 Recent success in determining the sequence of the human genome, the discovery of key genes responsible for devastating diseases, and developments in cloning or other means for manipulating the genetic makeup of our offspring, have focused our attention on the role of genes in determining who we are and who we want to become. However, genes are only part of the story, an important part, of course. But genes in their component DNA only indicate the potential of a living thing, not the realization of that potential. A genetic code is like a musical score, which maps the combination of notes and voices that musicians can use to create music. How that score is interpreted and brought to life is dependent on multiple decisions made by many individuals alone and in concert and in response to environmental forces such as the availability and expertise of the participants and rehearsal time. So too, a living organism is the realization of a genetic plan implemented by numerous cell divisions, migrations and processes of differentiation coordinated by a complex communication system and in response to external stimuli. How do cells orchestrate the miraculous transformation from a handful of cells to a living species? How do individuals learn complex tasks like playing music? Today's first speaker, Dr. Edmund Fischer, has contributed to answering questions of this sort by his co-discovery of a key process used by cells to translate extracellular signals into biochemical processes within cells. The controlled communication among cells is imperative so that individual cells of the heart pump in synchrony, so that memories are retained, and so that growth and metabolism are controlled. For discovering reversible protein phosphorylation, a chemical mechanism used by cells to respond to external messengers, he shared the 1992 Nobel Prize in Physiology or Medicine with his longtime collaborator, Professor Edwin Krebs. This work has led to new insights into the biochemical mechanisms associated with cancer, blood pressure, inflammatory reactions, and brain signals. Dr. Fischer was trained in chemistry, receiving the equivalent of his PhD in 1947 from the University of Geneva. He then traveled to the United States to pursue a postdoctoral research appointment at Caltech. En route to California, he presented several seminars at universities, collecting job offers at each stop along the way. Dr. Fischer discovered that the mountains, forests, and lakes surrounding Seattle reminded him of his native Switzerland, so he decided to accept a position at the University of Washington. It is here that he began his collaboration with Ed Krebs, studying phosphorylase enzymes. He quickly advanced to the rank of full professor as he earned various international awards and honors throughout his academic career. He was elected to the American Academy of Arts and Sciences in 1972 and the National Academy of Sciences in 1973. In addition to being an accomplished and insightful scientist, Dr. Fischer is a talented pianist. He was admitted to the Geneva Conservatory of Music in high school and he considered pursuing a career in music. Like others participating in this conference, Professor Fischer is worldly, witty, artistic, and fluent in several languages. He loves to tell stories and he seems to have one to fit every situation. Today, his story will concern how proteins speak with one another in cell signaling. Please join me in welcoming Dr. Edmund Fischer. Thank you. Like all the other speakers, I'm very grateful and honored to have been invited to talk at this symposium and I would like to thank President Sturrier and Tim Robinson and the other members of the faculty for inviting me to be part of this absolutely stunning event and celebration. So thank you. Today I would like to tell you something about cell signaling. That is the extremely complex reactions that induces cell to grow, to proliferate, and eventually to die in response to a multitude of internal or external signals. And let me give you an example of what I'm speaking about. You all have heard about stem cells and the extreme potential they have in curing various diseases because of their ability to develop and differentiate into any type of tissues. And indeed, they can go into a heart cell that will beat, a pancreatic cell that will produce insulin, a brain cell that might help us one day to cure Alzheimer's disease or a nerve cell that will repair a spinal cord injury. But we don't know the commands that will direct those cells to go in those different directions. We know that they involve a very intricate and precisely time set of signals coming in good part from other cells that stem cells need to grow, on which they are layered, the addition or removal of a maze of hormones and growth factor plus many other contributors. But we don't know what those signals are, though we begin to understand how they are received by the cell and the mechanism by which they are processed by the cell. And that's what I want to discuss. Now, the subject belongs to the field of proteomics that concerns itself with all the proteins that are generated during the lifetime of an organism, the structure, their properties, how they interact with one another to perform all the tasks that organism needs to stay alive. In a way, then, proteomics represents the other face of the coin, so to speak, of genomics, of which again you heard a lot about with respect to the Human Genome Project. Now, we all agree that the near completion of the Human Genome Project plus the genetic unraveling of maybe three, four dozen, five dozens of other organisms represents an achievement of enormous proportion, one that holds tremendous promise for the future biomedical research and medicine in general. Nevertheless, this huge accumulation of DNA-based information can