Tag Archives: J. Michael Bishop

Tony Hunter and the Serendipitous Discovery of the First Known Tyrosine Kinase: the Rous Sarcoma Virus Src Protein

In 1911 Peyton Rous, at the Rockefeller Institute, discovered the Rous sarcoma virus; the first virus known to cause solid tumors (1). Although Rous’ eponymous virus also would be known as the prototype retrovirus, his discovery generated only scant interest at the time, and would not be recognized by the Nobel Committed until 65 years later! [Nobel prizes are not awarded posthumously. Fortunately, Rous had longevity on his side. He died 4 years after receiving the prize, at age 87.]

In 1976 Harold Varmus and J. Michael Bishop, then at the University of California San Francisco, discovered that the Rous sarcoma virus oncogene, v-src, as well as the oncogenes of several other tumorgenic retroviruses, actually were derived from cellular genes that normally play an important role in controlling cell division and differentiation (2). Moreover, Varmus and Bishop showed that these cellular “proto-oncogenes” can be altered by mutation, to become “oncogenes” that contribute to cancer. [Varmus and Bishop received the 1989 Nobel Prize in Physiology or Medicine for their discovery of proto-oncogenes.]

But what is the actual activity of the protein coded for by the normal cellular c-src, and by v-src as well? The story of that discovery is rather delightful and begins as follows.

In 1978, Raymond Erikson and coworker Marc Collette, then at the University of Colorado Medical Center, were the first researchers to isolate the Src protein. They accomplished this by first preparing lysates from avian and mammalian cells, which had been transformed in culture into tumor cells by Rous sarcoma virus. Next, they precipitated those lysates with antisera from rabbits that bore Rous sarcoma virus-induced tumors. The premise of their strategy was that antibodies from the tumor-bearing rabbits would recognize and precipitate proteins that were specific to cells transformed by the virus .

With the Src protein now in hand, Ericson and Collette next sought its function. They initially asked whether Src might have protein kinase activity (i.e., an activity that adds a phosphate group to a protein.). This was a reasonable possibility because protein phosphorylation was already known to play a role in regulating various cellular processes, including cell growth and differentiation.

Ericson and Collette tested their premise by incubating their Src immunoprecipitates with [γ-32P] ATP (i.e. 32P-labelled adenosine triphosphate). In agreement with their proposal, they found that the antibody molecules in the Src immunoprecipitates had been phosphorylated. [Note that Src’s protein kinase activity was simultaneously and independently discovered by Varmus and Bishop.]

Ericson and Collette also carried out control experiments that were particularly revealing. When the same rabbit antisera was used to immunoprecipitate extracts from normal cells, or extracts from cells infected with a transformation-defective mutant of Rous sarcoma virus, no signs of protein kinase activity were seen in those immunoprecipitates. What’s more, the protein kinase activity was found to be temperature sensitive in immunoprecipitates from cells infected with a mutant Rous sarcoma virus that was temperature-sensitive for transformation.

These control experiments confirmed that the protein kinase activity in the immunoprecipitates was coded for by the virus. What’s more, they confirmed that the kinase activity of the retroviral Src protein plays an essential role in transformation. Furthermore, when taken with the earlier findings of Varmus and Bishop, they implied that the kinase activity of the cellular Src protein plays a key role in the control of normal cell proliferation.

While Erickson and coworkers were carrying out the above experiments in Denver, Walter Eckhart and Tony Hunter, at the Salk Institute, were looking into the basis for the transforming activity of the mouse polyomavirus middle T (MT) protein. [Unlike Rous sarcoma virus, which is a retrovirus, the mouse polyomavirus is a member of the Polyomavirus family of small DNA tumor viruses. SV40 is the prototype Polyomavirus.]

Tony Hunter
Tony Hunter

Since Erickson’s group was finding that Src expresses protein kinase activity, Eckhart and Hunter asked whether the polyomavirus MT protein might likewise be a protein kinase. Thus, as Erickson and Collette had done in the case of Src, Eckhart and Hunter examined immunoprecipitates of MT to see if they too might express a protein kinase activity, and found that indeed they did.

