Welcome to FOCUS In Sound, the podcast series from the FOCUS newsletter published by the Burroughs Wellcome Fund. I’m your host, science writer Ernie Hood.
One of the keys to ultimately conquering disease and extending the healthy lifespans of human beings is going to be to advance and refine our understanding of the machinery of living cells.
My guest on this edition of FOCUS In Sound, Dr. John York, has done as much as any scientist in the last two decades to contribute to knowledge of cellular processes, particularly in the area of inositol signaling, which has emerged as a critical factor in the regulation of a wide variety of biochemical phenomena, including calcium release, membrane trafficking, channel activity, nuclear function, and more.
John holds a dual appointment at Duke University, where he is a Cancer Biology Professor of Pharmacology and a Professor of Biochemistry. He is also a Howard Hughes Medical Institute Investigator. In 1995, he was one of the original recipients of the Burroughs Wellcome Fund’s Career Award in the Biomedical Sciences.
John, welcome to FOCUS In Sound…
Thank you, Ernie.
To begin, please give us a broad overview of inositol signaling – what is it, and why is it so important to our understanding of cellular mechanisms?
I think I’ll start with the idea of a fundamental problem in cell biology, and that is to understand how signals are interpreted by a cell or an organism. So if the outside of the cell gets a signal, how is it that it transfers or transduces that signal across the membrane, and then is decoded and expanded within the intracellular machinery? And one of the most widely activated, if not the most widely activated intracellular communication pathways is the inositol signaling cascade. And it is a pathway that we believe is combinatorially complex; it is a pathway that involves more than one species of inositol, and if you consider the idea that there is an inositol ensemble or code of species, then by getting different patterns in response to different signals, you could enhance the signaling complexity and specificity of the cell or the organism. So it’s a very wide-ranging, widely activated pathway. We believe it is centrally important, not only at the level of the cell, but also at the level of the development of the organism.
I see…So this really is a classic example of, the more you look at something the more complicated it becomes, isn’t it?
The more we know, the more we don’t know. So we started out knowing something, as you mentioned, about calcium signaling. It was learned early on that calcium was one of the major effectors of the pathway, so you turn on the signal in response to a photon of light, in response to a growth factor, in response to a hormone, and you turn on this wonderful expansion of signaling within the cell to release calcium. We now know that the inositol code is much more complex than just that one signal that generates calcium. So it’s been our mission to try to identify what these other molecules of inositol species might be doing, and that’s where we’ve made the most progress.
And that’s a great deal of work involved, because as I understand it, originally the thought was that there was one species of inositol and it did one thing, but the picture has gotten much larger.
I think early on, people try to simplify a process. They want to understand a component of it so that they can find a paradigm, and that paradigm has been extremely useful. In fact, second messenger paradigms have emerged from a number of things, beyond inositol signaling. For instance, cyclic AMP is a critically important activator of cell signaling and the protein kinase A family. So by taking these small molecules that emerge in response to an extracellular signal and generate them inside the cell, the paradigm is there to try to say, OK, well, if there’s more than one or two of these species, then you can start to enhance complexity exponentially. And that’s what really defined our quest to understand what these other species were doing, and so that’s what we’ve spent most of our time on.
I understand, John, that you’ve also discovered that cells also use inositol codes for regulation of proteins. Why was that of such great interest?
Obviously, once you identify a pathway, you want to know what it might be doing. So we’ve taken an approach where we actually take away the enzymes that make and break down these new codes, and then look in a cell or an organism to identify whether or not these actually are important for some process within the cell. So with respect to proteins, one of the most amazing things we’ve discovered recently is that the inositols actually bind to proteins in a way that goes beyond allosteric control. That is to say, if you imagine a switch in a cell, and you take a molecule and you generate it. It touches some kind of receptor and it changes the function…what if that receptor actually depends on that metabolite to actually fold or to be stable within the cell? In that way we think that the inositol code could then change as a cell differentiates, so in one pattern you might have a hand that has four fingers held up; in another pattern of code you might only have your thumbs sticking out, and it’s that change in patterns that could then lead to a very different set of interpretations within the cell’s machinery. So obviously a major question we still want to understand is: how is this signal decoded? What’s the decoder ring? And that’s where we’ve made some very interesting progress.
John, how did you originally come to be interested in and ultimately specialize in this particular branch of biochemistry?