only take us so far. The genome, which represents the ensemble of all the genes, can predict, of course, the proteins that can potentially be generated in a cell, but it cannot take into account the enormous diversification of structure that result from gene reorganization, gene switching, gene insertion, recombination and other types of rearrangements as one sees, for instance, in the immune system, where less than a total of a thousand genes can potentially give rise to something like 500 million different proteins and antibodies. This is why you can make antibodies against just about any antigen. Genomics cannot predict the mutations that will result, for instance, from radioactivity, chemical carcinogens, or the alterations by enzymatic cleavage processing or countless chemical modifications. So, in other words, the proteome contains far more proteins than the genomes contain genes, while the genome represents a rather fixed fixture. The proteome is always in a state of flux. It's changing constantly under the influence of development or even environmental factors. And finally, the genome cannot tell us how protein interact with one another for signaling, or the signals that will determine their translocation in the cell or binding to subcellular element. And Günter Blohbell will tell you all about that because this is really his bag. Or the genome won't tell us how proteins, the mechanisms by which they are regulated. And that's what I want to tell you now. Now, for those of you who might not be conversant on the field of cell signaling, let me try to put that on the basis of a notion that is very familiar to all of you. May I have the first slide, please? Now, don't you get excited. This is a television set. You all know what that is. All right, here in black is the picture tube on which the image is displayed. And here in the back is where you plug it in because you need a source of energy to activate all those elements. Biological cells are powered by nutrients, carbohydrates, amino acids, fats, that are metabolized here to give rise to ATP, that molecule that Harry Croto spoke of yesterday morning. This is the universal energy currency of all living organisms that they go standard if you want. Now, in the back of the cell, you have two receptors here to capture external electromagnetic signals coming from the antenna, the VCR, the satellite. And these signals now will be transduced by a whole series of elements, transducers, resistors, capacitors to give a physical response that is the image on the picture tube. And with maybe, I don't know, 150, 200 of those elements, you can generate some tens of thousands or hundreds of thousands of interactions by which not only can you display the image, but can you vary at will, its tint, color intensity, brightness, sharpness, and sound. This is what we call signaling or signal transduction. The conversion of some kind of external signal into some kind of a response. Now, those television sets have evolved in the last, I don't know, 75, 80 years maybe. Biological cells have evolved over more than three and a half billion years. And therefore, rather than having just a few hundred elements, there are well more than a hundred thousands of these. And the number of interactions among these must be in the order, I don't know, something like 10 to the 20th power. And this is what makes a biological cell autonomous, self-sufficient, allows it to synthesize itself, all its elements, regulate its growth, repair its damages, and even program its own death at the appropriate time. Biological cells differ from TV sets in that rather than having just two receptors here in the back, they have well more than a thousand of these receptors. And responding to tens of thousands of external signals. And they will come in the form of hormones or growth factors, neurotransmitters, drugs, also electromagnetic radiation that is light in the visual system, or UV irradiation or heat among various stress signals, odorance in the olfactive system. Animals have close to 1,000 different olfactive receptors, only about 300 left in humans. But this is what would allow, for instance, a dog to follow the trail of a single person in a large crowd by computing the response of a combination of a few odorant molecules coming in the atmosphere, or a honey bee to be attracted by the proper flower. Now, biological cells differ from TV sets in a much, much more important way, in that in all higher organisms, millions or billions of cells come together in tissue and organs. And the switch that occurred between a single cell and a multi-cell system represents undoubtedly one of the most determining, most consequential event of biological evolution. Because before that, let's say three and a half billion years, a single cell had to compete with one another for food, for micro-nutrients, for light, for heat, for vital space, whatever. But the day cells began to associate with one another, for the first time, they had to cooperate for the good of the whole. They had to share their resources. They had to synchronize their growth in response to internal or external demand. And they did this by adhering to one another through a variety of cell adhesion molecule, either directly or with this network of matrix, extracellular matrix proteins with which cells are surrounded, so that a crosstalk could be established in those systems. In those systems, messages have to be sent constantly back and forth, every cell having to know not only what happens in all the other cells, but having to be able to define what should happen in all the other cells. And the switch, this transition from single to multi-cell systems occurred very recently in geological times, something like 530 million years ago, or just before the pre-Cambrian border. But this is what allowed in