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GREAT EXPERIMENTS

The discovery of protein kinase C

Yasutomi Nishizuka

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Background

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Proteins have been known to contain phosphate since late in the 19th century, when chemists began to examine the chemical composition of biological macromolecules. The observation which led to my own interest in kinases was made early in the 20th century by Pheobus Levene, who had been a pupil of the great German chemist Emil Fischer. Levene, working at the Rockefeller Institute of Medical Research, was studying a class of acidic macromolecules present in the cell nucleus. This material proved to be protein containing a large amount of phosphorus, and was, in retrospect, presumably a mixture of highly phosphorylated transcription factors and other nuclear proteins. Levene was interested in determining in what form the phosphate was present in the protein, and in 1932 he and Fritz Lipmann discovered that it was covalently attached to serine residues. Levene and Lipmann thought that it could be a storage form of high-energy phosphate bonds. Over the next several decades, enzymes capable of transferring phosphate from ATP to egg yolk protein (phosvitin), milk protein (casein), and nuclear proteins (histone) were identified. These proteins were used only as convenient in vitro substrates, however, allowing no physiological function to be assigned to the enzymes that phosphorylated them.

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Protein phosphorylation emerged in a more physiologically meaningful context from studies of the action of hormones. Hormones were known to stimulate the breakdown of glycogen in liver and muscle. Work by the Coris and their disciples in the 1940s and early 1950s led to the conclusion that this was due to the conversion of glycogen phosphorylase, the enzyme which initiates the breakdown of glycogen, from an inactive to an active form in response to the hormone. A phosphate group was found to be added to the enzyme when it was activated, and by the mid 1950s it had become apparent from the work of Eddy Fischer and Ed Krebs that the activity of the enzyme was controlled by reversible phosphorylation. The activity of one of the enzymes required to synthesize glycogen was also controlled by phosphorylation.

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These studies revealed phosphorylation to be an essential part of regulating a very important physiological response, and raised the question of how general a mechanism it is. Many proteins were known to contain phosphate, so its potential seemed significant. At the time, however, the enzymes that added and removed the phosphates were unknown. It was obvious, though, that among the most interesting questions about them once they were discovered would be how they are controlled.

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In the mid 1960s I had the opportunity to spend a year as a postdoctoral fellow in the laboratory of Lipmann and worked on the elongation factors of Escherichia coli. To give a feeling for the times, it was soon after Ted Rall and Earl Sutherland proposed cyclic AMP as a second messenger of hormone action, leading somehow to the phosphorylation of an enzyme; and Marshall Nirenberg had recently announced that poly U encodes polyphenylalanine synthesis, the first step in deciphering the genetic code. One of the most hotly investigated and debated topics of the era was how enzymes are induced in bacteria. Cyclic AMP, phosphorylation, and gene expression were on everyone's mind, and one day a possible relationship between the induction of bacterial enzymes by cyclic AMP and the phosphorylation of nuclear proteins in eukaryotes was discussed in a lunch seminar. For me this discussion sparked a lifelong interest in how protein kinases mediate responses to hormones. In the mid 1960s, however, no one could predict whether cyclic AMP would act directly or indirectly on protein kinases, nor how widespread kinases would turn out to be.

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Figure 1  
The modes of activation of cyclic AMP-dependent protein kinase (PKA), cyclic GMP-dependent protein kinase (PKG), and protein kinase C (PKC). R indicates the regulatory subunit or region of the kinase, and C the catalytic subunit or region.

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At the end of the 1960s, I moved to Kobe and started to investigate the biological role of phosphorylation of nuclear proteins. At about this time, Ed Krebs and his colleagues discovered a kinase that cyclic AMP activated directly, and the evidence strongly suggested that this was the way that cyclic AMP stimulated glycogen breakdown physiologically (4290). Because it was dependent on cyclic AMP, this kinase was called protein kinase A, or PKA. H. Yamamura, a graduate student in my lab, had isolated a functionally unidentified kinase from rat liver using histone as a phosphate acceptor, and he confirmed that cyclic AMP greatly stimulated its enzymatic activity. He also observed that two enzyme peaks, cyclic AMP-dependent and independent, resulted when he subjected the enzyme preparation to column chromatography, although the two peaks showed otherwise identical catalytic properties. Most interesting was that cyclic AMP shifted the former form to the latter (4291). Soon, in 1970, four laboratories (Krebs, Lipmann, G. N. Gill, and ours) concurrently reported that PKA consists of catalytic and regulatory subunits, and that cyclic AMP activates the enzyme by dissociating these subunits, as shown on the left in Figure 1.