Interestingly, it was not known at the time of these experiments that MT actually does not express any intrinsic enzymatic activity of its own. Instead, MT interacts with the cellular Src protein to activate its protein kinase activity. See Aside 1.

[Aside 1: For aficionados, MT is a membrane-associated protein that interacts with several cellular proteins. Importantly, the phosphorylation events carried out by MT-activated Src cause a variety of signal adaptor molecules [e.g., Shc, Grb2, and Sos] and other signal mediators [e.g., PI3K and PLCγ] to bind to the complex, thereby triggering a variety of mitogenic signaling pathways. These facts were not yet known when Eckhart and Hunter were doing their experiments.]

At the time of these experiments, serine and threonine were the only amino acids known to be phosphorylated by protein kinases. In fact, Erikson and Collette, as well as Varmus and Bishop, believed that threonine was the amino acid phosphorylated by the Src kinase (see below). Consequently, Hunter asked whether the polyomavirus MT protein likewise would phosphorylate threonine. [Recall that MT actually does not express any intrinsic enzymatic activity of its own.]

Hunter’s experimental procedure was relatively straightforward and reminiscent of Erikson’s and Collette’s. It involved incubating immunoprecipitates of MT with [γ-32P]ATP, hydrolyzing the immunoglobulin, and then separating the amino acids in the hydrolysate by electrophoresis. But, to Hunter’s surprise, the position of the labeled amino acid in his electropherogram did not correspond to that of either threonine or serine.

Hunter was well aware that tyrosine is the only other amino acid with a free hydroxyl group that might be a target for the MT kinase activity. And, while there was no precedent for a tyrosine-specific protein kinase, Hunter proceeded to ask whether the polyomavirus MT protein indeed might phosphorylate tyrosine.

Hunter began by synthesizing a phosphotyrosine molecule that could be used as a standard marker against which to compare the labeled amino acid in a repeat of his earlier experiment. And, to his pleasure, Hunter found that the amino acid that was phosphorylated by the MT kinase activity ran precisely with the phosphotyrosine standard marker in his new electropherograms.

But why had other researchers not detected tyrosine phosphorylation earlier? It was partly because phosphotyrosine accounts for only about 0.03% of phosphorylated amino acids in normal cells. The remaining 99.97% are phosphoserine and phosphothreonine. But, again, that is not the entire explanation. The rest is truly precious.

In Hunter’s own words, he was “too lazy to make up fresh buffer” before doing his experiments. Had the buffer been fresh, its pH would have been the usual 1.9; a pH that, unbeknownst to all at the time, does not separate phosphotyrosine from phosphothreonine during the electrophoresis procedure. The pH of the old buffer that Hunter used in his experiment had inadvertently dropped to 1.7; a pH at which phosphotyrosine is resolved from phosphothreonine. That fact enabled Hunter to discriminate phosphotyrosine from phosphothreonine for the first time. Thus, Hunter attributes his hugely important discovery to his laziness.

The finding that tyrosine is the amino acid phosophorylated  by the polyomavirus MT protein kinase activity led Hunter and his Salk Institute-colleague Bart Sefton to ask whether Src too might phosphorylate tyrosine, rather than serine or threonine (4). Indeed, they found that the retroviral Src protein, as well the normal cellular Src protein, function as tyrosine-specific protein kinases. [Recall that it became clear only later that MT actually has no intrinsic enzyme activity of its own and that it acts through Src.] Moreover, the levels of phosphotyrosine were 10-fold higher in cells infected with wild-type Rous sarcoma virus than in control cells, consistent with the premise that Src’s protein tyrosine kinase activity accounts for the altered growth potential of those cells.