I was an undergraduate at the University of Iowa, and then I did my graduate work with Dr. Phil Majerus at Washington University in St. Louis, and Dr. Majerus has long been interested in how blood cells undergo changes. So during hematopoiesis, when a blood cell differentiates, or upon exposure of a platelet to something like thrombin, there are signal transduction cascades that are at play, and one of the most important pathways in those processes is the inositol pathway. So that’s what I chose to study as a graduate student, and it hooked me from the very beginning. My training is in biochemistry and molecular biology, and so it was very natural for me to want to understand the enzymes that regulate the production of inositols within the cell, and so what we did then is when I started my lab at Duke, we wanted to use a genetic system such as the budding yeast Saccharomyces cerevisiae as a way to expand on that. And we expanded on it by taking those new genes that we identified, the new kinases and phosphotases that manipulated the inositol code, and we started deleting them and gaining their function within cells. And that’s where some things really opened up.
So how does this work, which as you mentioned mainly takes place in a yeast model, translate into insights into human biology?
What we believe is that of the 6,000 genes, not we, the field believes that the 6,000 genes that are found in the yeast genome, and this is baker’s yeast, we use it to make bread in our kitchens, right? This is laboratory reagent now. We know the sequence of the genetic code of yeast, and when we look at that sequence, we know there’s about 6,000 proteins made. Of those 6,000, about 98% of them are also found in humans. So model organisms are viewed to be in some ways a simplified version of the human cell, and because you can genetically manipulate yeast in a way that is much more efficient than a human cell, by learning something about that through a simplified model, we can then translate it into a human cell, or into a mammalian cell. So in our case what we’ve done is, anything we’ve discovered in yeast, we immediately then begin to look at it in the fruit fly, Drosophila melanogaster, which is also a very easily manipulated genetic system, but has the advantage that if you sacrifice a few fruit flies, the world may be a better place, actually. So there’s not the stigma related to genetic manipulation. And then we also use a mouse model system. So we try to use the different systems to identify the core, or the most fundamental aspect of it, and surely there are differences between the way signaling occurs in yeast and the way signaling occurs in humans, but we think that there is a fundamental aspect that is shared.
So how might these discoveries in this particular field potentially translate into implications for human health?
We already know that there are some major connections to human health in the case of inositol signals. So part of this code, for instance, is a lipid code. These are codes that are inositol phosphates that are generated on the membrane of the cell, the inside of the membrane of the cell, and when those codes emerge, it tells the cell to grow or to survive. And so one of the major players in the pathway is the PI 3-kinase signaling cascade, and one of the most frequently mutated genes in cancer are mutations in either the tumor suppressor p10, which is a phosphatase that’s like a brake of the system, so when you mutate the brake and turn it off, you’ve now got a runaway train. This train is just continually telling cells to survive and to proliferate. And likewise, in human cancers there are mutations in the kinase, the accelerator pedal. And instead of inactivating the accelerator pedal, they actually push it to the floor, they tell it to go even faster. And these kinds of mutations are probably found in anywhere from 20-80% of the human cancers that we know of today. So there’s already a precedent for inositol signaling pathway perturbation and human disease, and in fact we wrote an article on a series of phosphatases that are part of the lipid cascade that are involved in human disease, and that was done recently with Phil Majerus, who’s pioneered many of the lipid phosphatases. And it turns out that not only cancer, but there’s defects in oculocerebrorenal syndrome or Lowe syndrome, there’s myotubular myopathies, Charcot-Marie-Tooth syndrome…So it’s interesting that changes in these inositol lipids can really lead to disease states, and in many cases it’s mutations in the brakes of the system, the ones that sort of attenuate those signals, that give rise to disease. And part of that is because the kinases, if you were to lose their function, the organism cannot survive. So we believe that by studying the new pathways that are downstream of these lipids, such as the inositol phosphates, which are water soluble molecules like IP-3 that are free to move about the cabin of the cell, these molecules may play equally important roles in the regulation of cellular processes that when perturbed would lead to human disease.
So ultimately down the road at some point—I know the research right now is very basic, but as things progress, could some of those molecules be targets for therapy?
