Cell Signaling and Control

Medicine
Anthony “Tony” R. Hunter
American Cancer Society Professor, Molecular and Cell Biology Laboratory, and Renato Dulbecco Chair, Salk Institute for Biological Studies, and Adjunct Professor, Section of Molecular Biology, University of California San Diego, La Jolla, CA USA
Anthony “Tony” J. Pawson
Distinguished Scientist and Apotex Chair in Molecular Oncology, Samuel Lunenfeld Research Institute of Mount Sinai Hospital, and Professor, Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada

Hunter is suggested as a possible Nobel Prize winner “for the discovery of tyrosine phosphorylation and contributions to understanding protein kinases and their role in signal transduction”

Pawson is suggested “for identification of the phosphotyrosine binding SH2 domain and demonstrating its function in protein-protein interactions”

Hunter and Pawson have been selected here for two closely related fields of research. Hunter discovered tyrosine phosphorylation, the basis of almost all cellular signaling pathways, and Pawson for identification of the SH2 protein domain, which connects phosphorylated signal receptors with the rest of their pathway.

Hunter and Pawson tackled different parts of a key aspect of multicellular organisms: how do cells communicate with one another? How do they know when to grow, and when to slow? Cancers, after all, are cells that either have their accelerator stuck down, or broken brakes. What causes those malfunctions?

In the late 1970s, many researchers focused on Rous sarcoma virus, which contains an oncogene, v-SRC, that causes cancer in chickens. Hunter decided to identify the protein encoded by v-SRC. He came a close second to a team led by Ray Erikson at Harvard, who showed that the protein added a phosphate molecule to proteins; it was a phosphorylation enzyme. At the time, only two amino acids were known to be a target for phosphorylation: serine and threonine. Hunter showed that v-SRC’s target was a third amino acid: tyrosine.

Because phosphorylation of tyrosine was unprecedented, part of Hunter’s breakthrough was to devise a method to separate serine phosphate, threonine phosphate, and tyrosine phosphate. Chromatography showed that Src phosphorylated tyrosine, not threonine or serine. But when Hunter checked his results, with fresh reagents, he could not replicate them. It turned out that one of the buffer solutions he used in the original separation was old and more acid than was indicated on the label. With fresh buffer, tyrosine phosphate and threonine phosphate moved together and could not be distinguished. Only with deliberately aged buffer did tyrosine reveal itself.

Hundreds of protein kinases have now been discovered, and they are involved in growth, differentiation, cell division, cell movement, synaptic transmission, and much else besides. Most (but not Src) are signal receptors. One part of the molecule sits outside the cell as a kind of antenna. A signal molecule, such as a growth factor or a hormone, attaches to the antenna and this causes the kinase to autophosphorylate, activating itself and starting a cascade of activation that eventually turns on specific target genes. When the signal moves off the antenna, the cascade quickly stops. That is in the normal cell; but more than half of the known tyrosine kinases are implicated in cancers, the result of mutations that make them “on” all the time.

Pawson’s research helps to understand the details of the signal transduction that begins with the activation of a protein kinase. He too started this work by looking at an oncogene, Fps from the Fujinami sarcoma virus. Pawson identified three separate domains within the protein coded by Fps. One was a tyrosine kinase domain, related to the Src tyrosine kinase. Another domain interacts both with phosphorylated tyrosine and with other protein domains, thus linking the Fps protein with its targets, which it phosphorylates. This linking domain is similar to a corresponding domain in the Src protein, so Pawson called it the Src Homology 2 domain, or SH2. More than 100 human proteins contain an SH2 domain, and it is the specificity of the SH2 domain for a particular protein that ensures the specificity of the cell’s response to the extracellular signal.

Commentary on the Medicine Laureates by Jeremy Cherfas, Biology correspondent, ScienceWatch

Interview with Anthony “Tony” R. Hunter, American Cancer Society Professor, Molecular and Cell Biology Laboratory, Renato Dulbecco Chair, Salk Institute for Biological Studies, and Adjunct Professor, Section of Molecular Biology, University of California, San Diego

For the discovery of tyrosine phosphorylation and contributions to understanding protein kinases and their role in signal transduction

Please provide a brief overview of your field of research and explain what led you to focus in this area?

Our goal is to understand how signal transduction pathways that transmit signals through post-translational modification of proteins, such as phosphorylation, regulate cell behaviors, including proliferation and cell cycle progression. I started to work on animal tumor viruses as models for human cancer, when I joined the Salk Institute as a postdoctoral fellow in 1971. I was studying a small DNA tumor virus called polyomavirus, and ultimately this led to our 1979 discovery that middle T antigen, one of the three polyomavirus transforming proteins, is associated with a protein kinase activity, which had the unique ability to phosphorylate tyrosine in proteins.  This was quickly followed by our discovery that the Src transforming protein encoded by Rous sarcoma virus was also a tyrosine kinase, implying an important general role for tyrosine phosphorylation in cancer. Ever since then, I have been deeply interested in the functions of protein phosphorylation. When the first protein kinases were cloned and sequenced in the early 1980s, I began to catalogue protein kinases based on the relatedness of their catalytic domains, and this led to my 1987 prediction that mammals might have as many as 1001 protein kinase genes. When the human genome sequence was reported in 2001, we were able to define the complete human kinome, which has over 530 protein kinases, including 90 tyrosine kinases.

What did you want to accomplish when you began your research?

When I began studying tumor viruses, I hoped to learn more about the mechanisms of cellular transformation at the molecular level that might ultimately lead to better cancer therapies.  We have certainly been successful in that regard, but my research has not taken a linear path and we have diversified in many directions, and based on my interest in phosphorylation, which is involved in most cellular processes, we have moved into many new areas.

What notable problems, challenges, or obstacles did you face? Conversely, have there been particular sources of enjoyment, satisfaction, or pride?

In the early days of our work on tumor viruses, molecular biology techniques were very primate, and the lack of molecularly characterized reagents and genome sequences made our efforts to define the mechanisms of transformation extremely challenging. My identification of phosphotyrosine as the novel phosphorylated compound in middle T antigen generated by in vitro phosphorylation is a particular source of satisfaction, and it is extremely gratifying that this discovery has played a role in the development of new class of cancer drug. Finally, I would add that even though the discovery of tyrosine phosphorylation was unprecedented, the scientific community very quickly accepted it, and within a year of the first publication in 1979 many groups had begun working on tyrosine kinases.

How would you assess the importance and influence of your work?

The discovery of tyrosine phosphorylation and the general role that unregulated tyrosine phosphorylation plays in cellular transformation and human cancer has ultimately led to a new generation of cancer drugs that target activated tyrosine kinases, such as imatinib/GleevecTM, a very successful treatment for chronic myelogenous leukemia.

Has this research found wider application in industrial or commercial areas? If so, what are some possible future developments?

Tyrosine kinase inhibitors have become an important new class of cancer drug, and the success of imatinib became the stimulus for the development of inhibitors of other types of kinase that play a role in cancer. Kinase inhibitor drugs are now a multibillion dollar industry in the US.