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In the decade of the 1970s, other protein kinases regulated by second messengers were found. Among them was cyclic GMP-dependent kinase (PKG), found first in arthropoda and later in mammalian tissues by Jef Kuo and Paul Greengard in 1970. M. Inoue, another graduate student in Kobe, found that, in contrast to PKA, PKG was a single polypeptide chain that was activated when cyclic GMP bound to a regulatory region within it. An inhibitory interaction between different regions of the polypeptide was suggested because limited proteolysis by trypsin or by calpain (Ca2+-dependent neutral protease) generated a constitutively active fragment of the enzyme which was no longer sensitive to cyclic GMP (4266)  (Figure 1). This was a key observation leading to our subsequent discovery of protein kinase C (PKC; C stands for its requirement for Ca2+).

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The discovery of protein kinase C

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In the course of studying PKG, we made the observation that rat brains which had been frozen contained a very active protein kinase which was independent of any cyclic nucleotide. The only requirement we could detect for this enzyme was for Mg2+, so we provisionally called it PKM. We considered the possibility that it was derived from PKG by limited proteolysis during storage, because it was not found in the brains of freshly sacrificed rats. Freezing and thawing fresh brain resulted in the appearance of PKM, suggesting the existence of such a proenzyme susceptible to proteolysis. Before long we found the proenzyme, which we could convert to PKM by limited proteolysis, but which was insensitive to any cyclic nucleotides (4267). This observation was curious since at the time we anticipated that the proenzyme would be PKG. Instead, the proenzyme appeared to be a new kinase which could be activated by limited proteolysis. We thought the protease involved was worth pursuing, and Y. Takai in my group began looking for it in different subcellular fractions by asking if they were capable of activating the proenzyme. He found that the particulate fraction — which is largely composed of the cell's membranes — was very effective at stimulating kinase activity, but all his attempts to purify the active component were unsuccessful. After much effort we finally realized that this was because the component was not a protein, but simply anionic phospholipids, especially phosphatidylserine. Rather than finding a protease, as we had intended, we had discovered that the intact "proenzyme" could be activated by lipids. We had found a new protein kinase, which we would later call PKC (Figure 1).

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Figure 2  
A difference in the ability of phospholipids from brain and erythrocytes to activate purified PKC in an in vitro reaction (top) led to the identification of diacylglycerol as an essential activator of the enzyme under physiological conditions. The data at the bottom show the PKC activity in a mixture of the purified enzyme with phosphatidylserine, diacylglycerol and a physiological concentration of Ca2+ ("complete"), and the results when each component is omitted individually. EGTA is an organic compound which chelates Ca2+ ions, making them unavailable to the enzyme.

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Even more curious was that phospholipids extracted from brain could activate the enzyme alone, whereas those from erythrocyte membranes could not produce any activation unless the reaction mixture was supplemented with a high concentration of Ca2+. These results are shown in the graph in Figure 2. Analysis of minor components in the brain phospholipids led us to conclude that diacylglycerol is an indispensable activator at physiological Ca2+ concentrations (4288), as shown in the table at the bottom of Figure 2. A small amount of diacylglycerol dramatically increased the apparent affinity of PKC for Ca2+, fully activating the enzyme without change in Ca2+ levels. Triacylglycerol, monoacylglycerol, and free fatty acid were totally inactive in this capacity. Despite knowing these details about how the enzyme worked, however, we knew nothing about its physiological role. Here the activation of the enzyme by diacylglycerol provided a clue because of a series of observations that had been made about how diacylglycerol is generated in membranes.

