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historical perspective The origins of

Philip Cohen

The reversible phosphorylation of is central to the regulation of most aspects of cell func- tion but, even after the first protein was identified, the general significance of this discovery was slow to be appreciated. Here I review the discovery of and give a per- sonal view of the key findings that have helped to shape the field as we know it today.

he days when protein phosphorylation was an abstruse backwater, best talked Tabout between consenting adults in private, are over. My colleagues no longer cringe on hearing that “ kinase phosphorylates phosphorylase” and their eyes no longer glaze over when a “”kinase kinase kinase” is mentioned. This is because protein phosphorylation has gradu- ally become an integral part of all the sys- tems they are studying themselves. Indeed it would be difficult to find anyone today who would disagree with the statement that “the reversible phosphorylation of proteins regu- lates nearly every aspect of cell ”. Phosphorylation and , catalysed by protein and protein , can modify the function of a protein in almost every conceivable way; for Carl and Gerty Cori, the 1947 Nobel Laureates. Picture: Science Photo Library. example by increasing or decreasing its bio- logical activity, by stabilizing it or marking it for destruction, by facilitating or inhibiting movement between subcellular compart- so long before its general significance that catalysed the phosphory- ments, or by initiating or disrupting pro- was appreciated? lation of casein3. Soon after, Fischer and tein–protein interactions. The simplicity, Krebs4,5, as well as Wosilait and Sutherland6, flexibility and reversibility of phosphoryla- found that the interconversion of phospho- tion, coupled with the ready availability of Regulating by phosphorylation rylase b to phosphorylase a involved a ATP as a phosphoryl donor, explains its In the late 1930s Carl and Gerty Cori dis- phosphorylation/dephosphorylation mech- selection as the most general regulatory covered that there were two forms of glyco- anism. In particular, Fischer and Krebs4,5 device adopted by eukaryotic cells. gen phosphorylase (called b and a), the demonstrated that the b form could be con- It is thought that perhaps 30% of the enzyme that catalyses the rate-limiting step verted to the a form in the presence of Mg- proteins encoded by the genome of . Phosphorylase b was only ATP and an enzyme they termed phospho- contain covalently bound , and active in the presence of 5′ AMP, whereas rylase kinase4,5. was abnormal phosphorylation is now recog- phosphorylase a was active in the absence of subsequently shown to catalyse the transfer nized as a cause or consequence of many this nucleotide. They reasoned (incorrectly) of the γ-phosphoryl group of ATP to a spe- human diseases. A number of naturally that phosphorylase a must contain tightly cific residue on phosphorylase b 7. occurring toxins and tumour promoters bound 5′ AMP, and that the enzyme that The reconversion of phosphorylase a to exert their effects by targeting particular converts phosphorylase a to phosphorylase phosphorylase b was therefore catalysed by protein kinases and phosphatases. A topical b, discovered in 1943 (ref. 2), must catalyse a ‘phosphate-releasing’ (or PR!) enzyme, example is the cyclic heptapeptide micro- the removal of 5′ AMP.The effect of 5′ AMP today called protein 1 to reflect cystin, which has just been listed as a “noti- on phosphorylase b was the first example of its much wider use in cell regulation8. fiable dangerous substrance”, along with allosteric activation, but, because this term In 1950, Earl Sutherland showed that anthrax, in the Anti-terrorism, Crime and would not be coined for another 20 years, glycogenolysis could be stimulated if liver Security Act of 2001 recently approved by they called the a-to-b converting enzyme slices were incubated with adrenalin or the British parliament. Microcystin, pro- ‘prosthetic-group-removing’ (or PR) ; he subsequently showed that the duced by toxic blue-green algae, is a potent enzyme2. But the Coris’ never demonstrated activity of phosphorylase a was increased hepatotoxin and liver carcinogen that that PR enzyme released 5′ AMP from under these conditions (reviewed in ref. 9). inhibits members of one of the major fam- phosphorylase a and, although they received This was the first demonstration that a hor- ilies of protein phosphatases1. a Nobel Prize in 1947 for “discovering the mone could influence the activity of a spe- In view of these developments, it seems course of the catalytic conversion of glyco- cific enzyme, although the response was lost timely to reflect on the early days of gen”, many years passed before the true if the liver slices were homogenized. But, research on protein phosphorylation. How nature of the reaction was discovered. when the activation mechanism of phos- was this phenomenon originally discovered activity was first observed phorylase was discovered, it became obvi- as a control mechanism and why did it take in 1954 when Kennedy described a ous that Mg-ATP would be necessary for

