by Tony Hunter, Professor of Molecular Biology, The Salk Institute, Recipient of the 1994 Charles S. Mott Prize, General Motors Cancer Research Foundation lectures, June 15, 1994
The phosphorylation of tyrosine residues in proteins was discovered in 1979 during our analysis of the protein kinase activity that phosphorylates the middle T antigen of polyoma virus in vitro. The presence of phosphotyrosine in the middle T antigen was the first indication that tyrosine could be a target for phosphorylation by a protein kinase. Shortly thereafter, we found that v-Src, the Rous sarcoma virus (RSV) transforming protein, and c-Src, its cellular progenitor, also have tyrosine kinase activity. Moreover, we showed that v-Src- transformed cells have elevated levels of phosphotyrosine in protein. Other retroviral transforming proteins were soon found to be tyrosine kinases, and the epidermal growth factor (EGF) receptor was shown to have tyrosine kinase activity that is stimulated by EGF binding.
This evidence immediately implied that tyrosine phosphorylation plays a role in cell growth and transformation. There are now more than 80 known vertebrate tyrosine kinases, and about half of them are receptor tyrosine kinases activated by their ligands. In the past few years significant progress has been made in identifying key substrates for activated receptor tyrosine kinases whose phosphorylation leads to the mitogenic response. These substrates include enzymes, structural proteins, and proteins called adaptors. Most of these substrates have a specialized domain, called Src homology 2 (SH2), that binds to phosphotyrosine in a sequence-specific fashion. Adaptors are SH2 proteins that lack enzymatic activity but that have other protein-binding domains that bind effector proteins. Ligand binding to the extracellular domain of receptor tyrosine kinases induces its dimerization and autophosphorylation, which creates binding sites for cytosolic SH2 adaptor proteins, whose binding and activation triggers signaling pathways leading to the nucleus. One of the best-understood pathways is the Mitogen-Activated Protein (MAP) kinase pathway, in which activated MAP kinase moves into the nucleus, where it phosphorylates and activates transcription factors, leading to gene expression. MAP kinase is activated via a series of protein kinases which is triggered by activated, GTP-bound Ras protein. The loading of GTP onto Ras is stimulated by Sos, a guanine nucleotide exchange factor that is bound to the Grb2 adaptor protein. The binding of the Grb2-Sos complex to a specific phosphotyrosine residue in an activated receptor tyrosine kinase translocates Sos to the membrane, where it can convert Ras-GDP to Ras-GTP. Over the past 15 years, research has revealed that tyrosine phosphorylation, in addition to its role in mitogenic signaling pathways, is involved in many fundamental cellular processes including differentiation, regulation of the cell cycle, and neuronal function.
Q: What was your starting point in this research, and how have your questions evolved?
A: In 1977, we were studying polyoma virus, a small DNA tumor virus that causes tumors in rodents. We were trying to identify the viral gene products responsible for transformation. Interest had centered on a single protein called middle T (or tumor) antigen, which is encoded by one of the three alternatively spliced mRNAs generated from the so-called early region of the polyoma virus genome. As soon as Marc Collett and Ray Erikson, then at University of Colorado in Denver, reported in 1978 that the RSV-transforming protein, v-Src, had protein-kinase activity when assayed in an immunoprecipitate, we began to test whether polyoma virus middle T antigen also had such activity. We were excited to find that middle T antigen became phosphorylated in immunoprecipitates, and by the middle of 1979, we had shown through the use of viral mutants that the presence of this protein kinase activity correlated well with the ability of middle T antigen to transform mammalian cells. In the course of analyzing by acid hydrolysis which amino acid was phosphorylated in middle T antigen, we discovered that the phosphate was not linked to serine or threonine but to another amino acid. We guessed that this might be tyrosine, quickly made some phosphotyrosine, and showed that the product of acid hydrolysis of phosphorylated middle T antigen co-migrated with the synthetic phosphotyrosine. At the outset, we thought this might be a unique property of this viral protein. However, as a control, we were testing v-Src and found that it, too, had tyrosine kinase activity. In this case, we were able to show that c-Src, the cellular progenitor of v-Src, also had tyrosine kinase activity, suggesting that a normal, vertebrate protein could act as a tyrosine kinase. This finding was quickly followed by the demonstration by others that the v-Fps and v-Abl retroviral transforming proteins are tyrosine kinases, and that the EGF receptor also has tyrosine kinase activity that is stimulated by EGF binding. By the end of 1980, we knew of four different cellular tyrosine kinases, and it was apparent that tyrosine phosphorylation played a role in cell growth and malignant transformation. Over the next 15 years, it became apparent that tyrosine phosphorylation functions not only in growth control but also in many other cellular processes, including differentiation, the cell cycle, transcriptional regulation, and synaptic transmission.
Q: Which findings have been most surprising to you or to other scientists?
A: Even though phosphorylation of proteins on serine and threonine residues had been known for many years, no one had reported phosphorylation of a protein on tyrosine residues. Thus, it was totally unexpected that the protein kinase activity associated with polyoma virus middle T antigen should be able to phosphorylate tyrosine. The second major surprise was that v-Src and several other retroviral transforming proteins and their cellular counterparts proved to be tyrosine kinases as well. This led to the realization that tyrosine kinases are a major family of cellular enzymes that play important roles in cell growth and many other cellular processes. Indeed, over 80 tyrosine kinase genes are currently known in the human genome, and the true number appears likely to be much larger, once more of the human genome is sequenced.