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In the early 1950s, when radioactive phosphate became available for experimental use in biology, M. and L. Hokin had observed that acetylcholine induced rapid labeling of acid-precipitable materials in some exocrine tissues such as pancreas. They later identified these materials as inositol phospholipid and phosphatidic acid (4263). It was clear that the rapid labeling of these lipids resulted from the acetylcholine-induced hydrolysis and subsequent resynthesis of inositol phospholipid, but its biological significance remained unclear for more than two decades. One suggestion was that this phospholipid hydrolysis might open Ca2+ gates because many hormones that elicit inositol phospholipid hydrolysis require Ca2+ for their actions (4275). The properties of the kinase that we had discovered suggested the additional possibility of a critical link between protein phosphorylation and the hormone-induced hydrolysis of inositol phospholipid, because the immediate lipid product of hydrolysis is diacylglycerol.

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Figure 3  
The chemical structures of a tumor-promoting phorbol ester and a membrane-permeable diacylglycerol (OAG) capable of activating PKC when added to cells.

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To test for such a link, we needed a method of introducing diacylglycerol into the membranes of live cells and a convenient in vivo system in which it was likely that diacylglycerol was mediating a signaling event. Experimentally adding purified natural diacylglycerols to cell membranes is not possible because the forms of diacylglycerol found in vivo have two long chain fatty acids. This makes them too insoluble in water to be transferred into membranes. We found that if we replaced one of the fatty acyl moieties with an acetyl group, then the resulting diacylglycerol (such as 1-oleoyl-2-acetyl-glycerol [OAG], shown on the right in Figure 3) is sufficiently water soluble to allow it to be dispersed into the lipid bilayer of a membrane, where it activates PKC directly. That solved the first problem. In the meantime, several reports in the literature had attracted our attention and suggested a cellular system that looked advantageous. In one, Susan Rittenhouse-Simmons reported how in thrombin-stimulated platelets diacylglycerol accumulated transiently, possibly as a result of inositol phospholipid hydrolysis. In two others Phil Majerus and Dick Haslam independently described how upon stimulation of platelets with thrombin, two endogenous proteins with molecular weights of 20 and 47 kDa were heavily phosphorylated.

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Figure 4  
Human platelets isolated from blood were suspended in medium containing radioactive phosphate and then thrombin (a natural stimulant of platelets), a soluble form of diacylglycerol (OAG), or phorbol ester was added. Each sample was then subjected to gel electrophoresis to show the proteins into which phosphate was incorporated. The 47 kDa protein that serves as an indicator of PKC activation is marked. The 20 kDa protein is phosphorylated by a Ca2+-dependent kinase. It is phosphorylated when the platelets are stimulated with thrombin, but not when either of the two compounds that stimulate only PKC is used instead.

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Figure 5  
Two convenient types of cells were examined to determine if PKC plays a role in their response to hormones. In each case both stimulation of PKC (by addition of the permeable diacylglyecerol to the cells) and elevation of Ca2+ in their cytoplasm (by addition of a Ca2+-ionophore) was required to evoke the response produced by the natural stimulant.

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Platelets looked to us like an excellent system to use to test if PKC activation or Ca2+ increase or both are needed for some cellular responses to hormones. Platelets can be isolated in biochemical quantities from blood, and undergo a dramatic response to release serotonin when stimulated with thrombin. It was already known that the 20 kDa protein is myosin light chain, and the enzyme responsible for this phosphorylation is a specific Ca2+-dependent myosin light chain kinase. The enzyme responsible for the phosphorylation of the 47 kDa protein, however, was unknown. Through extensive analysis in vitro and in vivo, for instance using the peptide fingerprinting technique, Y. Takai, Y. Kawahara and other colleagues in my lab were able to show that the 47 kDa protein, called pleckstrin today, is a substrate specific to PKC, and that this protein can be phosphorylated heavily by the addition of OAG (as shown in Figure 4). In short, PKC activation and Ca2+ increase could be induced separately and independently by the addition of OAG and Ca2+-ionophore, respectively, and the two endogenous proteins served as excellent markers for the activation of PKC and the increase in Ca2+ concentrations. In the spring of 1980, we were able to show in this way that Ca2+ increase and PKC activation were both essential, and together activated platelets as well as thrombin did (4269). Similarly, neutrophils required both PKC activation and Ca2+ increase for their activation. Figure 5 shows these results. These experiments clearly established a role for PKC in a cellular response to hormones and provided an important clue for investigating the biochemical mechanism of hormone actions within the cell.

