A catalytic career: Studies spanning glutamine synthetase, phospholipase C, peroxiredoxin, and the intracellular messenger role of hydrogen peroxide

I learned biochemistry from P. Boon Chock and Earl Stadtman while working on the regulation of Escherichia coli glutamine synthetase as a postdoctoral fellow at the National Institutes of Health. After becoming a tenured scientist at the same institute, my group discovered, purified, and cloned the first three prototypical members of the phospholipase C family and uncovered the mechanisms by which various cell-surface receptors activate these enzymes to generate diacylglycerol and inositol 1,4,5-trisphosphate. We also discovered the family of peroxiredoxin (Prx) enzymes that catalyze the reduction of H2O2, and we established that mammalian cells express six Prx isoforms that not only protect against oxidative damage but also mediate cell signaling by modulating intracellular H2O2 levels. To validate the signaling role of H2O2, we showed that epidermal growth factor induces a transient increase in intracellular H2O2 levels, and the essential cysteine residue of protein-tyrosine phosphatases is a target for specific and reversible oxidation by the H2O2 produced in such cells. These observations led to a new paradigm in receptor signaling, in which protein tyrosine phosphorylation is achieved not via activation of receptor tyrosine kinases alone but also through concurrent inhibition of protein-tyrosine phosphatases by H2O2. Our studies revealed that Prx isozymes are extensively regulated via phosphorylation as well as by hyperoxidation of the active-site cysteine to cysteine sulfinic acid, with the reverse reaction being catalyzed by sulfiredoxin. This reversible hyperoxidation of Prx was further shown to constitute a universal marker for circadian rhythms in all domains of life. I learned biochemistry from P. Boon Chock and Earl Stadtman while working on the regulation of Escherichia coli glutamine synthetase as a postdoctoral fellow at the National Institutes of Health. After becoming a tenured scientist at the same institute, my group discovered, purified, and cloned the first three prototypical members of the phospholipase C family and uncovered the mechanisms by which various cell-surface receptors activate these enzymes to generate diacylglycerol and inositol 1,4,5-trisphosphate. We also discovered the family of peroxiredoxin (Prx) enzymes that catalyze the reduction of H2O2, and we established that mammalian cells express six Prx isoforms that not only protect against oxidative damage but also mediate cell signaling by modulating intracellular H2O2 levels. To validate the signaling role of H2O2, we showed that epidermal growth factor induces a transient increase in intracellular H2O2 levels, and the essential cysteine residue of protein-tyrosine phosphatases is a target for specific and reversible oxidation by the H2O2 produced in such cells. These observations led to a new paradigm in receptor signaling, in which protein tyrosine phosphorylation is achieved not via activation of receptor tyrosine kinases alone but also through concurrent inhibition of protein-tyrosine phosphatases by H2O2. Our studies revealed that Prx isozymes are extensively regulated via phosphorylation as well as by hyperoxidation of the active-site cysteine to cysteine sulfinic acid, with the reverse reaction being catalyzed by sulfiredoxin. This reversible hyperoxidation of Prx was further shown to constitute a universal marker for circadian rhythms in all domains of life. In May 1961, a month after I entered Seoul National University College of Liberal Arts and Sciences as a chemistry major, the fledgling democracy in Korea ended with a military coup. A series of intense antigovernment demonstrations ensued and persisted throughout the 1960s. Our college campus was the center for the student antigovernment movement. Streets surrounding the campus were frequently filled with soldiers and shrouded in clouds of tear gas, and many classes were canceled. However, the classes taught by Professors Kyu Won Choi (analytical chemistry) and Se Hun Chang (physical chemistry) were seldom canceled. These professors, like many others since the Korean independence in 1945, had been teaching without Ph.D. degrees, but they had recently returned to Korea after earning their doctorates in the United States. Chemistry majors were mocked as the most subservient students on campus. Textbooks used in class were in English, but most students could not afford to buy these books printed in the United States or even cheaper versions printed with the copyright in Japan. Instead, they bought pirated versions printed in Taiwan. The professors encouraged us to go to the United States for graduate studies. Many of us who attended the chemistry department in the 1960s recall how lucky we were to be taught by two such passionate professors with a vision for the future. I took the Korean equivalent of ROTC courses