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The scientific career and life of Edmond H. Fischer—A personal tribute

2022; Wiley; Volume: 75; Issue: 4 Linguagem: Inglês

10.1002/iub.2695

1521-6551

Philip Cohen,

Biochemical and Molecular Research

I am delighted to have been invited to write the introductory article of this special issue of IUBMB Life on Protein Phosphorylation dedicated to Edmond Henri Fischer, known simply as Eddy to all his friends, who died on 27 August 2021, at the age of 101. Eddy was a great scientist with a passion for music, history and art. He had an exciting, eventful and fulfilling life, but he was also a kind and generous person. The 2 years I spent in his laboratory from 1969 to 1971 were among the most formative of my life, and my career and life would have been entirely different had I not met and worked with him. A number of obituaries about Eddy have appeared since his death, and so will only mention his early life briefly. Readers are referred to another article for a detailed account of his early life and upbringing.1 Briefly, Eddy was born in Shanghai, China in 1920. His family moved to Switzerland when he was seven and he was educated near Geneva, graduating from high school in 1939. He then studied chemistry and biology at the University of Geneva, from where he received a Bachelor of Science degree in Organic Chemistry and then a PhD in 1947 for research on the structure of polysaccharides and alpha-amylases under the supervision of Kurt Meyer. Eddy moved to the USA in 1953 to begin postdoctoral research at Caltech but on arrival was unexpectedly offered an assistant professorship in biochemistry at the University of Washington, Seattle, which he accepted, spending the rest of his life there. During the 1930 and 1940s, Carl and Gerty Cori discovered that glycogen phosphorylase existed in two forms, which they termed a and b. Phosphorylase b was only active in the presence of adenylic acid (5' Adenosine Monophosphate(5′-AMP)), whereas phosphorylase a was active in the absence of this molecule. The Cori's reasoned (incorrectly as it turned out) that phosphorylase a probably contained tightly bound 5′-AMP and that another enzymatic activity they had detected, probably converted phosphorylase a to phosphorylase b by catalysing the removal of 5′-AMP. However, despite being awarded the Nobel Prize in Physiology or Medicine in 1947 for ‘their discovery of the course of the catalytic conversion of glycogen’, they never managed to discover the molecular difference between phosphorylase a and phosphorylase b and eventually dropped the problem. When Eddy Fischer arrived in Seattle, he found that one of his colleagues was Edwin (Ed) G. Krebs who had joined the Department 5 years earlier, after completing a postdoctoral fellowship at Washington University, St Louis, with Carl and Gerty Cori, where he had worked on glycogen phosphorylase. Eddy Fischer also worked on glycogen phosphorylase in potatoes and on its role in starch breakdown during his PhD with Kurt Meyer. Eddy and Ed discussed why glycogen phosphorylase from potatoes did not require 5′-AMP for activity and what the difference might be between phosphorylase a and b and decided to take a crack at the problem. The story of how Eddy and Ed solved the problem within 18 months has been described in detail by Eddy Fischer in his biographical memoir of Edwin Krebs.2 Briefly, when they removed the structural muscle proteins from muscle homogenates by centrifugation, they found that glycogen phosphorylase was entirely in the b form, whereas Carl and Gerty Cori, who had removed the structural muscle proteins by passing the homogenates through filter paper (preparative centrifuges having not yet been invented), found that phosphorylase was entirely in the a-form. Fischer and Krebs then found that the b-form could be converted to the a-form if they passed the muscle extracts through filter paper. They found that the key ingredients required for the b to a conversion were Mg-ATP in the extracts and calcium ions, which leached from the filter paper during the filtration process. The requirement for Mg-ATP was suggestive of a phosphorylation reaction, which they confirmed by showing that 32P-phosphate was incorporated into glycogen phosphorylase from [γ-32P] adenosine trisphosphate (ATP) during the b to a conversion, leading them to propose that the b to a converting enzyme was a phosphorylase kinase.3, 4 Fischer and Krebs showed subsequently that phosphorylase kinase transferred the phosphate from ATP to a specific serine residue on glycogen phosphorylase b,5 which meant that the a to b converting enzyme had to be a phosphorylase phosphatase. Identifying the molecular mechanism by which calcium ions activated phosphorylase kinase turned out to be surprisingly difficult and the solution to the problem only emerged after the relatively specific calcium chelator 3,12-Bis(carboxymethyl)-6,8-dioxa-3,12-diazatetradecane-1,14-dioic acid (EGTA) became available in the 1960s. It then became clear that calcium ions were activating phosphorylase kinase in two quite different ways. First by an indirect mechanism catalysed by a calcium-dependent proteolytic enzyme, which Fischer and Krebs termed Kinase-Activating-Factor (KAF), now known to