Reversible Protein Phosphorylation as a Regulatory Mechanism • iBiology

00:00:06.25 My name is Ed Fischer, and I'm a retired professor of biochemistry
00:00:10.29 at the University of Washington. Today I would like to give you
00:00:15.04 a sort of rapid historical overview of the discovery of reversible
00:00:20.24 protein phosphorylation as a regulatory mechanism.
00:00:25.24 Now this came about in the course of a study that
00:00:30.17 we had undertaken with my colleague and longtime friend,
00:00:35.00 Ed Krebs, on glycogen phosphorylase 60 years ago. The
00:00:42.11 original reaction we described was really embarrassingly
00:00:46.27 simple. And nobody would have paid much attention to it
00:00:50.25 if it didn't turn out to be absolutely crucial for the regulation
00:00:56.02 of cellular processes. On the other hand, it's fair to say that when we
00:01:00.26 came up with it, it came as a total surprise because in those days
00:01:05.22 one knew essentially nothing about cell and enzyme regulation.
00:01:11.15 The prevailing idea was that enzymes were regulated
00:01:17.24 by the rate at which they were synthesized and then degraded.
00:01:23.12 And that stood until the late '40s and early '50s, when it was
00:01:28.14 realized that this couldn't be the case. That cells had to have
00:01:33.10 ways of manipulating the activity of their enzymes once they
00:01:38.12 had been produced and liberated inside the cell. And that
00:01:43.28 was really one of the big problems that faced the biologists in those
00:01:49.18 days and that was the problem that we decided to tackle
00:01:53.15 with Ed. We wanted to know how the activity of an important
00:02:00.00 enzyme called glycogen phosphorylase was regulated.
00:02:05.08 It was very important because it was already known to catalyze
00:02:10.27 the rate limiting step of carbohydrate metabolism. The first step in the
00:02:16.06 degradation of glycogen. And furthermore, it was under complex
00:02:21.08 hormonal regulation, being activated by adrenaline and
00:02:27.25 inhibited by insulin. When first discovered in the early '30s,
00:02:35.22 by Yakov Parnas in Poland and Carl and Gerty Cori in the United States,
00:02:42.07 the muscle enzyme was known to be totally inactive in the absence
00:02:48.05 of AMP, adenylic acid. And automatically, the Coris assumed that
00:02:54.29 AMP served as a coenzyme, and that stood until about '43,
00:03:02.11 when Arda Green and Coris crystallized the native form
00:03:09.01 of the active form of the enzyme. But she crystallized it in a new form.
00:03:17.22 In that it was fully active in the absence of AMP. So they called that
00:03:26.24 form phosphorylase a and logically assumed that it had to contain
00:03:33.26 AMP bound covalently to the protein as a prosthetic group.
00:03:39.09 And furthermore, they assumed that that had to be the native
00:03:45.09 form of the enzyme in muscle, because when muscle extracts
00:03:49.26 were left standing, it was rapidly converted to the old form
00:03:55.11 that they now call phosphorylase b, the form that required AMP
00:04:00.25 for activity. But they could show no formation of AMP in the reaction
00:04:08.10 and furthermore, they could show no AMP in the native
00:04:13.21 enzyme. So they were stuck. The Coris knew that phosphorylase
00:04:20.15 existed in two forms, active and inactive, but they did not
00:04:27.16 know in which way those two forms differed. And as strange as it would
00:04:33.22 seem today, they actually dropped that problem. Ed and I became
00:04:40.26 involved in that problem when we found ourselves in the same department
00:04:47.16 in Seattle. Ed had been a postdoc of the Coris and had some
00:04:54.05 hesitations to work on that problem. In those days, it should be said
00:04:59.29 that students and postdocs when they left the lab did not
00:05:04.08 work on the problem of their mentors. But then he reasoned that he had left
00:05:10.14 St. Louis five years ago and it should be permissible for us to work
00:05:15.25 together on that problem. Okay, so we cleared the bench in the lab and started
00:05:21.28 working, just the two of us side by side, much more like two friends than
00:05:26.26 like two colleagues in the department. And the first thing we had to do
00:05:31.28 of course was to purify phosphorylase, get the pure enzyme,
00:05:37.25 by Coris' classical procedure. Now Coris' procedure consisted in
00:05:43.26 taking rabbit muscle, grinding it up, extracting the hamburger in
00:05:50.11 water, throwing the whole thing in cheesecloth, squeezing