and was commissioned after graduation in 1965 as a second lieutenant in the Korean Army with a specialty in ammunition supply. I was assigned to an infantry division located near the Korean Demilitarized Zone. Given that I wanted to go to graduate school in the United States after my 2 years of military service, I had to take the Graduate Record Examinations (GRE) and the Test of English as a Foreign Language (TOEFL) while I was in the army. The last chance to take the tests to fulfill the 1967 admission requirements was September of 1966. In that month, there were several instances of North Korean agent infiltration through the area controlled by our infantry division, and all officers were ordered to stay near the military base. I could not take days off to take the tests in Seoul. Next January, I received letters denying me financial assistance from all the graduate schools to which I had applied. One of these graduate schools was Catholic University of America in Washington, D.C., where Gilbert Castellan, the author of a physical chemistry textbook used in our undergraduate course, was a faculty member in the chemistry department. I had a cousin, Jhoon Goo Rhee, who was widely recognized as the “Father of American Taekwondo” for introducing this martial art to the United States. After learning that I was not accepted to Catholic University, my cousin went there to meet Richard Timmons, a young assistant professor of chemistry in charge of graduate admissions. I was then admitted to a teaching assistant slot not taken by another applicant, and I arrived in Washington in June 1967. Richard Timmons later told me that he was most impressed with my cousin, who he had seen several times on local television. I chose to do my Ph.D. thesis under the direction of John Eisch, who had recently joined the department as head after moving from the University of Michigan and who was the author of a textbook titled The Chemistry of Organometallic Compounds. In the meantime, I married Young Kyu Park, another graduate student whom I met at Catholic University. (The infiltration of North Korean agents thus turned out to be a blessing in disguise for me.) The goal of my Ph.D. project was to elucidate the mechanism of the hydroalumination of alkynes, and the research progressed well. Early in 1971, my thesis advisor told me to write up my results and prepare for graduation in the fall. As my writing skills were not well-honed, this progressed at a painfully slow pace. I would constantly look for excuses to do more experiments. I gave up the prospect of graduating that fall and moved to the State University of New York (SUNY) in Binghamton with John Eisch, who joined the university as chair of the chemistry department. Although I had not formally received my Ph.D. degree, I was appointed to a postdoctoral fellow position. I finally and successfully defended my thesis in 1972, and my work in the Eisch laboratory resulted in eight publications in respected journals. While I was preparing for my thesis defense, John Eisch asked me about my future plans. I told him that I intended to return to Korea and to undertake the total synthesis of ginsenosides, triterpene saponins found in the ginseng root, which is considered an herbal panacea in Korea. At that time, I did not comprehend very well the importance of research in an academic career and was not well-prepared to answer Eisch's question. From time to time, I followed publications on ginseng coming out of a research institute at the University of Tokyo as well as a research institute in Vladivostok, in the former Soviet Union. Russia's interest in ginsenosides stemmed from the finding of its scientists that these compounds could improve the performance of astronauts and athletes. Knowing how poorly prepared I was for the difficult task of natural product synthesis, Dr. Eisch gently advised me to apply for a postdoctoral fellowship at the National Institutes of Health (NIH) to get more training in enzymology and natural products. The logic behind his advice was that the synthesis of complex molecules like ginsenosides is possible only through a combination of bulk chemistry and the stereospecific action of enzymes. He also mentioned that Richard Timmons from Catholic University was spending a sabbatical at a renowned enzymology lab at NIH, the Laboratory of Biochemistry, within the National Heart, Lung, and Blood Institute (NHLBI), headed by Earl Stadtman. When Richard Timmons heard of my desire to apply for a postdoctoral fellowship at NIH, he introduced me to P. Boon Chock, who had just joined the Laboratory of Biochemistry in a tenure-track position. The latter agreed to be a sponsor for my NIH postdoctoral fellowship application, and, with his generous help, I was accepted for the position. The regulation of glutamine synthetase (GS) 2The abbreviations used are: GSglutamine synthetasePrxperoxiredoxinPLCphospholipase CEGFepidermal growth factorPDGFplatelet-derived growth