an extremely short span of time, people say 10, perhaps 20 million years, some say as little as 5 million years, for the establishment of all the species one finds inscribed in the fossil record. And there is a real lesson to be learned here, because before that, for 3 billion years, one witnessed a slow evolution marked by countless small victories followed by so many defeats until that day when cells began to communicate with one another and cooperate with one another, only then could they develop and evolve in the more and more complex organism that finally led to the appearance of man. Now, as you can imagine, the regulation of all those processes requires myriads of commands, positive and negative, that have to be strictly coordinated in order to keep under control all the reactions that take place, to ensure that a crucial event would not occur out of phase or at an inappropriate time. And it so happens that most of the signals that are used to orchestrate those reactions, the switches that have to be turned on and off, rely on a process called protein phosphorylation, or reversible protein phosphorylation. Let me tell you a little bit about that, because really it's extremely simple. All proteins that control the metabolism, muscle contraction, nerve conduction and so forth can exist in two conformations, active or inactive. And they can switch from one to the other by the introduction or removal of phosphate groups depicted by the letter P here. So the kinases are the enzymes that introduce phosphate groups, the phosphatases are the enzymes that remove the phosphate group. It's a little bit like the traffic lights that control street intersections. When they are green, the way is open, everybody goes through. When they are red, everybody stops, except the Italians in Italy. And sometimes my wife when she's in a hurry. Now, the system depicted here is not quite correct because traffic light operate by all the non-reactions, they're either red or green. Whereas biological systems are regulated with exquisite sophistication. They can go past through all the stages of activation, all the levels of activation or inhibition, depending on the ratio of the activity of these two enzymes. As if your traffic lights were each modulated by a color modulator that allowed them to go from the red to the green by passing through all the colors of the spectrum. This is important because we know that many forms of cancer are due to the deregulation of those enzymes that modulate those changes. And I'll come back to that in a little while. In any case, this kind of very basic mechanism, phosphorylation, turned out to be the most prevalent mechanism by which several processes are regulated. It's involved in the regulation of metabolism, protein synthesis at the transcription, translation in the nucleus, in the cytoplasm, the immune response, differentiation, secretion, transport, cell death, and so forth. In fact, it would be difficult to find a physiological process that is not directly or indirectly regulated by phosphorylation. Now, many of you probably know that proteins are made of 20 different amino acids. And it so happens that most of those phosphorylation reactions occur on two amino acids called serine and threonine. But one of the most exciting developments in this field was the discovery just a little over 20 years ago that phosphorylation of a third amino acid, tyrosine, was intimately implicated in cell transformation. And this resulted from the discovery that the oncogenic product of one of the very powerful carcinogenic agents, rous sarcoma virus, capable of triggering the malignant transformation of cells 24 hours or 48 hours was precisely due to the deregulation of one of those kinase, a tyrosine kinase. By a mutation, the enzyme had become permanently active. We say constitutively active in such a way that it could no longer be regulated. And the cell becomes like a car in which the accelerator is stuck to the floor. It goes wild. And many forms of cancer are due to this kind of deregulation, either that the gas pedal is stuck to the floor or that the brakes, which we call tumor suppressors, don't function anymore. And indeed, we know now that more than 60% of all tyrosine kinases have this ability to become oncogenic when mutated. And this includes the very large family of growth factor receptors, which are all tyrosine kinases and which are some of the principal structures by which cells receive external signal. They are like the bells you have to ring to enter a house. And let me tell you a little bit about their structure, and I apologize if I have to delve a little bit into biochemistry. Like the receptors, the two receptors on your TV set, they sit on the surface of the cell. Here's the membrane. They are half in, half out. They have a single chance membrane segment that separates those blue boxes inside the cell that are the catalytic domain. This is the other tyrosine kinase. They have tyrosine kinase activity, and their function is to transduce the signal down the cell. They are the business end of the receptors. And then outside you have this great variety of classical structures whose function is to recognize and bind the external signal that come in the form of hormone or growth factors. Some of those motifs are very rich in amino acid. Other motifs have those so-called immunoglobulin-like loop that one finds in the immune system. And here you have the receptor for the epidermal growth factor, for insulin, for the fibroclast growth factor, for the nerve growth factor, and so forth. And as I said, all of these recognize hormones or growth factor that circulate in the blood, except for the very large family here of the F growth factors. They recognize membrane-bound ligands. And the main function is to help a developing nerve axon to reach