Subsequently, Stanley Cohen, at Vanderbilt University, discovered that the epidermal growth factor (EGF) receptor contains an intrinsic protein-tyrosine kinase activity, further underscoring the importance of protein-tyrosine kinases in the normal control of cell proliferation. [Cohen shared the 1986 Nobel Prize in Physiology or Medicine with Rita Levi-Montalcini for their discoveries of growth factors, including EGF.] Subsequent studies identified additional receptor protein-tyrosine kinases, such as the fetal growth factor (FGF) receptor, and non-receptor protein-tyrosine kinases, such as Abl, each of which activates a mitogenic intracellular signaling pathway.

Tony Hunter and coworkers went on to demonstrate that protein-tyrosine kinases play key roles in additional crucial cellular processes, including cellular adhesion, vesicle trafficking, cell communication, the control of gene expression, protein degradation, and immune responses. Moreover, discoveries regarding the role of protein-tyrosine kinases in cell transformation and cancer gave rise to a promising new rational approach to cancer therapy; i.e., the targeting of protein-tyrosine kinases. For example, the drug Gleevec, which inhibits activation of the Abl and platelet-derived growth factor (PDGF) tyrosine kinases, was approved by the U.S. Food and Drug Administration for the treatment of chronic myelogenous leukemia and several types of gastrointestinal tumors.

References:

  1. Howard Temin: “In from the Cold,” Posted on the blog December 14, 2013.
  2. Harold Varmus: From English Literature Major to Nobel Prize-Winning Cancer Researcher, Posted on the blog January 5, 2016.
  3. Collett, M. S. and R. L. Erikson, 1978. Protein kinase activity associated with the avian sarcoma virus src gene product. Proc. Natl. Acad. Sci. USA 75: 2021-2024.
  4. Hunter, T., and B. M. Sefton. 1980. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc. Natl. Acad. Sci. USA 77:1311–1315.

Harold Varmus: From English Literature Major to Nobel Prize-Winning Cancer Researcher

Harold Varmus and J. Michael Bishop changed cancer research in a fundamental way in the 1970s, when they discovered proto-oncogenes at the University of California at San Francisco (UCSF). Proto-oncogenes are cellular genes that normally play an important role in controlling cell division and differentiation. However, Varmus and Bishop found that proto-oncogenes can be altered by mutation, to become oncogenes that contribute to cancer. When Varmus and Bishop first began their collaboration in 1970, cancer research was, for the most part, focused on epidemiology (e.g., studies linking smoking to lung cancer) and empirical approaches to therapy (e.g., radiation and chemotherapy).

Harold Varmus, Cancer Researcher, Nobel Laureate, Director of the NIH
Harold Varmus, Cancer Researcher, Nobel Laureate, Director of the NIH

The discovery of proto-oncogenes is a pertinent topic for our Virology blog because it depended crucially on Varmus and Bishop’s earlier finding that retroviral oncogenes are mutated versions of cellular genes that retroviruses “captured” from their host cells. Varmus and Bishop hypothesized and then demonstrated that since retroviral oncogenes are versions of genes that actually are part of a normal cell’s genetic makeup, mutations in those genes, or their inappropriate expression, can lead to cancer. The v-src gene of Rous sarcoma virus was the first retroviral oncogene that Varmus and Bishop showed is derived from a cellular genome (1).

Varmus and Bishop continued searching for proto-oncogenes in the 1980s. Varmus also began investigating HIV (also a retrovirus and the cause of AIDS). In 1989 Varmus and Bishop were awarded the Nobel Prize in Physiology or Medicine for their discovery of proto-oncogenes.

In the early 1990s Varmus stepped out from his role as a research scientist to take up the cause of public funding for biomedical research. In 1993 President Bill Clinton acknowledged Varmus’ efforts in that regard, as well as his stature as a scientist, by appointing him to serve as Director of the National Institutes of Health (NIH). Thus, Varmus became the first Nobel laureate to head the NIH.