Q: What were the greatest stumbling blocks, and what new observations, techniques, reagents, or insights helped you get past them?
A: The initial problem was that there was no source of phosphotyrosine to test the idea that middle T antigen was phosphorylated on tyrosine. At that time, we did not realize that there was a published synthesis for phosphotyrosine, and tried mixing POCl3 with tyrosine. Not surprisingly, we ended up with a black tar, but luckily, we were able to purify a small amount of phosphotyrosine. In fact, we now use an efficient synthesis of phosphotyrosine that had been reported in 1941 by a British biochemist, although why he was making phosphohydroxyamino acids in the depths of World War II is not clear.
The next important technical step was to devise a reliable separation for all three phosphohydroxyamino acids. The separation of phosphotyrosine and phosphothreonine in our original experiments was fortuitous and was due to the use of an old stock of pH 1.9 buffer that had been reused many times for thin-layer electrophoresis. We realized later that upon repeated use, the pH of the buffer drops to 1.7, allowing resolution of phosphotyrosine and phosphothreonine, which co-migrate at pH 1.9. Through trial and error, we found a chromatography system that, in combination with electrophoresis at pH 1.7, gave reasonable separation of phosphoserine, phosphothreonine, and phosphotyrosine. This technique, however, was soon superseded by the technique that is still preferred today -- a two-dimensional, thin-layer separation using electrophoresis at pH 1.9 followed by electrophoresis at pH 3.5.
The other key problem was to find methods for identifying proteins with phosphorylated tyrosines in the intact cell -- the candidate substrates for tyrosine kinases. We developed a method for identifying these proteins based on two-dimensional gel electrophoresis of proteins from 32P-labeled cells. Subsequent treatment of the gel with 1M NaOH hydrolyzes much of the phosphate from phosphoserine but leaves the phosphate on phosphotyrosine, which is rather stable at alkaline pH. As a result, phosphotyrosine-containing proteins are enriched among the remaining 32P-labeled proteins. This technique yielded some of the first substrates for the v-Src tyrosine kinase.
Because the structure of phosphotyrosine resembles that of a dinitrophenyl group -- an excellent hapten -- an obvious alternative method of finding phosphotyrosine-containing proteins was to try to develop antibodies to phosphotyrosine. We were successful in doing this, but another research group had the same idea and was the first to report the use of anti-phosphotyrosine monoclonal antibodies in detecting phosphotyrosine-containing proteins. Anti-phosphotyrosine antibodies that can be used for immunoprecipitation and immunoblotting have now been developed and are proving invaluable for identifying and analyzing tyrosine kinase substrates.
Q: Do you see any potential areas where this research might provide insight to clinical scientists?
A: The clear connection between tyrosine phosphorylation and malignant transformation provided by the RNA and DNA tumor viruses has been extended to human cancer. At least four of the known human oncogenes are mutated forms of tyrosine kinase genes, Ret, Abl, Trk, and the PDGF receptor. The corresponding oncogenes encode tyrosine kinases that are constitutively active. In addition, the EGF and ErbB2 receptor genes, which also encode tyrosine kinases, are commonly amplified and overexpressed in human tumors, and members of the Src protein family often show elevated tyrosine kinase activity in human tumors. For this reason, there has been a strong push to develop specific, low-molecular-weight inhibitors of tyrosine kinases that could be used as cancer therapeutics. A large number of inhibitors have been reported in the past 15 years, and some of these appear to be reasonably specific. One inhibitor that is specific for the EGF receptor has just been described and appears to have some inhibitory effect on the growth of human tumor cells that express high levels of the EGF receptor in mice. Several companies have active programs aimed at developing drugs targeted at specific tyrosine kinases involved in cancer and other human diseases.
Q: What does your work focus on now, and what questions would you ultimately like to answer?
A: My lab's work is now focused largely on the signal pathways activated by receptor and nonreceptor tyrosine kinases. Ligand-induced receptor autophosphorylation creates binding sites for substrates and target proteins that have SH2 domains. SH2 domains bind to phosphotyrosine in a sequence-specific manner. Thus, following ligand binding, the activated receptor tyrosine kinase binds a set of SH2-containing proteins that recognize the individual phosphorylation sites displayed by the receptor in question. These SH2 proteins are activated by phosphorylation, by binding to the receptor, or by translocation to the membrane. Some substrates are enzymes, such as phospholipase C[[gamma]], and others are structural proteins. We are concentrating on a group of targets known as SH2 adaptor proteins which lack enzymatic activity but contain additional protein-interaction domains, such as SH3, that bind effector proteins that propagate the signal. Specifically, we are trying to elucidate the roles that Grb2 and Nck play in mitogenic signaling. We are also studying protein-tyrosine phosphatases -- enzymes that reverse tyrosine phosphorylation -- because it is clear that these enzymes must also be essential for any process regulated by tyrosine phosphorylation.