Absolutely. We know in a developmental situation, so if we knock out some of the kinases or phosphatases that produce these signals, that disease states occur in the animal. So what we’re hoping someday is that someone gives us a phone call and says, I just mapped the disease gene to disease X, and it turns out it’s one of the kinases or phosphatases that you’ve discovered. And then the wealth of information, the basic science, curiosity-driven, discovery-based information that we’ve done will then automatically fit into place to understanding mechanistically how we might target this pathway as a therapeutic way to overcome the disease.
So you’re kind of preparing the roadway…
We are hoping we’re laying a nice foundation for that. It is not the driving force, but having worked at Merck before I went to graduate school, I feel very strongly that medically oriented research is extremely important and the NIH’s main mission. And so having funding from the NIH, we’re obligated to try to look for those windows. But we can’t manufacture those windows when we don’t know what we’re really doing. And so the first step was to actually start figuring out what are the players in this system. It’s a totally new system. We had no idea ten years ago that these molecules really existed or how were made. Now, we know how they’re made [and] the processes that they might regulate, so this is the beginning steps to, as you point out, putting the road down. We’re putting the gravel, we’re laying the sand, and maybe at some point the concrete and asphalt will actually provide us a very nice venue to human disease.
Before we move on, John, I wanted to return to the concept we’ve been kind of touching on, about, as you put it, curiosity-based research, very basic research as opposed to necessarily applied, medical targeted research. Elaborate on your thinking about that for us…
I think it starts with the idea that many, many basic discoveries that were made led to very, very important medical discoveries, and I’ll use a colleague of mine as an example, Paul Modrich, who’s also a Hughes Investigator here at Duke. He was studying how bacteria undergo mismatch repair, and basically when their DNA is damaged, there is a mechanism to repair that damage. It turned out through studying bacteria and the basic fundamentals of a very important process of DNA damage, that it turned out that these were genes—the mut genes, the mutS and mutL genes—that turned out to be very important for our understanding of mutations in cancer, that there are many tumors that have mutations in mutS and mutL so that they cannot repair their DNA. That leads to the genetic instability or the genomic instability that causes the disease. Without that basic, curiosity-driven research about understanding how cells respond to DNA damage, we wouldn’t have the therapeutic targets that we have today.
And so, as a basic scientist, I love applied research. I think it is fantastic. When I was at Merck I worked on vampire bats, because if a vampire bat feeds on a cow the wound bleeds for six hours, so we knew that there were powerful agents in the saliva that would be potentially very interesting therapeutic agents for blood disorders. So natural products and applied research, I think, is fantastic. But for me, I love to solve problems, and for that, the clarity of being able to follow our nose, to be curious about certain problems, and to be satisfied that we don’t have to have a disease to drive our research…we’re basically looking to understand systems and problems, and by doing that, that’s where we’re headed.
And the rest will flow…
That’s the assumption, but it’s not like this magically happens. Someone else has to make a connection. So we need help. A basic scientist cannot exist in a vacuum, and then lead to medical discoveries. I think you definitely need all types of research, and I think a place like Duke or many of the medical centers around have that combination. So it’s important to get curiosity-driven researchers to talk to medically oriented or applied researchers, translational researchers. And it’s not to say that in my lab if we ever did discover that a gene was involved in a disease state, we’ve done chemical screens, we would find drugs. We would love to do that. I mean, tomorrow I’m not going to change the focus of my lab just so I can do that, but I feel very privileged that I’ve been able to follow our nose through basic science questions and funding.
Well John, I did want to spend a few moments with you on a subject that I know is near and dear to your heart—and that is career development for young scientists. You actually took a non-traditional route to becoming an independent investigator…tell us about the career path you followed…
Well, it’s not the norm, let’s just say that. So what happened in our case, I met my wife Sally as an undergraduate in the biochemistry department at Iowa. The year I graduated, the day after it, we got married and a year later our first daughter was born. So that changed a lot of the conventional path. And so what I did is, I went to look for a job, and got a job at Merck, Sharp, and Dohme labs in Pennsylvania.
So you couldn’t afford to starve for a few years, you had to make a living.