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Figure 6  
PKC naturally acts as part of a branching pathway of signal transduction (on the left). In response to extracellular signals PIP2 is hydrolyzed to produce diacylglycerol (DG) and IP3. Diacylglycerol stimulates PKC, while IP3 interacts with a channel within the membrane of the ER to cause the release of Ca2+ into the cytoplasm. The same result can be achieved artificially without the extracellular signal (on the right) by adding a cell soluble form of diacylglycerol to activate PKC and Ca2+-ionophores to allow ions to enter from the exterior.

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On the way back from a meeting at the Royal Society in London in 1982, I stopped at Cambridge and met M. Berridge. We discussed where the Ca2+ in cells may come from, but this important question remained to be answered. A year later Berridge presented evidence that inositol-trisphosphate (IP3), the other half of inositol phospholipid, also acted as a second messenger by opening Ca2+ channels and releasing Ca2+ from an internal store within the cell (4286). The bifurcating pathway of signal transduction starting with inositol phospholipid hydrolysis emerged in this way in the early 1980s. This pathway, and the way that it can be experimentally short circuited by Ca2+-ionophores and permeable forms of diacylglycerol, is shown in Figure 6.

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Experiments for phorbol ester action

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At this point our work went in another direction, away from the actions of hormones and toward carcinogenesis. It had been known since 1941 that oil from the seeds of a small plant called a croton had a property called tumor promotion. The oil was not itself carcinogenic, but greatly enhanced the ability of other chemicals to cause tumors. The active component in this oil was later identified as phorbol ester, which is shown on the left in Figure 3. Since its discovery, this compound had attracted great attention among biologists interested in hormone actions as well as in carcinogenesis since, when applied to the cell, phorbol ester elicits a wide variety of responses that are very similar to those of hormones. In most cases the responses require Ca2+, and a number of studies had suggested that the primary site of action of phorbol ester is located on the plasma membrane, presumably its own specific receptor.

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In the summer of 1980, I attended the 4th International Conference on Cyclic Nucleotides in Brussels and was invited to a garden party at Prof. H. de Wulf's home. It was just after I had given a talk about a possible role of PKC in cell signaling. People were excited, and I discussed with Monique Castagna a potential connection between PKC activation and phorbol ester. Monique had spent the previous summer in the laboratory of P. Blumberg, who characterized the phorbol ester receptor (4260). In August of 1981, she joined us in Kobe for a month to examine a possible mechanism of phorbol ester action. Our idea was that phorbol ester would work much like a hormone, stimulating a receptor to cause diacylglycerol production and PKC activation. My group in Kobe had already established all the procedures needed to test this.

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The result, however, was extremely disappointing. In platelets, phorbol ester did not show any sign of producing diacylglycerol. The tumor promoter did, however, produce marked phosphorylation of the endogenous 47 kDa protein (shown in Figure 4) and massive release of serotonin in the presence of Ca2+-ionophore. This fact meant to us that our already-published idea that diacylglycerol is the mediator of hormone action could not be correct. This conclusion was, of course, very unsettling, particularly because I had been invited to be a speaker at a meeting in the middle of forthcoming September and had already sent an abstract clearly stating that diacylglycerol is an indispensable intracellular mediator for cellular responses.

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One sleepless night after this result, reading the review article by Blumberg, I noticed, to my surprise, that the phorbol ester contains a diacylglycerol-like structure very similar to the membrane-permeable diacylglycerol, OAG, that we routinely used. The structures of the two compounds are shown side-by-side in Figure 3. What would happen if phorbol ester could activate PKC directly? This insight occurred near the end of August. It turned out that I was right: a series of experiments that fall would show how phorbol ester mimics the action of diacylglycerol by increasing the affinity of PKC for Ca2+ and for phosphatidylserine, thereby activating the enzyme directly (4261). These results strongly suggested that PKC itself is the phorbol ester receptor.