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adipose tissue and how glucagon inhibits Adrenalin Electrical in the liver. But the widespread excitation distribution of PKA in animal tissues and other organisms suggested an even wider range of functions19. More substrates were cAMP identified, such as cardiac troponin I (ref. 20) and phospholamban21, which explained how adrenalin regulates the rate and force of heart-muscle contractility. This cAMP-dependent protein kinase extended the involvement of phosphoryla- tion to proteins that are not , although the demonstration by Tom Langan in 1969 that PKA phosphorylates 22 Phosphorylase Phosphorylase H1 at a specific serine residue had already kinase Kinase hinted at this possibility. (inactive) (active) The first -dependent protein kinases were identified in the late 1970s and included myosin light-chain kinase23, phos- 15 Ca2+ phorylase kinase and calmodulin-depend- ent protein kinases I and II in the brain24. The subsequent realization that calmod- Phosphorylase b Phosphorylase a ulin-dependent protein kinase II has multi- 2+ (active) (active) ple functions in Ca -signalling akin to PKA (ref. 25), and especially the discovery that (ref. 26) is activated by the second messenger diacylglycerol, broad- ened the concept of second-messenger- Glycogenolysis dependent protein kinases. Some of the major serine/- specific protein phosphatases were classi- Figure 1 The glycogenolytic cascade in mammalian . Adrenalin stimulates fied during the late 1970s and early 1980s the production of 3′ 5′ cyclic (cAMP) leading the sequential (ref. 8), and mechanisms by which they are activation of cAMP-dependent protein kinase and phosphorylase kinase. The latter con- regulated began to be identified. Prominent verts phosphorylase from the inactive dephosphorylated b form to the active among these was the characterization in phosphorylated a form, stimulating glycogenolysis in advance of an increased energy 1981 of the calmodulin-dependent protein demand. The activity of phosphorylase kinase also depends on ions and is phosphatase 2B (also termed calcineurin)27, therefore also switched on during muscle contraction. This provides energy (via the which 10 years later was shown to be the 28 breakdown of glycogen) to sustain muscle contraction. target for cyclosporin , the immunosup- pressant drug that made organ transplants possible. In 1975, PKA was shown to phosphory- activation; addition of Mg-ATP did indeed Ca2+ receptors of eukaryotic cells) was one late in proteolytic digests of restore the response to . The of its subunits15. These findings explained basic protein29, and this led to the realization reconstruction of a response in a how glycogenolysis and muscle contraction that PKA phosphorylates serine residues in cell-free system was a major breakthrough were synchronized (Fig. 1). But by the end specific amino-acid sequence motifs30,31. that led to the discovery that adrenalin of the 1960s, 15 years after phosphorylase These studies paved the way for the devel- exerted its effects by generating a small, kinase had been discovered, phosphoryla- opment of synthetic substrates that heat-stable factor later identified as 3′5′ tion was still thought of as a rather special- have been a key technical advance in the cyclic adenosine monophosphate (or cyclic ized control mechanism largely confined to study of protein phosphorylation. AMP). The remarkable story of how the first the regulation of one In retrospect, the determination of the ‘second messenger’ was identified is beauti- (glycogen ). amino-acid sequence of the first protein fully described in the first chapter of Cyclic kinase (PKA) in the early 1980s (ref. 32) AMP (ref. 9) published in 1970, the year was more significant than it seemed at the before Sutherland received a Nobel Prize. Phosphorylation develops time (at least to me!), because it allowed It took much longer before other impor- It was through the 1970s and early 1980s geneticists to understand the functions of tant missing pieces of the jigsaw were put in that the general significance of protein several regulatory that they had iden- place. These included the discovery of phosphorylation came to be appreciated. tified. In particular, cdc2, the cell-cycle con- cAMP-dependent protein kinase (PKA) Lester Reed’s discovery in 1969 that the trol gene identified by Paul Nurse, was and the finding that it activated phosphory- mitochondrial shown to be a protein kinase33, a discovery lase kinase10, the first example of a ‘cascade’ complex was inactivated by phosphoryla- recognized last year by a Nobel Prize. in which one protein kinase activates tion16 was one of the first hints that this The 1970s also furnished the first exam- another. PKA was also found to inhibit control mechanism might operate in other ples of proteins that are phosphorylated on glycogen synthase11,12, the first example of metabolic pathways and organelles. This two or more residues by two or more kinas- enzyme inhibition by phosphorylation. view was strengthened when PKA was es, termed multisite phosphorylation34, Another crucial finding was that phospho- shown to activate hormone-sensitive which we now know to be the norm rather rylase kinase activity also depends on lipase17 and to inhibit L-type pyruvate than the exception. Ed Fischer and Ed Krebs another second messenger, namely calcium kinase18. These observations also helped to have often said they were fortunate in ions13,14, and that calmodulin (one of major explain how adrenalin stimulates lipolysis in studying , because