It was a choice – you’re now responsible for a life. So it was a very easy decision to say that we were going to have to put our careers on hold for a little bit to get some money, and it turned out to be one of the best things that has happened to me. Going to Merck was one of the most amazing experiences of my life. Steve Gardell was my PI in that case, and he was inspiring. So I viewed going to Merck as sort of my Ph.D. thesis. I worked to discover a number of things involved in how vampire bat saliva might function to regulate fibrinolysis, which is a process by which clots are dissolved. This is now something that is being used in patients. And we also did some very basic science work, so that’s where I got this notion of doing the core fundamentals of understanding mechanism. And when my wife applied to the MD/PhD program at Wash U [Washington University in St. Louis] in 1988, about a year-and-a-half after we got to Merck, we had to make another life choice. So she took a year deferment, we then went to St. Louis in 1989. I didn’t know what I was going to do, I really liked industry, I liked the applied research. So I applied for two positions, one at Monsanto, and one with Phil Majerus, who turned out to be my advisor for graduate school. And Phil Majerus hired me, and I decided to go there instead of Monsanto, and it was another one of those terrific decisions. And it was through that and the work of a graduate student that things worked very well. I started my graduate work in 1990. I defended my thesis in October of 1993. I had done enough work that people thought it was really very, very interesting, so I spent about a year-and-a-half as an independent post-doc, and then eventually when I won the Burroughs Wellcome Career Award to transition to faculty, I got a junior appointment for about a year at Wash. U., and then I started looking for jobs at Duke and came to Duke in 1996. So I never did a conventional post-doc. But I had a lot of support. Phil Majerus and Stu Kornfeld at Wash. U. gave me what was about a 500-square-foot lab space, and with the money that we had from Burroughs and some money from a license agreement with Merck, I started a lab, hired two technicians, and that’s when we started our yeast biology…and the rest is history.
John, what do you see as the major career development challenges facing young researchers today, particularly given the perspective that you look at it from?
Well, I think that a challenge is that right now the funding environment is really diminishing the enthusiasm for a number of folks. What you have to have is a positive atmosphere that makes trainees believe that when they finish their work, they’re going to have a place to contribute. And so one of the things that I’m very concerned about right now is the notion that we are losing a generation because of the funding situation that we have. It makes it very difficult to keep morale up when our trainees are not able to find the positions that they’re looking for. So I think that’s a major challenge.
But from the perspective of creative science, I think because of some of the initiatives in funding agencies that are so medically relevant, sometimes I think it takes away that curiosity-driven research that we all like to do, and I’m also very concerned about the funding of basic science. The R01 grant is a very important component; it has been for many, many, many decades, and it’s getting harder and harder for people to get the R01 grant. There’s a lot of initiatives that are collaborative; these are terrific, but let’s not lose the R01 grant.
So, and then I think in terms of mentorship. Our trainees are extremely talented. They have reagents and tools and technologies available to them that we did not have. And the fields move faster. It’s amazing how there are no islands any more in science—people are working on things together. So our trainees really do have to have a sense of team spirit, but they have to maintain their individuality as well, and I’m concerned that we give them opportunities to maintain that individuality.
Do you think the path to independence today is easier or more difficult than in the past?
The favorite saying of scientists is, “well, in my day we had to walk uphill and downhill to work.” I do think that there are things that make it harder right now. The funding situation, the morale, the idea that there are many more conflicts of interest, it changes the way we do our business. But part of adaptation and the ability to survive is that flexibility, being fleet-footed, and I think that institutions that will thrive in this environment—these are opportunity times as well. We’ll make good choices, and that’s going to be very, very important. So I don’t think it’s harder now that it was. I mean, there’s no question that morale has changed, but I think that our best trainees are still going to be able to find incredible opportunities.
John, what pearl of wisdom would you share with our listeners who are early in their careers?
Let me tell you that for me, one of the most important things was something my dad taught me, and that was the key to inventiveness and problem-solving was to first identify a problem, and then come up with eight different ways to solve that problem. And I think that when you take multiple approaches to a problem, and they converge, it gives you a scientific confidence that you’re on the right track. So my piece of wisdom for anybody starting a career in this business is to converge on a decision or a discovery through multiple routes. It helps give you confidence that what you see is real. And it’s that ability to prioritize—there’s way more experiments to do than there is time in the day or people in your lab to do them, so you really do have to prioritize. So for me, it’s a lesson from my father, and it’s been one that we’ve tried to adhere to for all these years.
John, it’s been just fascinating to learn about your work and your professional philosophy, and we certainly wish you the best of luck for continued success…thanks so much for joining us today on FOCUS In Sound…
Thank you, Ernie.
We hope you’ve enjoyed this edition of the FOCUS In Sound podcast. Until next time, this is Ernie Hood. Thanks for listening!