factorATaseadenylyltransferaseUTaseuridylyltransferaseTrxthioredoxinSrxsulfiredoxinPTPprotein-tyrosine phosphataseRBCred blood cellSHSrc homologyGPxGSH peroxidaseACTHadrenocorticotropic hormone. in E. coli was a focus of study in Earl Stadtman’s laboratory around the time of my arrival (1Stadtman E.R. The story of glutamine synthetase regulation.J. Biol. Chem. 2001; 276 (11585846): 44357-4436410.1074/jbc.R100055200Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Many later-renowned scientists studied this subject while training in the Stadtman laboratory. I learned fast kinetics from P. Boon Chock, who had done postdoctoral research in the laboratory of Manfred Eigen—a recipient of the 1967 Nobel Prize in Chemistry for his work on measurement of fast chemical reactions—at the Max Planck Institute in Göttingen. I applied the stopped-flow technique to study the kinetics of the GS reaction. glutamine synthetase peroxiredoxin phospholipase C epidermal growth factor platelet-derived growth factor adenylyltransferase uridylyltransferase thioredoxin sulfiredoxin protein-tyrosine phosphatase red blood cell Src homology GSH peroxidase adrenocorticotropic hormone. Studies in the 1960s and early 1970s by Stadtman's group revealed that GS activity is regulated via a bicyclic cascade involving reversible adenylylation (attachment of an AMP moiety) and uridylylation (attachment of a UMP moiety) of protein tyrosine residues (1Stadtman E.R. The story of glutamine synthetase regulation.J. Biol. Chem. 2001; 276 (11585846): 44357-4436410.1074/jbc.R100055200Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). In the 1970s, GS and glycogen phosphorylase provided the two best-known examples of post-translational regulation, the former via nucleotidylation of tyrosine in bacteria and the latter via phosphorylation of serine and threonine in hepatocytes. As I gradually learned enzymology and became acquainted with a number of interesting discoveries made on the NIH campus, my ideas on research and a career began to change. In particular, the story behind how Stadtman’s group discovered the nucleotidylation of GS influenced me strongly. They observed that the catalytic activity of GS varied widely depending on the conditions of E. coli growth. From their observations that different GS preparations showed small differences in UV absorbance at 260 nm, they postulated the existence of a nucleotide adduct (2Shapiro B.M. Kingdon H.S. Stadtman E.R. Regulation of glutamine synthetase. VII. Adenylyl glutamine synthetase: a new form of the enzyme with altered regulatory and kinetic properties.Proc. Natl. Acad. Sci. U.S.A. 1967; 58 (4860756): 642-64910.1073/pnas.58.2.642Crossref PubMed Scopus (114) Google Scholar). This was confirmed by the demonstration that treatment of some preparations with snake venom phosphodiesterase induced the release of AMP and a change in the catalytic properties of GS, thus leading to the discovery of a novel regulatory mechanism based on the adenylylation of tyrosine. Subsequently, they found that adenylyltransferase (ATase) could catalyze both the adenylylation and deadenylylation of GS (3Kingdon H.S. Shapiro B.M. Stadtman E.R. Regulation of glutamine synthetase. 8. ATP:glutamine synthetase adenylyltransferase, an enzyme that catalyzes alterations in the regulatory properties of glutamine synthetase.Proc. Natl. Acad. Sci. U.S.A. 1967; 58 (4867671): 1703-171010.1073/pnas.58.4.1703Crossref PubMed Scopus (137) Google Scholar) and that the activity of ATase was modulated by the protein PII. They further showed that, like GS, PII exists in two forms: in this case, an unmodified form that in the presence of l-glutamine stimulates the adenylylation of GS, and a uridylylated form (PII-UMP) that in the absence of l-glutamine and in the presence of α-ketoglutarate stimulates the deadenylylation of GS (4Brown M.S. Segal A. Stadtman E.R. Modulation of glutamine synthetase adenylylation and deadenylylation is mediated by metabolic transformation of the P II-regulatory protein.Proc. Natl. Acad. Sci. U.S.A. 1971; 68 (4399832): 2949-295310.1073/pnas.68.12.2949Crossref PubMed Scopus (102) Google Scholar). I joined the research project at this stage of its development. GS is composed of 12 identical subunits, and GS activity is inversely proportional to the state of adenylylation, with the average number of covalently bound adenylyl groups per molecule (n) ranging from 0 to 12. The adenylylation and deadenylylation reactions are regulated by l-glutamine and α-ketoglutarate, which have pronounced reciprocal effects. Theoretical analysis predicted that, in the presence of all adenylylation–deadenylylation cycle components, the value of n will achieve a steady state that will vary as a function of effector concentrations, with at least 28 kinetic or equilibrium constants being required to describe the reaction cycle. I eventually determined these constants with the help of P. Boon Chock (5Rhee S.G. Park R. Chock P.B. Stadtman