In 2000 Varmus left the NIH to accept the presidency of the Memorial Sloan-Kettering Cancer Center in New York. In 2010 Varmus returned to the NIH, this time appointed by President Barak Obama to serve as director of the National Cancer Institute (NCI). In 2015 Varmus was back again in New York where he is the Lewis Thomas University Professor of Medicine at Weill-Cornell Medical College.

Varmus was featured in two earlier blog postings. The first of these described how he mediated the dispute between Robert Gallo and Luc Montagnier over the right to name the AIDS virus (2). The second posting covered some of the political and social dilemmas Varmus faced during his days leading the NIH (3).

Here, we relate first how Varmus opted for a career in biomedical science and, second, how his collaboration with Bishop came about. This is an interesting tale because Varmus’ remarkable career as a science researcher, administrator, and spokesperson happened despite his initial intention to become a teacher of English literature. Indeed, his career in science did not begin until after he earned an M.A. degree in English from Harvard University, and then spent four years in medical school preparing for a career in clinical medicine.

We begin our story in 1950 as Varmus recounts how, as a ten-year-old, he witnessed his physician father receive a call that conveyed shocking news: “one of my mother’s favorite cousins, a robust man in the middle of his life, had just been diagnosed with leukemia. Of course, I did not know very much about leukemia, but I did know immediately from my parents’ expressions–and within a few weeks, from our cousin’s death—that his disease was a veritable tidal wave.” [All quotations are from Varmus’ book The Art and Politics of Science (4), in which he reflects back on his entire career.]

His cousin’s leukemia actually resulted from a mutation in one of the genes that Varmus would discover more than two decades later. And Varmus notes just how far the science in general had progressed during that 25-year interim: “…when my father heard about our cousin’s leukemia, biologists were not even sure that genes were made of DNA, had no idea how genetic information could be encoded in genes, and, of course, had no way of knowing that cancers are driven by mutations.”

Varmus was urged by his father to prepare for a career in medicine. Nonetheless, when Varmus enrolled as a freshman at Amherst College he strongly favored studying the humanities. Thus, he “toyed with the idea of majoring in philosophy (ultimately too abstract), physics (ultimately too hard), and English literature (ultimately selected).”

Throughout his undergraduate days, Varmus envisioned preparing for an academic career teaching literature. Still, he dutifully fulfilled premed requirements to keep open the possibility of obliging his father’s wishes that he become a medical doctor. Yet he never considered majoring in biology. “I couldn’t understand how some of my close friends (among them, some now distinguished scientists) could spend long afternoons and evenings incarcerated in a laboratory, when they could be reading books in a soft library chair or reciting poetry on Amherst’s green hills.”

Varmus began having doubts about his career choice when his Amherst College classmate Art Landy (later a well-known molecular biologist at Brown University) won an Amherst biology prize that allowed him to attend a 1961 international biochemistry meeting in Moscow. Importantly, Landy invited Varmus to accompany him to the Moscow meeting, where Varmus learned that Marshal Nirenberg had deciphered the genetic code. “Even though I did not understand its meaning or its importance at the time, I was not oblivious to the excitement around me…Scientists seemed likely to discover new, deep, and useful things about the world, and other scientists would be excited by these discoveries and eager to build on them. Would this be true of literary critics and teachers?”

Notwithstanding these misgivings, Varmus continued on his path to a career in English literature after graduating from Amherst College in 1961, earning an M.A. in English from Harvard in 1962 (his focus was on Anglo-Saxon poetry). But his uncertainties about his future only grew stronger. “Despite outward signs that I had chosen a life of studying and teaching literature, soon after starting my graduate work at Harvard I began to suffer some further internal doubts about abandoning medicine. The graduate curriculum in English literature was not especially onerous, but it felt like a prolongation of college. Most of my courses were heavily populated with Harvard and Radcliff undergraduates.” Varmus leaves the impression that he looked upon much of his course work at Harvard as a tiresome chore.