its proper target. During embryonic development, the head of the nerve, we call them the nerve growth cone, must explore the immediate, the local environment and make the proper choice to go where they are supposed to go, sometimes over very long distances. And path finding involves very sophisticated mechanisms like soluble, membrane-bound molecules serving as attractant or repellent. In certain cases, for instance, in the optic tectum of the chicken brain, those target molecules arrange according to a gradient of concentration from back to front and top to bottom, forming a sort of a grid, a position coordinate that will guide the axon to where it's supposed to go. And indeed, it's the largest family of receptors and practically exclusively concentrated in the brain. Now, we know now how signal is transduced down from these receptors. In the resting state, the receptors are separated from one another and inactive. Upon binding of a ligand, in this case, for instance, the epidermal growth factor, the receptors associate with one another, and immediately, they are kinases, immediately they phosphorylate one another. And now the phosphate groups that have been introduced can serve as docking sites for the attachment of adapter proteins. By virtue of the fact that these have binding domains, in this case we call that one SH2, that have a very high affinity for those phosphate groups, for each of those different phosphate groups. So the activated receptor recruits those adapter proteins, brings them to the membrane, and the change in conformation in the adapter now unmasks a different set of binding modules. That can recognize the next element of this pathway and then perhaps the next. And this is how in a sort of a vast tinkatory sort of way, the signal can be transduced from the receptor down the cell. And today we know several dozen of those binding modules, and I have illustrated just a few on the next slide. Just look at the color. Forget their name, forget the groups they interact with. Just look at the color. For instance, this one, the SH2, I just showed, its main function is to bind to activated receptor. This one, for instance, binds to the membrane. They bring proteins to the membrane. This one, its main function is to organize or cluster ion channels and other kinds of receptors, neurotransmitter receptors on the membrane. So the complexity of the regulation of signaling pathway is well shown by this adapter protein, Vav. It looks like one of those Swiss army knives that can do anything. With its eight or nine different binding modules, it can bind to a variety of different proteins. It can link several metabolic pathways or mitogenic pathways together. And also, by the way, if you chop off one of its end, it becomes oncogenic. Vav, the gene for that is what we call a proto-oncogene. But this molecule, I think, already tells you something very important we have learned on the structure of proteins is that they all have this kind of modular or multi-modular structure. There are mosaics of different binding modules. They are like necklaces made up of a variety of beads whose choice, whose selection, and whose disposition on the structure of the molecule will ultimately determine the function of that molecule. Coming back to receptors in a very, very oversimplified way, then they can be viewed like the old hand-operated telephone switchboard in which the operator would link a call coming from the outside to the proper recipient by introducing the peg, the plug in the proper hole. And this is why activation of a receptor can result in an immune response, cell, program cell death, oncogenicity, differentiation, proliferation, and so forth. But we don't know how selectivity or specificity is introduced in those systems. Indeed, that view is incorrect because we know that no receptor alone can do all those things. It's only when several receptors can speak with one another can bring about a combinatorial system of responses that they can bring about one physiological event or the other. As you can well imagine, mutations in those receptors will result in pathologic condition of various degrees of severity. And I just want to show you two or three of them. For instance, mutation in the fibroblast growth factor receptor leads to skeletal disorders resulting in dwarfism or cranial anomalies as seen on this picture that I borrowed from a review by Monkey and Shell. And it shows an example of achondroplasia. This is the most common form of dwarfism. Those towering skulls that you see in this Aper syndrome, cruzon syndromes, downslanting and protruding eye due to shallow orbits, and then general facial asymmetry because of the premature fusion of the bones of the skull. Mutation in another receptor, the C-kit receptor, results in a interference with the development of the thymus and certain skin cell population including melanoblasts and melanocytes are giving those patchy areas of hyperpigmentation. And you see the same areas of hyperpigmentation in men. But notice that it also occurs in the mid-forehead. So much for those who still don't believe in evolution. The most investigated receptor mutations are those that involve the insulin receptor resulting in insulin resistant forms of diabetes. And the most severe, the most severe leads to early death and what we call leprechaunism in which the child has those very low set ears, a depressed nasal bridge and then there are severely glucose intolerant in spite of huge amounts of circulating insulin because in a sort of a desperate but futile attempt to overcome the deficiency the organism can produce concentration of insulin a hundred times above normal. But the most dramatic of these receptor mutations are those that lead to oncogenicity. And indeed we know that over expression of several of those growth factor