E.R. Allosteric regulation of monocyclic interconvertible enzyme cascade systems: use of Escherichia coli glutamine synthetase as an experimental model.Proc. Natl. Acad. Sci. U.S.A. 1978; 75 (28522): 3138-314210.1073/pnas.75.7.3138Crossref PubMed Scopus (46) Google Scholar), benefitting from his strong theoretical background. This steady work provided the path to my being granted a tenured senior scientist position in 1979 (Fig. 1). The determination of these constants required the purification of large amounts of GS, ATase, and PII proteins as well as the partial purification of uridylyltransferase (UTase) for preparation of PII-UMP. GS is a relatively abundant protein, and its purification had been well established. For isolation of the other proteins, however, we had to grow kilogram quantities of E. coli and subject the cell homogenates to industrial-scale column chromatography. The preparation of PII was especially difficult, given the extremely low abundance of this protein. We achieved a major breakthrough with the finding that most proteins, but not PII, in E. coli homogenates are precipitated by the addition of β-mercaptoethanol to a final concentration of 26% (v/v). This procedure, whose effectiveness was because PII does not contain any cysteine residues, allowed enrichment of PII by several-hundredfold. Although I was delighted with the discovery of this magical step for PII purification, I became highly unpopular in our building as a result of the unpleasant odor caused by the use of liter quantities of β-mercaptoethanol. Even though I tried to perform the attendant tasks under a chemical fume hood and during the weekend as much as possible, the centrifugation and dialysis of large volumes of material inevitably polluted the entire corridor, with the stench lingering into the workweek. Even my 4-year-old daughter held her nose and tried to stay away from me when I came home. I was warned several times by chemical safety officers on the NIH campus. Looking back, I feel I was somewhat ruthless but that I was lucky to have patient and understanding colleagues. Late in 1979, Emilio Garcia joined my laboratory as a postdoctoral fellow from the University of California at Davis. Taking advantage of the fact that PII was now readily available, Emilio and I purified UTase (6Garcia E. Rhee S.G. Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII uridylyltransferase and uridylyl-removing enzyme.J. Biol. Chem. 1983; 258 (6130097): 2246-2253Abstract Full Text PDF PubMed Google Scholar). The purification was facilitated by the use of an E. coli strain that harbors multiple copies of the UTase gene and overproduces the enzyme by a factor of 25. This strain was selected by screening the Clarke and Carbon collection of 2000 E. coli strains that carry ColE1 plasmids containing small random segments of the bacterial chromosome. Using the purified enzyme, which comprises a single polypeptide, we showed that it catalyzes the uridylylation as well as the deuridylylation of PII, similar to the bifunctionality exhibited by ATase (Fig. 2). One day in 1982, Earl Stadtman came to my office and casually mentioned that it might be time for me to move on to a new area of research, adding that he would continue to get undeserved credit for the work I published as long as I continued to work on GS. He also mentioned that he was gradually shifting his focus to the role of protein oxidation and degradation in aging. I sensed he was telling me that I had become too complacent with my work—around that time we had cloned all four genes for the protein components of the GS regulatory cascade (GS, ATase, PII, and UTase), and we continued to publish follow-up papers on the regulation of GS in E. coli. Appreciative of his generosity and wisdom, which he had shared with many of his trainees (Stadtman mentored two Nobel laureates, multiple members of the National Academy of Sciences, and many current leaders in the biomedical community), I took his advice seriously, and I decided to look for a new research topic. One easy choice was to study GS in yeast, which had been shown by others to be subject to complex regulation as in E. coli, but not through either nucleotidylation or phosphorylation. Kanghwa Kim joined my lab as a postdoctoral fellow from Korea in 1983 and devoted his full efforts to this topic. Although our studies in this area were largely unfruitful, an astute observation made by Kanghwa during the purification of yeast GS led us to discover a family of unconventional antioxidant enzymes, which we later named peroxiredoxins. This discovery led me to concentrate my last 20 years of research on the intracellular messenger function of hydrogen peroxide (H2O2) and its regulation by peroxiredoxins (more on this later). Ultimately, although intrigued by the similarities and dissimilarities between the GS enzymes of E. coli and Saccharomyces cerevisiae, I was not satisfied with our progress on regulation of the yeast