be the proteinase calpain. Activation by KAF required millimolar concentrations of calcium ions, was irreversible and may not have any physiological relevance.6 Second, by the direct and reversible binding of calcium ions to phosphorylase kinase at micromolar concentrations, a mechanism that explained how glycogen breakdown is synchronised with the onset of muscle contraction.7, 8 Much later, I found that calcium ions activate phosphorylase kinase by interaction with the calcium-binding protein calmodulin, which is an integral component of the phosphorylase kinase complex (its δ subunit), and which is closely related to troponin C, the protein that confers calcium sensitivity to the muscle contractile apparatus.9, 10 In the early 1950s, Earl Sutherland, who had also trained with Carl Cori, found that epinephrine (adrenaline) stimulated the conversion of phosphorylase b to a and initiated glycogenolysis by generating 3′,5′-cyclic adenosine monophosphate (cyclic AMP), the first ‘second messenger’ to be identified, and discovery for which Sutherland was awarded the Nobel Prize in Physiology or Medicine in 1970. Krebs and Fischer found that partially purified preparations of phosphorylase kinase were activated by incubation with Mg-ATP and that this reaction was accelerated by the addition of cyclic AMP.11 Later Donal Walsh working in Ed Kreb's laboratory discovered that the effect of cyclic AMP was mediated by a separate cyclic AMP-dependent protein kinase, traces of which contaminated the partially purified preparations of phosphorylase kinase that were being used at this time.12 The activation of phosphorylase kinase by cyclic AMP-dependent protein kinase was the first protein kinase ‘cascade’ to be discovered in which one protein kinase activates another. Soon after their seminal discovery, Eddy and Ed agreed that one of them should focus on phosphorylase kinase (Ed Krebs) and the other on phosphorylase phosphatase (Eddy Fischer). Eddy published the first papers on phosphorylase phosphatase in the 1960 and 1970s describing the partial purification and characterisation of the skeletal muscle enzyme13, 14 but this phase of Eddy's career was a less successful and the decisive breakthroughs in this area were made later by others. However, after oncogenes and growth factor receptors were identified as protein kinases that attach phosphate to the hydroxyl side chain of tyrosine, Eddy became fascinated by the as-yet-unknown protein tyrosine phosphatases that must exist and how they might be regulated. He began to tackle this project when Nick Tonks, who had been a graduate student in my lab, joined Eddy's laboratory as a postdoctoral researcher. In Eddy's lab, Nick isolated and characterised the first protein tyrosine phosphatases, revealing a new gene family that we now know comprises about 100 members and includes both cytoplasmic and transmembrane receptor tyrosine phosphatases.15-18 This topic will be covered in much greater detail by Nick Tonks in another article on this special issue. For many years, it was thought that protein phosphorylation was a specialised form of enzyme control mechanism confined to the regulation of glycogen metabolism. However, other examples where phosphorylation acted as a regulatory device gradually emerged during the 1970s and more rapidly during the 1980s. The protein products of cancer-causing oncogenes, such as src and abl, as well as the receptors for epidermal growth factor and insulin, were identified as protein kinases, and the overproduction or mutation of growth factor receptors was found to be a major cause of cancer. It also became clear that progression through the cell division cycle is driven by the actions of protein kinases and phosphatases. Then, in 1990, cyclosporin, the immunosuppressant drug that has permitted the widespread use of organ transplantation, was found to exert its effects by inhibiting a calcium/calmodulin-regulated protein phosphatase, highlighting the potential importance of both phosphatases and kinases as drug targets. It, therefore, did not come as much of a surprise when Eddy Fischer and Ed Krebs were jointly awarded the Nobel Prize in Physiology or Medicine in 1992 for their discoveries concerning ‘reversible protein phosphorylation as a biological regulatory mechanism’, 37 years after making their seminal finding (Figure 1). The award of a Nobel Prize frequently signifies that a field of research is about to explode, not that it has already peaked, and this has most certainly proved to be the case for protein phosphorylation. Indeed, it is remarkable how much more has been discovered about this process after 1992. More recent discoveries have included the identification of the mitogen-activated protein kinase cascades and the protein kinase cascade activated by the second messenger phosphatidylinositol-3,4,5-phosphate, which mediates the intracellular actions of insulin. The protein phosphorylation and dephosphorylation events that regulate innate and adaptive immunity have been elucidated, and the first protein kinase inhibitor entered clinical trials in 1998. Over 70 drugs that target protein kinases have subsequently been approved during the 21st century, and have transformed the clinical treatment of many cancers. For example, Imatinib, the