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In March 1982, I presented our results for the first time. This talk attracted a great deal of attention. Over the following year, several groups found that PKC and phorbol ester receptor can be copurified, confirming our results. In my laboratory Ushio Kikkawa showed the stoichiometric binding of phorbol ester to PKC using the enzyme in pure form. These results and our understanding of the role of PKC replaced the traditional concept of tumor promotion with an explicit biochemical explanation. Whereas the physiological mediator diacylglycerol is only produced transiently in response to hormones, phorbol ester is not metabolizable and chronically activates PKC, thereby putting the cells in a stimulated state for a prolonged period of time. This was the first molecular explanation of a process known to promote carcinogenesis, and the first concrete link between signal transduction and cancer. Since these studies, phorbol esters and membrane-permeant diacylglycerols have been employed as crucial tools for the manipulation of PKC in intact cells, and have allowed the determination of the wide range of cellular processes regulated by this enzyme (4278). It was realized much later, however, that phorbol ester can bind to several other proteins as well, and potentially affect cell functions through several targets of signal molecules (4271).

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Subsequent events and the future

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Our work definitively established a role for PKC in cellular signal transduction, but it was not clear how many different signaling events it might be involved in. After our discovery of PKC and phorbol ester action in the early 1980s, studies that focused on PKC became widespread in many research fields in physiology and medicine including biochemistry, pharmacology, endocrinology, oncology, neurosciences, and developmental biology (4279). Today, it is clear that PKC plays pivotal roles in the transmembrane control of a wide variety of cellular functions, including exocytosis, cell growth and differentiation, apoptosis, and morphogenesis.

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Figure 7  
Schematic representation of the PKC family and other related kinases. A catalytic domain common to all members of this group of kinases is on the right. The ATP binding site is indicated within it in yellow. The members of the family differ in the regulatory region attached at the N-terminal end of the catalytic region. Each regulatory region consists of a collection of sites (indicated in green) at which signaling components (e.g. Ca2+ ions or diacylglycerol) or cellular structures (e.g. actin) bind. Green is used to indicate a binding site for any factor; most of the sites are not homologous to one another.

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Although once considered as a single entity, molecular cloning and enzymological analysis in the mid 1980s revealed the existence of multiple isoforms of PKC. The mammalian PKC family consists of at least 10 isoforms encoded by 9 genes (4280). The PKC isoforms are conserved in a variety of species including yeast, nematoda, fly, fish, and frog (4282). As shown in Figure 7, the conserved region of the proteins is a serine/threonine protein kinase domain that is located in the C-terminal half of each molecule and shows similar enzymatic properties in all of the proteins. The N-terminal half of each enzyme contains several functional domains unique to that isoform (4265). Several protein kinases which share kinase regions closely related to the PKC family also exist (4274).

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The mechanism of activation of members of the PKC family is more complicated than was originally assumed. Newly synthesized PKC molecules are catalytically inert and mature by phosphorylation, either by PKC itself (autophosphorylation) or by other protein kinases (4277). Activation by other kinases is a form of interaction of PKC with other signaling pathways. Cross talk has been revealed between the signaling pathway of inositol phospholipid hydrolysis and the phosphatidylinositol 3-kinase pathway. Cross talk involving activation of PKC by tyrosine phosphorylation also occurs. Thus the signaling pathway in which PKC participates is often only part of a larger network of pathways.

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It is now clear that the isoforms of PKC play a large number of physiological roles. Some are found in multiple cell types and play roles required of many or all types of cells. The most general isoform is PKCa, which is expressed in all tissues and cell types and has been implicated in a variety of cell functions including proliferation and differentiation (4276). The PKCd isoform may play a role in apoptotic processes (4272), and another, PKC?/?, is important for generating cell polarity and plays an essential role during the development of multicellular animals (4287). Other isoforms are restricted to particular cell types or tissues and apparently play more specialized roles. For example, PKC? is expressed solely in the central nervous system and may modulate neuronal transmission (4284), and the PKCß and PKC? isoforms appear to be essential for B- and T-cell differentiation and functions, respectively (4270; 4259). The PKC isoforms and their specific functions are described in a series of 15 minireviews recently published by the Japanese Biochemical Society (Journal of Biochemistry, the October 2002 through January 2003 issues).