enzyme, and I continued to look for a new area of investigation. While attending a number of NIH and national conferences in 1984 and 1985, I noticed certain sessions were crowded and often overflowed to the entrance. Curious about what was drawing such interest, I stepped out of my immediate area of research and was introduced to the two newly crowned second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Through further reading, I came to comprehend that these two messengers are generated as a result of the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C (PLC) in response to the activation of various cell-surface receptors, that inositol 1,4,5-trisphosphate induces the release of Ca2+ from the endoplasmic reticulum, and that diacylglycerol activates protein kinase C (7Berridge M.J. Irvine R.F. Inositol trisphosphate, a novel second messenger in cellular signal transduction.Nature. 1984; 312 (6095092): 315-32110.1038/312315a0Crossref PubMed Scopus (4435) Google Scholar, 8Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion.Nature. 1984; 308 (6232463): 693-69810.1038/308693a0Crossref PubMed Scopus (5991) Google Scholar). I also learned that one critical question that remained regarding this bifurcating signaling pathway was how various receptors are coupled to PLC. I felt that purification of PLC and the cloning of its cDNA would be necessary to address this question. Several earlier studies suggested that the coupling of receptor function to PLC is mediated by an unspecified G protein, in a manner analogous to the modulation of adenylate cyclase via the G proteins Gs and Gi, which couple receptor function to synthesis of the second messenger cAMP (9Gilman A.G. G proteins and dual control of adenylate cyclase.Cell. 1984; 36 (6321035): 577-57910.1016/0092-8674(84)90336-2Abstract Full Text PDF PubMed Scopus (1162) Google Scholar). Alfred Gilman's finding that adenylate cyclase is an intrinsic membrane protein strongly suggested that the association of PLC with the plasma membrane would also be a prerequisite for its regulation by cell-surface receptors. Indeed, PLC activity had been detected in various membrane preparations by several investigators. Nevertheless, we decided to purify PLC from the cytosolic fraction of bovine brain because the cytosolic fraction showed a much higher phosphoinositide-hydrolyzing activity than did the membrane fraction. Several people I later met at various meetings asked me why I pursued the seemingly unrewarding purification of cytosolic PLC. The answer was ignorance—I simply did not know enough about G proteins and adenylate cyclase at that time to try to account for the coupling of PLC function to cell-surface receptors. Just as I was launching this project, two postdoctoral fellows, Sung Ho Ryu (in late 1985) and Pan-Ghill Suh (in early 1986), joined my laboratory. They were not at all familiar with G proteins, adenylate cyclase, or, for that matter, PLC. I am sure that if they had been fully informed about what they were getting into, they would have said no to my suggestion. Fortunately, I at least had the prior experience of purifying GS, and I felt both physically and mentally prepared to embark on the large-scale purification of PLC. Early each Monday morning, one of us would drive 50 miles round trip to purchase 16 fresh bovine brains from the J. W. Treuth & Sons slaughterhouse in Catonsville, MD, near Baltimore. It was usually past midnight by the time we had prepared a cytosolic fraction and started to apply it to an ion-exchange chromatography column, which yielded three partially resolved peaks of PLC activity. After processing 400 bovine brains through five chromatography steps, we succeeded in 1986 to 1987 in purifying to homogeneity three distinct forms of PLC with molecular masses of 150, 145, and 85 kDa. We initially named these bovine brain enzymes PLC-I, PLC-II, and PLC-III, respectively (10Ryu S.H. Suh P.G. Cho K.S. Lee K.Y. Rhee S.G. Bovine brain cytosol contains three immunologically distinct forms of inositolphospholipid-specific phospholipase C.Proc. Natl. Acad. Sci. U.S.A. 1987; 84 (3477795): 6649-665310.1073/pnas.84.19.6649Crossref PubMed Scopus (178) Google Scholar). Two small incidents (although perhaps not small from the perspective of the NIH safety officers) occurred at this time. We use to homogenize one brain at a time in a Waring blender with 1 liter of buffer containing two protease inhibitors, phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. After a number of weeks of this operation, we all began to complain that it was rather dark in the cold room and asked building maintenance to change the fluorescent light bulbs. One of us then noticed that our pupils were all markedly constricted. Although we had thought that we were dealing with the neurotoxic diisopropyl fluorophosphate (a cholinesterase inhibitor) with extreme care, we were performing the fume-generating homogenization process 16 times in 1 day in a poorly