receptors or mutations that would lock them in the active form results in oncogenicity. And the first to be discovered was this V herb B, my three independent groups in an avian erythroblastosis retrovirus and it results from the massive deletion of practically the entire external domain of the epidermal growth factor. In other cases the mutation can be incredibly discreet. The simple point mutation of one amino acid in the transmembrane of a related receptor the new HER2 receptor leading to, it's under the influence of carcinogens leading to the appearance of neuroblastoma in rats and its human homologue has been implicated in the progression of human mammary carcinoma. Again, oncogenicity can result by the fusion of part of the gene of the receptor with part of another gene and this is the case of one of the mutations for the nerve growth factor receptor originally isolated from a human colon carcinoma. In all these cases those mutations allow those receptor to associate with one another and then bind with one another and then undergo unrestricted spontaneous phosphorylation and activation. So you see, even though we know that cancers are characterized by a multiplicity of oncogenic events that together contribute to the advanced form of cancer we do begin to understand now how cancer proceeds. 20, 30 years ago the dream of everybody was to find out the agents responsible for the initiation or development of cancer. We were totally in the dark. Today most of these agents are known. We have characterized the major pathways that lead to tumor geneticities, tumor progression, metastases and so forth. We know their genes, we know the molecules involved and I personally am convinced that sooner rather than later we'll be able to put all this information together and devise the means to control most type of cancers. Now with so much evidence then that oncogenicity results from an increased tyrosine phosphorylation of protein, it's not surprising that any groups including our own became interested in the protein tyrosine phosphatases with the assumption that if oncogenicity would result from an over expression of protein kinases well then necessarily over expression of a phosphatase that would remove numbers would block or reverse transformation. And I'll tell you that unfortunately this assumption was incorrect. That's not the way it works. About 12 years ago a very bright post-doctoral fellow, Nick Tonks and a very able technician succeeded in isolating the first protein tyrosine phosphatase from human placenta. And to our surprise it had no homology with any of the other known phosphatase but a computer search of the database told us that our molecule was strictly related, closely related to a surface antigen already well known by the immunologist. They had called it the leukocyte common antigen because it's present on practically all hematopoietic cell that is blood cells. And it had a big structural relationship with the phosphatase we had isolated like the growth factor receptor that I spoke of before. It's a chance membrane molecule, one single chance membrane domain that separates inside the catalytic activity except that it's a phosphatase rather than being a kinase outside great diversity of structure for ligand recognition. It's not the place to go into the immune reaction. Suffice it to say that when an immune T cell recognizes an antibody presented by another cell this triggers immediately an activation of several tyrosine kinases inside the cell and that results in an explosion of tyrosine phosphorylation in the cell. Within seconds a dozen protein become phosphorylated on tyrosine residue but nothing happens in the absence of this phosphatase. If you use a leukemic cell that lacks this molecule is also called CD45 there is no kinase activation, no tyrosine phosphorylation and no immune response. What this tells you is that you need a phosphatase to activate the kinase that trigger the reaction. Since then a great variety of receptor linked protein tyrosine phosphatases have been isolated and they all have the same general structure. The catalytic activity inside and a variety, a mosaic of motifs outside. But what was absolutely surprising is that all those structural motifs have all the elements have all the structural characteristics of cell adhesion molecules. In other words, if in a cell the growth factor receptors can be represented by those kind of molecule that responds to external signals no circulating ligand has been found for the phosphatase receptor which means that they have to participate in or be regulated by cell-cell interaction or cell matrix interaction. They are cell adhesion molecules with a very exciting possibility that they might be directly involved in what we call contact inhibition. Many of you probably know that if you grow cells in a dish they will grow if they have a solid support on which they can adhere and provided with a number of growth factors and they will grow until they reach confluency. The moment the cells touch one another, growth is arrested by contact inhibition. Only cancerous cells will continue to grow and this is why they can grow one on top of the other and form tumors. So this is a characteristic of oncogenic cells. Their cell represents the changes that cells undergo when they no longer abide by the restrictions under which normal cells must operate. They don't need a solid support to grow, they will grow. On soft media they don't need growth factors and they are no longer contact inhibited. And there is the possibility, exciting possibility that one of the functions of the growth factor phosphatase is to act as oncogenic compounds by maintaining contact inhibition in cells. Just rapidly, those are the intracellular tyrosine phosphatase. They all have a great diversity of structure either preceding