first protein kinase inhibitor approved for clinical use in 2001, has transformed chronic myelogenous leukaemia from a rapidly fatal disease to a manageable condition (reviewed19). As I found out when I joined Eddy's lab in 1969, he treated his research team as if they were his own family, meeting them personally at the airport on arrival, and insisting that they stay at his house until they found a suitable apartment to rent. Soon after my arrival, he took me to watch the University of Washington ‘Huskies’ against Stanford University to kickstart my education in American football. An accomplished skier, he would take the lab to the Cascade Mountains in the winter and to his vacation home on Lopez Island in the San Juan Island chain in the summer. He bought an aeroplane and learned to fly at the age of 60 so that he could get to Lopez faster on weekends, and he continued to fly until he was 80. My one and only flying lesson was taken on ‘Air Fischer’ when Eddy flew me from Ed Kreb's 65th birthday symposium on Orcas Island in the San Juans' to Seattle in his four-seater Cessna, insisting that I sit in the cockpit and help him to fly the plane and land it on Boeing Airfield. Eddy had a 1958 Cadillac of which he was very fond and affectionately referred to it as ‘the Bathtub’ because of the shape of its very broad trunk. When my car broke down 6 weeks before I was due to leave Seattle to return to the UK, Eddy insisted that I use the Bathtub for the remainder of my time in Seattle and said that Bev (his second wife Beverly) would drive him to and from work over this period. Fortunately, I managed to return the bathtub intact to Eddy at the airport prior to departure! Eddy had great knowledge and a love of history and art, kindled during the many summers he had spent in Venice during his youth. At a meeting in Venice in 2004 to celebrate the 50th anniversary of the discovery of the first protein kinase by Gene Kennedy,20 Eddy treated me to a personal walking tour of the city, amazing me with his encyclopaedic knowledge of the history of many buildings that we passed, even in obscure backstreets not usually frequented by tourists. He also enjoyed painting, signing his works in the maiden name of his mother (Tapernoux). I am proud to have an original hanging in the study of my house. Eddy spent a sabbatical leave at the Weizmann Institute of Science, Israel in 1963 during which time he became a good friend of Ephraim Katzir, then known as Ephraim Katchalski, a well-known biophysicist, who was later elected by the Knesset to serve as the fourth President of Israel. When President Katzir heard that his friend Eddy was attending a conference near the Dead Sea in 1978 he invited everyone at the conference to a reception at his Palace in Jerusalem, which included me! Unfortunately, not having been forewarned and knowing it would be rather hot, I had failed to bring any clothes with me that were suitable for this grand occasion, but Eddy came straight to my rescue and lent me one of his jackets and a tie (Figure 2). Eddy was an outstanding pianist and had at one time thought about becoming a professional musician (Figure 3). However, he gave recitals at many scientific meetings and continued to play the piano daily until a few weeks before his death. In June 2021 at the age of 101, he sat at his piano and played the opening lines of Beethoven's ‘Ode to Joy’ from memory as part of the Lindau organization's virtual orchestra and on 31 July 2021 he played the piano at the wedding of Leo, one of his grandsons (Figure 4). Speaking about science Eddy once said:- ‘As to what has always attracted me toward scientific research … I believe it's the systematic way one has to proceed in science, the kind of logic one has to apply to solve a given problem. Science builds on science, where every result obtained suggests a number of questions, and every question asked suggests the next experiment. One must follow those leads just like a detective follows different leads to solve a murder mystery, never knowing whether it will lead you to a dead end or to the next big breakthrough. Because in science, one cannot order at will a great discovery or buy it at any cost because there is no way of predicting when and from where it will come’. Eddy Fischer had two sons and four grandchildren but only his granddaughter Elyse became a scientist. To my great surprise, Eddy informed me in 2013 that Elyse would soon be arriving in Scotland to become an undergraduate at the University of St Andrews, only a 30-minute drive from my house. Elyse became very interested in Structural Biology at St Andrew's and mentioned that she was interested in staying in Europe to study for a PhD in this area. My wife Tricia and I introduced her to David Barford at the MRC Laboratory of Molecular Biology, Cambridge, UK and in 2021; she received a PhD under David's supervision becoming Dr E. Fischer at the age of 27, exactly like her Grandpa in 1947! During her PhD. Elyse solved the molecular mechanism by which Mad1 is targeted to kinetochores through direct interaction with phosphorylated Bub1,21 an important advance for which she received the first Whelan Award from IUBMB. Fittingly, the next article in this special issue of IUBMB Life is contributed by Elyse herself!