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The most recent developments with PKC concern its location within the cell. Considerable evidence has accumulated supporting the concept that the protein components of signaling pathways are organized into complexes through their ordered association with scaffolding, adaptor or chaperone proteins. Such juxtaposition of signaling proteins and their substrates facilitates their tight regulation, specificity of action, and their compartmentalization within the cell. This is true of PKC. The proper functioning of PKC isoforms appears to depend on their specific intracellular localization. Upon stimulation of cells, different PKC isoforms are translocated or targeted to particular compartments within the cell, including the plasma membrane, the Golgi complex, mitochondria, and the cell nucleus (4265; 4285). Such specific localization is directed by the different functional domains in the various PKC isoforms, as well as by lipid mediators which are generated in membranes when cells are stimulated by various extracellular signals (4281). It is plausible that, in addition to diacylglycerol, many other lipid products produced transiently in membranes by the action of specific phospholipases play roles in such translocation and targeting of PKC family members to distinctly different intracellular compartments. Translocation and targeting appear to activate each isoform to phosphorylate substrate proteins and to perform specific functions at its destination (4285). Further exploration of the dynamic aspects of such lipid-protein interaction may unveil more of the molecular basis of transmembrane control of a wide range of physiological and pathological cellular processes.

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The author

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Dr. Yasutomi Nishizuka was Professor Emeritus at the Biosignal Research Center of Kobe University, Kobe, Japan. He was born in Ashiya, Japan, in 1932 and received both an M.D. and a Ph.D. from Kyoto University, finishing in 1963. From 1964 to 1968 he was an associate professor on the Faculty of Medicine of Kyoto University. In 1969 he moved to the Kobe University School of Medicine to become Professor and Chairman of the Department of Biochemistry, a position he held until 1995 when he became President of Kobe University. From 1992 to 1995 he also held a joint appointment as the Director of the Biosignal Research Center there. Exhausted, he retired in 2001 to become Professor Emeritus.

Throughout his career Dr. Nishizuka focused on mechanisms of signal transduction and how they are coordinated to regulate the responses of cells. For his work he was awarded membership in national scientific academies in the United States, Great Britain, Japan, France, Spain, and Germany, and was also an honorary fellow of the Asiatic Society of Calcutta, India, and an Associate Fellow of the Third World Academy of Sciences. He won a similar number of prominent awards worldwide for his work.

In addition to his scientific achievements, Dr. Nishizuka had the unusual experience of seeing his laboratory destroyed by an earthquake.

Dr. Nishizuka died late in 2004.

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Last Revised on February 1, 2005