Varmus was also aware of the enthusiasm of former Amherst College classmates who were then studying at Harvard Medical School. “Occasionally, on Saturday mornings, I traveled across the Charles River to join some Amherst classmates at Harvard Medical School, while they sat in the Ether Dome at Massachusetts General Hospital, entranced by diagnostic dilemmas discussed at the weekly pathology conference. These stories struck me as far more interesting than those I was reading, and my medical school friends expressed general excitement about their work. They also seemed to have formed a community of scholars, with shared interests in the human body and its diseases and common expectations that they would soon be able to do something about those diseases…These Saturday excursions probably account for an influential dream I had one night about my continuing indecision. In that dream, my future literature students were relieved when I didn’t turn up to teach a class, but my future patients were disappointed when I didn’t appear. It seemed I wanted to be wanted.”

So, Varmus finally came to grips with his qualms about a career teaching English literature, hastily preparing an application to Harvard Medical School and biking across the frozen Charles River to deliver it just in time to meet the deadline. But it was to no avail, since the dean of admissions thought Varmus was “too inconstant and immature” for medical school.

Varmus next sent off an application to Columbia University’s College of Physicians and Surgeons (P&S). His interviewer at Columbia was an esteemed physician named David Seegal, who also happened to be rather literate. Seegal asked Varmus if he might translate the Anglo Saxon phrase Ich ne wat. “This was easy; it simply means ‘I don’t know.’” Seegal used his question as a lead-in to discuss why a physician might admit fallibility to a patient. In any case: “By the fall of 1962, I was happily enrolled at P&S, helped for the first, but not the last, time by someone’s exaggerated appreciation of my competence in two cultures.”

Now ensconced at P&S, Varmus thought he might become a psychiatrist; an ambition stoked by an interest in Freud and by his winning of an essay prize at P&S in psychiatry. But, he found his “first hour alone in a room with a psychotic patient to be more difficult and less interesting than an hour reading Freud.” So, Varmus’ interests in medical school turned from the “elusive mind” to the physical brain and then, more generally, to diseases that might be explained by known physiology and biochemistry.

When graduation from medical school was impending, Varmus had to consider his career options more deliberately than he had in the past. A key factor was the Vietnam War, which was in progress, and which he and many others of that era vehemently opposed. “I was determined not to serve in it. Medical graduates were subject to the draft; however, we did have the more palatable option of two years training at one of the agencies of the Public Health Service. For most of my classmates with academic ambitions similar to my own, the NIH was the favored choice. As the largest biomedical research campus in the world, it offered unequaled opportunities to learn virtually any form of biomedical research…”

Varmus confesses that he had a “woeful lack of laboratory credentials.” Nonetheless, he entered the competition for one of the coveted research slots at the NIH. But, because of his lack of research experience, he was not encouraged by most of the NIH laboratory chiefs who interviewed him. However, one of them, endocrinologist Jack Robbins, suggested to Varmus that he speak to Ira Pastan; a young endocrinologist who, at that time, was interested in the production of thyroid hormones.

As Varmus relates, “The recommendation proved to be wise and fateful. My schooling in literature turned out to be more important than my interest in endocrinology, Ira’s field, because Ira’s wife Linda, a poet, had often complained that Ira’s colleagues seldom talked about books…When matches were announced I was told I would become Ira’s first clinical associate, having been passed over by the more senior investigators. This outcome could not have been more fortunate.”

But, before Varmus could take up his position at the NIH, he received a “shocking phone call” from Pastan, to the effect that he (Pastan) was giving up his work on thyroid hormones because he and colleague Bob Perlman “had made a shocking discovery about gene regulation in bacteria.” Pastan and Perlman found that cyclic AMP is a major regulator of bacterial gene activity, and that it plays a similar role in animal cells—findings which led Pastan to pioneer the field of receptor biology in animal cells.

The discovery by Pastan and Perlman had important consequences for Varmus. First, it immediately forced him to give up his plan to train in endocrinology. Instead, Pastan assigned Varmus to find out whether cyclic AMP augments bacterial gene expression by increasing transcription of mRNA. Second, as explained below, Pastan’s new research direction led to Varmus’ introduction to and fascination with virology.

