I was
pretty much specialized in science from then on.
That was very young for such a decision. Were you happy with it?
I could have been happy either way, I expect. Clearly, I was good at science. I was
not so good at math, which was an issue and still is. But science was easy, so I never
really questioned the decision.
Did you ever think about following in your father's footsteps?
I did, but he strongly discouraged me. He felt that the National Health Service leveled
all doctors, so that the talented ones really weren't given the due they deserved.
I don't think I would have been a very good doctor. I don't have the people skills
or the necessary compassion.
So you chose biology when you went off to Cambridge?
I read natural sciences at Cambridge and specialized in biochemistry for my final
year. But I didn't go to university straight after my A levels. I was still only 16
and I didn't feel ready. Most people going to university are 18, and that's the drinking
age, so I think it makes sense to go when you're 18 to enjoy it!
You then stayed on at Cambridge?
Someone in the biochemistry department suggested that I apply for an MRC studentship
to do graduate work. I thought, “Well, it seems like a reasonable thing to do, and
the path of least resistance.”
I opted to join Asher Korner's lab, which was the one lab in the department doing
anything resembling molecular biology at the time. He actually left in the middle
of my Ph.D. to take up the chair in biology in Sussex, but I decided not to move with
him.
Was that rather disrupting?
Not at all. He never spent much time talking to us. He let us do what we wanted.
We each had our own projects that we developed, so it didn't make a lot of difference.
Nevertheless, he was very important in creating a great lab environment and recruiting
the best graduate students. Four out of the nine students who were there at the same
time as me are now fellows of the Royal Society. It was really a very eminent group
of young scientists.
What was it about molecular biology that appealed to you?
It was the mid-1960s, and the genetic code was just being solved. The first protein
structures were just beginning to emerge. The structure of DNA had been solved. It
seemed like if you really wanted to understand how cells or organisms worked, you'd
have to understand how the molecules worked inside the cells.
It was a really exciting time, because we were right there at the cutting edge, even
as students. In the department in the center of town, we were very much the poor cousins
to the Laboratory of Molecular Biology (LMB) up the road, where all the high profile
molecular science was going on. But we went up there for seminars, and people from
there, like Fred Sanger, Sydney Brenner, and Max Perutz, would come and lecture to
us, which was fantastic.
UK TO USA TO UK…
If it was going so well, what made you head off to the States?
After my Ph.D., I stayed on as a college fellow for four years and was planning on
staying longer, but I married Pippa Marrack, who was a grad student at the LMB. She
was a couple of years behind me and wanted to come to UCSD to do a postdoc with Dick
Dutton.
Obviously, we had to stick together, so Alan Munro, who had done a one-year sabbatical
here at the Salk Institute suggested that I work with Walter Eckhart, a new faculty
member at Salk who worked on polyoma virus, a small DNA tumor virus. I thought, “Sounds
interesting,” and I met Eckhart at a meeting in London, and he agreed to take me into
his lab. So in 1971, Pippa and I came out to San Diego.
I teamed up with a postdoc in Walter Eckhart's lab, and we produced a bunch of papers
together, setting up polyoma virus DNA synthesis as an in vitro model for DNA replication.
That worked pretty well. But during this time, Pippa and I split up, and I decided
I would go back to Cambridge.
Then six months later, back in Cambridge, we burned the lab down. The origin of the
fire is still obscure, but it was probably caused by ether. It was pretty bad. We
lost most of our stuff, but luckily the liquid nitrogen canister containing all our
precious biologicals survived.
Phew!
Yeah. We were homeless, but luckily a new university building had just been built
right opposite the LMB, and we were offered space there on an empty floor. We actually
had a functioning lab again in less than six weeks.
Max Perutz, director of the LMB, generously offered us dining rights in the LMB canteen—the
famous canteen where everyone was supposed to sit at tables that did not have any
of your lab mates, in order to promote scientific discussion. As a result, we hooked
up with a group working on tobacco mosaic virus and had a very fruitful collaboration.
During this time, I applied for a couple of faculty positions in the UK but had no
luck. So I wrote to Walter Eckhart, who had offered me a position before I left the
Salk. He told me it was still open, so I moved back to San Diego in February, 1975.
…AND BACK
A few years later, you discovered that tyrosines could be phosphorylated. What led
up to that?
When I got back, I had begun working on polyoma virus again and by this time we knew
that a protein called middle T antigen, which gets expressed immediately after the
virus infects cells, could by itself transform fibroblasts. We wanted to know how
but were at a bit of a loss.
A postdoc then joined my lab and started trying to identify the transforming protein
for a different tumor virus: Rous sarcoma virus. We were beaten to the punch by Ray
Erikson's group, who identified the Src protein. They also discovered that Src was
a kinase.
That led us to test whether the polyoma virus middle T antigen was also a kinase.
And it was. Erikson had reported that Src phosphorylates threonine. So I started routine
hydrolysis experiments to identify the amino acid target of the polyoma kinase. One
evening I ran a hydrolyzed sample of labeled middle T antigen from polyoma-infected
cells together with markers for phosphoserine and phosphothreonine—the only known
phosphoamino acids at the time. The next day, it was clear that the target amino acid
was neither phosphoserine nor phosphothreonine.
Figure 2
A recent study from Hunter's lab reveals how the action of kinases affects cell movement
(colored lines) in a model of cancer.
My biochemistry training came in useful, because I knew that there was a third hydroxyl
amino acid, tyrosine, that could potentially be phosphorylated. I crudely synthesized
some phosphotyrosine, ran it against the polyoma sample, and found that indeed tyrosine
was the polyoma kinase target.
To run the thin layer plates, I had been using an old bottle of pH 1.9 buffer. Then,
rather foolishly, I made up some fresh buffer to repeat the experiment. To my horror,
I discovered that phosphotyrosine and phosphothreonine migrated together! I spent
some time aging the buffer, and it turns out that this causes its pH to drop slightly,
allowing the two phosphoamino acids to run separately.
I later ran a sample of the Src kinase product as a control, and much to my amazement,
this turned out also to phosphorylate tyrosine. Erikson had been misled by of the
comigration of phosphotyrosine and phosphothreonine when he reported that Src is a
threonine kinase.
Ah, he should have been using old buffer!
Yes! When the story broke, the word spread incredibly fast. I spoke about it in December
1979 in Basel, and soon everyone knew about it and started testing their favorite
transforming proteins. Within a year, we knew that tyrosine phosphorylation was important
for normal cells, and within three or four years, it was clear this was a major regulatory
system. Then in the early '80s, the first mutations in tyrosine kinases and its link
with cancer began to be reported.
Since then, you've worked on all sorts of kinases. What's the next big question in
kinase biology?
Several published studies say that there are thousands of different phosphorylation
events in a typical cell. So, the key questions are, What do they all do? And how
many of them are noise? For some proteins, we know they're phosphorylated under particular
conditions, but it's proved difficult to figure out how that changes their function.
Also, for proteins with multiple sites of phosphorylation, do different combinations
of phosphates mean different things?
Then, of course, there's the other side of the coin: the phosphatases. There are over
500 kinases and maybe 150 or so phosphatases. So there's a lot of interest in trying
to build networks of phosphorylation events—the systems biology approach. I think
that's certainly going to be a very important area.