or following a highly conserved catalytic core with phosphatase activity. A very interesting enzyme is this one, a yoke enzyme. Jack Dixon at Michigan has shown that this phosphatase is responsible for the very powerful virulence of bacteria of the genus Yersinia. And this includes Yersinia pestis, the agent responsible for bubonic plague, the black death that wiped out a good proportion of the human population in years past. And Jack has shown that the pathogenicity of Yersinia is due to this phosphatase because if he replaces it by a dead enzyme, the bug is no longer virulent, no longer pathogenic. Now the interesting thing is that Yersinia itself has no tyrosine kinase and no tyrosine phosphorylation. Therefore, this phosphatase serves absolutely no internal purpose. Its only mission is to go into the host cell and trigger a catastrophic set of tyrosine phosphorylation that obliterates the immune defense of the cell. Okay, I want to stop here and just conclude. The data that I gave you, plus others that I didn't have time to describe, tell us now that phosphatases cannot be viewed as the off switch in an on-off kinase phosphatase system. They are not there simply as scavenger enzyme to remove the phosphate group introduced. In certain cases, they can act synergistically with the kinase to increase the kinase activity. In other cases, they can act in the reverse. The difference whether they act synergistically or negatively is not due to the activity, it's due to where they localize in the cell and how they are regulated. Secondly, as you have seen, the structure of the phosphatase, particularly the transmembrane phosphatase, are so different than those of the kinase that they must have a mission of their own in controlling cell-cell interaction. And thirdly, and perhaps the most important, most importantly, I mean, where do we go from there? What has to be now still discovered in this field? What can we expect in the years to come? We have identified, I think, the major mitogenic pathways in the cell and characterize the molecules involved. But clearly, those molecules are only the words that cells use to carry out their daily chores. We know many of those words. We know bits and pieces of phrases that they spell out to bring about a given response. We don't know the language that has to be spoken among receptors, among pathways to coordinate all the reactions that take place, to introduce the specificity and selectivity that cells require. Furthermore, through more than 3 billion years of evolution, cells have had all the opportunity in the world to introduce this vast array of secondary pathways and parallel pathways and shunts and fail-save mechanisms and so forth. They require to regulate their growth and to protect themselves against all kinds of adversity and, as I said, to program their own death when the time comes. So we don't know the cross-talk that the cells have to use to sort out all those reactions. And much more importantly, we don't know the cross-talk that cells have to use to communicate with one another, to synchronize all their reactions. And this cross-talk, this inter-cell communication has been crucial for the establishment of those very sophisticated networks of communication when encounters, for instance, during embryonic development, during organogenesis in our immune system, in the infinitely more complex central nervous system where more than 1,000 billion cells speak with one another through more than a million, billion synapses leading ultimately to the establishment of thought and memory and consciousness. And solving this problem will be one of the major challenges that will confront the biologists in the years to come. Thank you for your attention. We'll be having our Q&A here in just a moment. So if you'd like to write a question for Dr. Fisher on the card, pass it to the center. We'll assemble our panel here in just a minute, and then we'll begin our discussion. So if our panel would join us up here, we can get started in a few minutes. Survey, or do you still want to meet? I'll do it right now. Okay. Is Dr. Bobel here? Well, we should probably begin. Thank Dr. Fisher for that marvelously elegant description of some incredibly complicated processes. Let's once again begin by asking members of our panel if they'd like to comment on the talk or if they have specific questions that they'd like to raise. Dr. Maddox. If I might ask a question of Dr. Fisher, what is this? ATP, he mentioned, is the currency of energy that transports in cells. Phosphorylation is crucial to activating proteins. Phosphorus obviously plays a very big part. This mechanism obviously goes back a long way. You mentioned, Professor Fisher, that multicellular organisms began 500 million years ago, or terabytes. But how far further back does the phosphorylation go? Can anything be said? Can you guess even, letting your hair down at what role this had in the very early stages of life? You don't find tyrosine phosphorylation in bacteria, but you find it in archaebacteria. You find, of course, regular phosphorylation in bacteria. People have been wondering, since the original Earth was in a reducing atmosphere, whether or not rather than having phosphate, they had phosphites, perhaps hypophosphite. Paul Boyer, whom you mentioned, was worried about that at some time, but no phosphitease had been found. I don't know at what time phosphate became the main transporter, let's say, for changing conformation in proteins. As I say, we find it in bacteria. We find it in plants, of course. I don't know when it originated. People wonder why phosphate. It's abundant, it's very stable. To be more specific about it, would you guess that phosphorus was a component of the very first self-replicating organisms? Whatever they were. I wish I knew, John. I don't know. I