00:05:55.08 the cheese cloth, and then clarifying the very turbid
00:05:59.27 extract by passing it through a battery of filter papers.
00:06:05.00 And of course the filter paper became clogged up right away,
00:06:09.01 so with a glass rod, they would punch a hole and go to the next
00:06:12.24 paper, and then the next one. It wasn't unusual for them to use
00:06:20.07 maybe 15-20 filter papers for one purification. The method
00:06:26.25 was totally archaic and in fact, here's a picture of our lab.
00:06:33.21 So, we decided with Ed, enough with that jazz. Let's replace
00:06:40.13 paper filtration by a centrifugation. In defense of the Coris,
00:06:46.02 it should be said that in those days there was no good
00:06:49.10 lab centrifuge. But then, when we centrifuged our extract,
00:06:54.07 we could never obtain active phosphorylase a, only the
00:06:59.28 so-called "degraded b form." Until finally, in desperation,
00:07:05.17 we said, you know we have to go back to Coris' procedure.
00:07:09.22 Following it to the letter, paper filtration, and all. Measuring
00:07:14.17 every step to see what's going on. And to our immense
00:07:20.11 surprise, this is what we found. That the very first extract from
00:07:26.13 the muscle, which should have contained phosphorylase a,
00:07:29.15 contained the inactive form of the enzyme. And then when it passed
00:07:34.28 through the filter paper step, it was converted to active
00:07:39.24 phosphorylase a. Speaking of a let down would be to speak
00:07:48.01 it easily, because paper filtration as a method to activate an enzyme
00:07:53.12 was really the pits. Pretty below the worst. But, it soon happened
00:07:59.26 that it was not the filtration perse that did the trick, but
00:08:04.05 the fact that all filter papers in those days were contaminated
00:08:09.09 by calcium ions. And enough was picked up during those multiple
00:08:16.16 filtrations to convert inactive to active phosphorylase. And then
00:08:22.08 quite rapidly, we found that ATP was always present in crude muscle
00:08:29.17 extract was also needed. And that strongly suggested that we
00:08:33.19 were dealing with a phosphorylation reaction. Of course we didn't know what
00:08:39.03 had become phosphorylated, because in those days you had
00:08:42.09 no ATP 32, you had to synthesize the damn thing yourself.
00:08:49.03 But we knew that Art Kornberg in St. Louis had made some for his
00:08:54.11 beautiful studies of DNA synthesis. So we called Art and he
00:08:58.23 right away sent us a sample of gamma-labeled ATP 32,
00:09:03.19 with which we could show that radioactivity was incorporated
00:09:08.15 in a protein fraction, which we could isolate and which turned out
00:09:14.07 to be phosphorylase. So then for the first time, we could then
00:09:19.14 suggest that the activation of phosphorylase occurred like so.
00:09:24.15 That the inactive enzyme was converted, was phosphorylated,
00:09:31.22 converted to the active form in a reaction that required calcium,
00:09:37.15 magnesium, and ATP, and an enzyme of course, which we called
00:09:42.05 phosphorylase kinase. And then the reverse reaction happened to be
00:09:47.20 catalyzed by a phosphorylase phosphatase. And immediately
00:09:53.21 we shipped the paper to the Journal of Biological Chemistry.
00:09:59.04 And we knew that Carl Cori was one of the editors, so you know,
00:10:07.08 perhaps a question, but then we had faith that Cori would not
00:10:12.13 take advantage of that situation. And he did not, in fact he was very
00:10:17.26 excited by our results and he never touched that problem.
00:10:23.04 Now, the reaction that I showed earlier became slightly more complicated
00:10:31.09 when we began to use partially purified elements. Particularly
00:10:38.19 when we used partially purified phosphorylase kinase.
00:10:43.17 Because it became clear that calcium could not act in
00:10:50.16 that reaction. And yet, it was absolutely needed when crude
00:10:56.19 muscle extracts were used. So the only possibility is
00:11:01.13 that in crude muscle extract, calcium had to act at an earlier
00:11:07.02 step. And we hypothesized that perhaps phosphorylase kinase
00:11:13.09 just like phosphorylase, might also exist in an inactive and
00:11:20.01 active form. And perhaps calcium was required for that reaction,
00:11:24.22 and that turned out to be correct. In the presence of calcium
00:11:30.15 ions, phosph kinase is rapidly phosphorylated and activated.
00:11:36.09 At that same time, Earl Sutherland, Ted Rall, and his group