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reviews
  • 4259 Altman, A., and Villalba, M. (2002).  Protein kinase C-theta (PKCtheta): a key enzyme in T cell life and death.  J. Biochem. (Tokyo) 132, 841-846.  PubMed  
  • 4260 Blumberg, P. M. (1980).  In vitro studies on the mode of action of the phorbol esters, potent tumor promoters: part 1.  Crit. Rev. Toxicol. 8, 153-197.  PubMed  
  • 4265 Hurley, J. H., and Misra, S. (2000).  Signaling and subcellular targeting by membrane-binding domains.  Annu. Rev. Biophys. Biomol. Struct. 29, 49-79.  PubMed   Journal
  • 4270 Kawakami, T., Kawakami, Y., and Kitaura, J. (2002).  Protein kinase C beta (PKCbeta): normal functions and diseases.  J. Biochem. (Tokyo) 132, 677-682.  PubMed  
  • 4271 Kazanietz, M. G. (2000).  Eyes wide shut: protein kinase C isozymes are not the only receptors for the phorbol ester tumor promoters.  Mol. Carcinog. 28, 5-11.  PubMed  
  • 4272 Kikkawa, U., Matsuzaki, H., and Yamamoto, T. (2002).  Protein kinase C delta (PKCdelta): activation mechanisms and functions.  J. Biochem. (Tokyo) 132, 831-839.  PubMed  
  • 4274 Mellor, H., and Parker, P. J. (1998).  The extended protein kinase C superfamily.  Biochem. J. 332, 281-292.  PubMed   Journal
  • 4275 Michell, R. H. (1975).  Inositol phospholipids and cell surface receptor function.  Biochim. Biophys. Acta 415, 81-147.  PubMed  
  • 4276 Nakashima, S. (2002).  Protein kinase C alpha (PKCalpha): regulation and biological function.  J. Biochem. (Tokyo) 132, 669-675.  PubMed  
  • 4277 Newton, A. C. (2001).  Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions.  Chem. Rev. 101, 2353-2364.  PubMed  
  • 4278 Nishizuka, Y. (1984).  The role of protein kinase C in cell surface signal transduction and tumour promotion.  Nature 308, 693-698.  PubMed  
  • 4279 Nishizuka, Y. (1986).  Studies and perspectives of protein kinase C.  Science 233, 305-312.  PubMed  
  • 4280 Nishizuka, Y. (1988).  The molecular heterogeneity of protein kinase C and its implications for cellular regulation.  Nature 334, 661-665.  PubMed   Journal
  • 4281 Nishizuka, Y. (1995).  Protein kinase C and lipid signaling for sustained cellular responses.  FASEB J. 9, 484-496.  PubMed  
  • 4282 Ohno, S. and Nishizuka, Y. (2002).  Protein kinase C isotypes and their specific functions: prologue.  J. Biochem. (Tokyo) 132, 509-511.  PubMed  
  • 4284 Saito, N., and Shirai, Y. (2002).  Protein kinase C gamma (PKCgamma): function of neuron specific isotype.  J. Biochem. (Tokyo) 132, 683-687.  PubMed  
  • 4287 Suzuki, A., Akimoto, K., and Ohno, S. (2003).  Protein kinase C lambda/iota (PKClambda/iota): a PKC isotype essential for the development of multicellular organisms.  J. Biochem. (Tokyo) 133, 9-16.  PubMed  
reviews
  • 4261 Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982).  Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters.  J. Biol. Chem. 257, 7847-7851.  PubMed  
  • 4263 Hokin, M. R., and Hokin, L. E. (1953).  Enzyme secretion and the incorporation of P32 into phospholipides of pancreas slices.  J. Biol. Chem. 203, 967-977.  PubMed  
  • 4266 Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1976).  Guanosine 3':5'-monophosphate-dependent protein kinase from silkworm, properties of a catalytic fragment obtained by limited proteolysis.  J. Biol. Chem. 251, 4476-4478.  PubMed  
  • 4267 Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977).  Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. II. Proenzyme and its activation by calcium-dependent protease from rat brain.  J. Biol. Chem. 252, 7610-7616.  PubMed  
  • 4269 Kawahara, Y., Takai, Y., Minakuchi, R., Sano, K., and Nishizuka, Y. (1980).  Phospholipid turnover as a possible transmembrane signal for protein phosphorylation during human platelet activation by thrombin.  Biochem. Biophys. Res. Commun. 97, 309-317.  PubMed  
  • 4285 Sakai, N., Sasaki, K., Ikegaki, N., Shirai, Y., Ono, Y., and Saito, N. (1997).  Direct visualization of the translocation of the gamma-subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein.  J. Cell Biol. 139, 1465-1476.  PubMed  
  • 4286 Streb, H., Irvine, R. F., Berridge, M. J., and Schulz, I. (1983).  Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate.  Nature 306, 67-69.  PubMed  
  • 4288 Takai, Y., Kishimoto, A., Kikkawa, U., Mori, T., and Nishizuka, Y. (1979).  Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system.  Biochem. Biophys. Res. Commun. 91, 1218-1224.  PubMed  
  • 4290 Walsh, D. A., Perkins, J. P., and Krebs, E. G. (1968).  An adenosine 3',5'-monophosphate-dependant protein kinase from rabbit skeletal muscle.  J. Biol. Chem. 243, 3763-3765.  PubMed  
  • 4291 Yamamura, H., Takeda, M., Kumon, A., and Nishizuka, Y. (1970).  Adenosine 3',5'-cyclic phosphate-dependent and independent histone kinases from rat liver.  Biochem. Biophys. Res. Commun. 40, 675-682.  PubMed  

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