ventilated cold room. Fortunately, after we stopped using diisopropyl fluorophosphate, our vision returned to normal. A second incident, which took longer to resolve, occurred while we were preparing 32P-labeled phosphoinositide for use in our PLC assay. While harvesting yeast grown in a medium containing [32P]orthophosphate, a bottle broke inside the centrifuge. My license for isotopes was suspended, and the centrifuge was sealed for several months. By 1988, we had cloned the cDNAs corresponding to the three bovine brain PLC enzymes (11Suh P.G. Ryu S.H. Moon K.H. Suh H.W. Rhee S.G. Cloning and sequence of multiple forms of phospholipase C.Cell. 1988; 54 (3390863): 161-16910.1016/0092-8674(88)90548-XAbstract Full Text PDF PubMed Scopus (262) Google Scholar). As we and others purified and cloned more PLC enzymes, it became apparent that bovine PLC-I, -II, and -III were the first three prototypical members of a PLC family, with each type of PLC actually comprising two to four different proteins. Conversely, a 62-kDa PLC purified from guinea pig uterus in 1987 appeared to be distinct from the three types of PLC purified from bovine brain (12Bennett C.F. Balcarek J.M. Varrichio A. Crooke S.T. Molecular cloning and complete amino-acid sequence of form-I phosphoinositide-specific phospholipase C.Nature. 1988; 334 (3398923): 268-27010.1038/334268a0Crossref PubMed Scopus (217) Google Scholar). We therefore started to use Greek letters to designate the PLC enzymes with distinct primary structures, assigning the letters according to the chronological order of their purification—α for the 62-kDa enzyme, β for the 150-kDa enzyme, γ for the 145-kDa enzyme, and δ for the 85-kDa enzyme—and we assigned Arabic numerals to be placed after the Greek letters to designate subfamily members (13Rhee S.G. Regulation of phosphoinositide-specific phospholipase C.Annu. Rev. Biochem. 2001; 70 (11395409): 281-31210.1146/annurev.biochem.70.1.281Crossref PubMed Scopus (1243) Google Scholar). Comparison of the predicted amino acid sequences of PLC-β, PLC-γ, and PLC-δ revealed that, although these three types of enzyme showed a low overall sequence similarity, they shared marked similarity in two regions designated X (∼150 amino acids) and Y (∼120 amino acids). We therefore predicted that the X and Y regions constitute the catalytic site, with this prediction ultimately being corroborated when the crystal structure of PLC-δ was determined. Whereas PLC-β and PLC-δ contain short sequences of 50–70 amino acids separating the X and Y regions, PLC-γ has a long sequence of 400 amino acids separating the two regions that contain both Src homology (SH) 2 and SH3 domains, the domains first identified as noncatalytic regions common to a variety of Src-family tyrosine kinases (14Suh P.G. Ryu S.H. Moon K.H. Suh H.W. Rhee S.G. Inositol phospholipid-specific phospholipase C: complete cDNA and protein sequences and sequence homology to tyrosine kinase-related oncogene products.Proc. Natl. Acad. Sci. U.S.A. 1988; 85 (2840660): 5419-542310.1073/pnas.85.15.5419Crossref PubMed Scopus (153) Google Scholar). PLC-γ was the first nontyrosine kinase protein found to harbor SH2 and SH3 domains. The subsequent identification of SH2 and SH3 domains in several other proteins that do not possess tyrosine kinase activity contributed substantially to our understanding of the role of protein–protein interactions in signal transduction pathways. Surprisingly, the sequence of PLC-α showed no similarity to those of the other PLC enzymes. The isolated cDNA was subsequently found to encode a protein-disulfide isomerase devoid of PLC activity. The putative PLC-α cDNA was obtained with the use of an antibody directed against the 62-kDa uterus enzyme. Apparently, however, the enzyme preparation was contaminated with a protein-disulfide isomerase that is highly antigenic. The PLC-α protein was subsequently shown to be a fragment of PLC-δ1 (15Taylor G.D. Fee J.A. Silbert D.F. Hofmann S.L. PI-specific phospholipase C “α” from sheep seminal vesicles is a proteolytic fragment of PI-PLCδ.Biochem. Biophys. Res. Commun. 1992; 188 (1445352): 1176-118310.1016/0006-291X(92)91355-TCrossref PubMed Scopus (29) Google Scholar). Thereafter, the designation PLC-α ceased to exist. As I have already mentioned, an important issue in the area of receptor signaling concerned the mechanism by which PLC activity is coupled to various receptors. Although we had the PLC clones in hand and not being familiar with cell-surface receptors, we were unsure how best to tackle this question. Luckily, as the result of fruitful collaboration with Graham Carpenter, Joseph Schlessinger, Tony Hunter, and Gordon Guroff, we were able to show that treatment of a number of cell types with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or nerve growth factor induced the tyrosine phosphorylation of PLC-γ1, but not that of PLC-β1 or PLC-δ1, and that the tyrosine phosphorylation of PLC-γ1 correlated well with the increa