00:11:43.21 had isolated cyclic AMP, which they showed had served as
00:11:49.02 a second messenger, intracellular messenger, for the action of
00:11:53.19 adrenaline. So Earl immediately sent us a sample of his
00:11:58.20 material, and we could show that it acted on the activation
00:12:04.29 of phosph kinase. Either by accelerating the rate of autophosphorylation
00:12:12.10 or perhaps even, by acting on yet another enzyme, which for want of a better
00:12:20.02 term, we called a kinase kinase. Some people thought it was a funny type
00:12:26.15 of name, others thought it was a dumb name. And we never forget the
00:12:30.26 comments of the reviewer who looked at this paper, first of all, he
00:12:36.18 didn't believe that another enzyme would be involved. He said,
00:12:40.24 when will it end? But then we said, if really another enzyme
00:12:46.11 is involved, I wish that the authors would think of a better name than
00:12:51.27 a kinase kinase. Anyway, that hypothesis was confirmed 4 years
00:12:58.26 later by the isolation, by Ed and Don Walsh, of the cyclic AMP
00:13:04.26 dependent protein kinase. And since by that time, Earl and his
00:13:10.18 group had clarified the production of cyclic AMP at the membrane,
00:13:16.01 the whole cascade for the phosphorolysis of glycogen was
00:13:23.11 elucidated. We have been often asked by Ed whether we realized
00:13:30.03 at the beginning that we were dealing with a very ubiquitous
00:13:34.11 and therefore very fundamental reaction. Absolutely not.
00:13:39.29 We knew that it was a nice reaction, we knew that it was an
00:13:43.13 important and exciting one, we stayed with it, but we could
00:13:48.10 never have guessed the incredible developments that followed.
00:13:52.04 Should be said that 4-5 years later, everybody was thinking in terms of
00:13:59.18 allostery. Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux
00:14:04.25 had come up with a model of allosteric activation of enzymes.
00:14:09.28 And it was very clear to me that Jacque Monod was a very
00:14:13.15 close friend of mine, never believed one second that covalent
00:14:19.03 modification by protein phosphorylation could serve a fundamental
00:14:24.12 role in the regulation of cellular processes. We know today
00:14:30.15 that phosphorylation is one of the most prevalent mechanisms of regulation,
00:14:37.02 and it is clear that it would be very difficult to find a physiological
00:14:46.00 reaction that was not directly or indirectly affected by protein
00:14:52.18 phosphorylation. Another question that was often asked
00:14:58.11 was how was it for you guys to work for more than 50 years
00:15:03.23 together? Well, it was very easy and most enjoyable.
00:15:11.13 At first, Ed would make fun, would laugh at my English. Every morning,
00:15:18.20 I would say, "let's go take a coffee." Because in French we say,
00:15:22.05 prendre un cafe. And he really made fun of that.
00:15:26.18 What Ed didn't realize was that in the following year,
00:15:31.18 while my English didn't improve very much, his deteriorated
00:15:36.07 completely. Anyway, those were marvelous, marvelous days.
00:15:41.16 And during all the time we worked together, we never considered
00:15:47.16 what we were doing as work. It was never work.
00:15:52.16 It was fun. Thank you.

This Talk

Speaker: Edmond Fischer

Audience:

General Public
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Recorded: February 2015

Talk Overview

Edmond Fischer tells an engaging story of how he and Edwin Krebs were the first to observe reversible protein phosphorylation, a mechanism so ubiquitous, it influences almost every cellular process. This Nobel prize winning discovery happened over 60 years ago when Fischer and Krebs collaborated to study how the enzyme glycogen phosphorylase is activated in cells. Working side-by-side at the bench, they isolated the phosphorylase from the muscle tissue of rabbits, and, in subsequent experiments, revealed that the enzyme was activated by the transfer of phosphate groups from adenosine triphosphate (ATP) to the protein itself.

Speaker Bio

Edmond Fischer

Edmond Fischer

Edmond Fischer is Emeritus Professor of Biochemistry at the University of Washington. He, along with his colleague Edwin Krebs, demonstrated for the first time the mechanism of reversible protein phosphorylation on the enzyme glycogen phosphorylase. Fischer and Krebs were awarded the Nobel Prize in Physiology or Medicine in 1992 for this discovery. Since their initial… Continue Reading