So, Varmus was now a budding molecular biologist. But, since he had no prior research experience, his first days in the Pastan lab were a near disaster, leading Pastan to half jokingly ask, “Now remind me why I took you into the lab.”

In any case, Varmus worked closely with Pastan to develop a molecular hybridization assay to measure transcription of E. coli lac mRNA. [Their specific the goal was determine whether the mechanism by which cyclic AMP reverses catabolite repression of the E. coli lac operon is by enhancing transcription of lac mRNA.] And, they used an E. coli phage, which had incorporated the lac operon into its genome, as their source of isolated lac operon DNA. Thus, Varmus was introduced to virology. [Aficionados, note, “These experiments with the lac operon proved to be analogous in several ways to experiments that revealed the first proto-oncogene a few years later.”]

The satisfaction that Varmus derived from his research in Pastan’s lab caused him to reconsider his aspirations for a career in clinical medicine, and instead to think about a future in biomedical research. He thought he might next try his hand at cancer research, motivated in part, by his mother’s breast cancer, first diagnosed in 1968, to which she succumbed two years later. But there were other factors at work as well. In particular, Varmus’ use of the E. coli phage in Pastan’s lab led to his fascination with virology. And his interest in virology was relevant to his new plans because the DNA and RNA tumor viruses held immense potential for cancer research. 1970s technology could not identify which one of the tens of thousands of cellular genes might have mutated to result in a cancer. However, that technology was potentially able to identify which of the handful of a tumor virus’ genes might underlie its ability to transform a normal cell into a tumor cell.

That line of thought led Varmus to apply for a research position in Renato Dulbecco’s lab at the Salk Institute. [Dulbecco would win a share of the 1975 Nobel Prize in Physiology or Medicine for his pioneering studies of the DNA tumor viruses (5).] However, reminiscent of Varmus’ unsuccessful application to Harvard Medical School, he was “rebuffed by not one but two letters from his (Dulbecco’s) secretary.”

While the rejection from Dulbecco was a disappointment, it would be another of the seemingly providential happenings in Varmus’ career. In the summer of 1969 he chanced to visit Harry Rubin, an eminent Rous sarcoma virus researcher at U Cal Berkeley. Rubin, who had earlier introduced Howard Temin to virology (another auspicious happening; see reference 6), told Varmus about a new group at UCSF that had begun to study retroviruses. Importantly, the goal of the UCSF group was to discover cancer-causing genes. Thus, Varmus stopped over at UCSF, where he met members of the group, including a smart young virologist named Mike Bishop. Varmus reports, “we recognized from the first moments that we were destined to work together.”

Varmus came to Bishop’s lab in 1970 as a postdoctoral fellow. However, their relationship quickly evolved to one of equals, and they made all of their major discoveries in the 1970s and 1980s as a team, and they rose together through the UCSF academic ranks. Bishop relates that their bond formed not just by a shared fascination with cancer viruses but “by our mutual love of words and language.” Varmus, for his part, notes that “after many years of ambivalence and indecision…I appeared to be headed in a clear direction, even if not towards medicine or literature.”

References:

1. Stehelin D, Varmus HE, Bishop JM, Vogt PK., 1976. DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature 260:170-173.

2. How the Human Immunodeficiency Deficiency Virus (HIV) Got Its Name, posted on the blog February 4, 2014.

3. The Politics of Science: Vignettes Featuring Nobel Laureate Harold Varmus during his Tenure as Director of the NIH, posted on the blog June 2, 2014.

4. Varmus, H. 2009. The Art and Politics of Science, (W. W. Norton & Company).

5. Renato Dulbecco and the Beginnings of Quantitative Animal Virology, posted on the blog December 3, 2013.

6. Howard Temin: “In from the Cold”, posted on the blog December 14, 2013.