don't know when it first was introduced. For instance, carbohydrate metabolism I think existed very early. And sugars, for instance, in glycolysis and the Krebs cycle, come in the form of phosphate derivatives. And this is why when we first found that reaction we thought it might be restricted to carbohydrate metabolism. We wondered if nitrogen metabolism might not be regulated by anodation, amination, fat metabolism, acetylation and things like that. Dr. Hoffman. There's just a comment on this which I think might be interesting to people. That is, if you have a choice of chemical reactions to choose to construct some complexity in a system, do you choose, and you have the workings of evolution, of selection, do you choose something which gives you a lot of little variation, or do you choose something that's very big? That is, we're all of a sudden 10 kilojoules or 30 kilojoules are pumped in. I think one reason the phosphorylation system was chosen is because it's tunable in small amounts. That is, you can, by modifying the things, you can have the workings of evolution optimally so you can build anything that you want. And that's true, I think, throughout a lot of the biological systems with a few exceptions, like photosynthesis, you have to get in a large amount of energy in one jar. And then, all of a sudden, but then you reduce it in all kinds of 100, 200 chemical reactions, you tune it and you get it out in little steps. But it, by and large, things that are variable, tunable over small amounts are chosen by the workings of evolution to be the components of biological systems. Phosphate has the big advantage that its bond is very stable. I mean, we store all our energy in ATP. Why not acetic and hydride? It's much more, it's much more higher energy except that it hydrolyzes immediately in water, whereas ATP is stable for at least a year if you keep it at that neutral pH or more. So this is why we bank our energy in the form of ATP rather than something else. Anyone else? I'll take a question from the audience here. Are we at the stage at which tyrosine phosphorylation can be prevented in prophylaxis versus cancer development? Not that I know of. It's a very general mechanism. At first we thought it was a mechanism that was restricted to cell transformation and differentiation. Now you find it in every system. It wasn't found because it represents no more than a tenth or a hundredth of a percent, 0.01 percent of phosphorylation on serine and threonine. This is why it was so difficult to find out. Yeah, people are, as you can imagine, working like crazy to try to find inhibitors of tyrosine phosphorylation or inhibitors of the tyrosine kinase. There's something very interesting and perhaps disturbing that happened very recently in the last few months. An excellent inhibitor of a kinase that is responsible for chronic myelogenous leukemia, CML, the oncogenic agent is called BCR-able. Able is the tyrosine kinase. And the excellent inhibitor of this tyrosine kinase was found, which really blocked specifically the activity of this oncogenic product, BCR-able. Within less than a year, cells have become resistant to that inhibitor. And it's enormously disturbing. I mean, you can imagine that bacteria would mutate in such a way that they become resistant to antibiotics to find out now that mammalian cells can resist that. And they found out that what happens is that there are some mutations. There are some mutants. A single group is mutated and it's a group that forms an important bond with the inhibitor and therefore the inhibitor doesn't work anymore. And probably those forms, those mutated forms exist in all the cells, maybe one per thousand. And you don't know them until all the normal cells are wiped out and then the resistance cell take over. So of course now one has to find ways of changing those groups. It's always you find something that's in advance and then it's blocked by another reaction. So you have to find another advance and it's blocked by the next reaction. I have another question from the audience here. If we have only a rudimentary understanding of the proteomic responses to a cell's genomic makeup, is it not risky to introduce foreign genetic material into a cell vector or a viral vector? It is always risky, but it has to be done under certain conditions. All you have to do is to pay enormous attention, introduce all the safeguards you can. But because of the risk you cannot stop this approach, this technique. And by the way, on your list, Tim, on the list that you had, you didn't put genetic engineering, which in my opinion is one of the greatest advances. I was going to ask people who have taken the survey if they want to change their vote after hearing Dr. Fisher's talk, the phosphorylation. It's still possible to do that. We have another question here. Would it be possible to eliminate or neutralize the receptor that tells the cells to die? Would it be possible to prolong life through a process similar to this? Well, no, cell death has not much to do with aging. Cell death, apoptosis, as we call it, is a way of getting a ring rid of a cell without letting it break open so that it would spill all its proteolytic enzymes and create havoc. So during apoptosis, the cell that is designed to die will auto-digest itself without destroying the cells around it. On the contrary, in cases of cancer, they wonder if they can trigger apoptosis, specific apoptosis of cancerous cells so that the cells would destroy themselves and people are, as you can imagine, working like crazy in those directions. Where is there a multitude of cancers? Are there just a few common mechanisms for cancer growth and metastasis? Can cancer therapy focus on a common spreading mechanism of some sort? No, this is really not my field. I'm no expert in cancer, but there are many, many forms of cancer. There are many, many oncogenic agents. As I said, many of the growth factor receptors have the possibility of mutated or becoming cancerous, and this is where all their genes are called proto-oncogenes, genes that under certain circumstances can become oncogenic. So as I said, the cancers require multiple oncogenic agents that collectively work together to allow for the progression of various forms of cancer. Unfortunately, it will not happen like you see in the movies where you have the mad scientist with the test tube and then some white fumes coming out and he said, I have found a cure for cancer. That will not happen. But when one tries to block specific types of cancer, in fact, there have been already some very successful ways of controlling certain kinds of leukemias and so forth, very successful, and people are trying cancers that are triggered by the mutation of a given receptor. They try to target indeed that receptor, find inhibitors that would inhibit that receptor and not the others. You can't block everything. So people are working. It's a very sexy topic for biotechnology companies and for investigators if they want to get money from the NIH. All right, here's a question that comes up occasionally. I'm concerned about the lack of female role models for young women in the audience. Tell us about women doing important work in this field. Oh, in my particular field, there was Gertie Corey, the wife of Carl Corey, and she was a tremendous investigator. She died unfortunately very, very young of Hodgkin's disease, but she was a superb investigator. Oh, there are many. I could give you a long list of people who are very able to first-last investigate this. Dr. Hoffman. I have a question for our protein guys. First, we had the genome at the center of attraction, and now it's the proteome. Now, part of that is that science creates new questions as it answers others. Part of that is words of hype. And do those come externally? Do they come from the media, from the scientific media? Do they come from the workers themselves? How do you feel about the succession of these words, you guys? You mean the word proteomics? I mean the effects of that word on the shift. I mean, why didn't we hear about the proteome so much before? They see the light. No, indeed, there are some organizations that try to do with the proteome what Celera has done with the genome. They try to identify all the proteins around in one system, for instance in the plasma. Some of those companies, I can tell you, are incredibly well-equipped. I know one. It has no less than 52 mass spectrometers, six-mile detox instruments that work 24 hours a day. They have computers larger than the one Celera had to sort out the human project. So they are working with the hope of discovering new proteins that might be targets for drugs. So yes, this is the direction in which it is going, but the huge difficulty is, I don't know how many proteins there are, leaving out those of the immune system. I said in the immune system, those reorganization of those V, D, J genes that control the immune and where you can potentially make something like 500 million different proteins. Leave this out. How many other proteins you have for the usual working of the cell? I don't know what it is. My guess is probably around a million and coming 500,000 to a million different proteins due to all the modifications, due to the alternative splicing and so forth. Many, many of all those receptors, they exist in several forms. Some are as I showed with an external domain and internal. Most of these are also processed. They are split. So you have the inside without the outside. And several of those receptors can exist in three, four different isoforms having different properties. So this is one gene and you have five proteins. Yes, John, you probably... Can I ask another version of this slightly skeptical question? It is this. You talk with us for sure of there being 100,000 proteins at all. Many other variables affecting the life of the cell. It just were a problem in physics. It was a problem like the problem of predicting the climate by coupled atmospheric and oceanic models. People would build models. Hasn't the time come when this very, very complex bit of chemistry, cellular chemistry were made into a model too so that we would have a chance not simply of understanding it better but also telling which bits of the explanations that one gives are incomplete. Is it a fair question? It is. I think it's a very fair question and I agree with you. I think it should be asked. The problem is that even in E. coli very simple cell, half of the proteins, the function of half of the proteins is not normal. So you are limited in your model building. And it's even worse, of course, in the human genome because of the eternity-displaced forms and many, many other complications. In answer to Waouge, term proteomics is a recent creation but people have been excited about proteins for a very long time and it's just that the event of getting the human genome sequenced put the genome on the platform for a short while and now it will stay there for a while but proteins will always be very important. Do we have any other closing comments? I remember a conversation with my colleague the other day I was telling him about proteomics a bit and what we could expect to hear and he said, well, this reminds me a bit of the movie The Graduate in 1960 where at the cocktail party the guy comes up to the young graduate and I have one word for you in the 60s, plastics. In the next millennium it's going to be proteins. Alright, well, thanks to all of our panelists here. We will have our next talk. Let's have a round of applause. We'll assemble once again about 1245. Our next scheduled talk is from Dr. Stanley Prusner at one o'clock.