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it is one of the few proteins phosphorylated kinase cascade was worked out during the at a single site by a single kinase. Had this early 1990s through the efforts of a num- enzyme been as complex as, say, glycogen ber of laboratories. This was followed by synthase (which eventually turned out to be the dissection of many other MAP kinase phosphorylated at nine serine residues by at cascades that are important in protecting least six different kinases35), it might have cells against cellular stresses, cell-damag- taken much longer to sort out what was ing agents and infection by pathogenic going on. organisms. The 1980s also led to the realization that A phosphatidylinositol (PtdIns) protein phosphatases and kinases do not 3-kinase, or PI(3)K, activity associated with always find their substrates by simple diffu- Src and the platelet-derived growth factor sion, but are frequently directed to particu- (PDGF) was identified in the late lar subcellular locations by ‘targeting’ pro- 1980s (ref. 54). After the discovery of teins with which they interact. The glyco- PtdIns(3,4,5)trisphosphate (PIP3) in neu- Ed Krebs and Eddy Fischer in 1992, after hearing gen-targeting subunit of protein phos- trophils as an inositol phospholipid pro- phatase-1 (ref. 36) and proteins that inter- about their award of the Nobel Prize in Physiology duced in response to fMet–Leu–Phe act with the RIIβ regulatory subunit of PKA and Medicine. (ref. 55), it became clear that this com- (ref. 37), later called A-kinase anchoring With permission from the University of Washington. pound was formed from PtdIns(4,5)bis- proteins (AKAPs; reviewed in ref. 38) were phosphate by the action of class 1 PI(3)Ks the first examples of this important and (reviewed in ref. 56). The importance of widespread phenomenon. The late 1980s and early 1990s saw the PIP3 as a second messenger in and cloning of the JAK kinases, a new family of growth-factor signalling emerged when rel- protein kinases. JAK is an acronym atively specific inhibitors of PI(3)K Protein tyrosine kinases of ‘just another kinase’ (in recognition that, (refs 57,58) were found to block many of The discovery by Ray Erikson that v-Src, the like many other kinases, it emerged from a the effects of these signals. A PIP3-depend- protein encoded by the transforming gene PCR-based screen) or (in ent protein kinase cascade that has a major of Rous sarcoma virus, was a kinase39 was a recognition of their two-kinase domain function in mediating cellular responses landmark event in the late 1970s. This led structures reminiscent of the two-faced triggered by insulin and growth factors was Tony Hunter to the surprising finding that Roman god Janus)47. These enzymes are identified in 1995 (refs 59–61), culminating v-Src phosphorylates tyrosine residues in activated at the plasma membrane after in the discovery of 3-phosphoinositide- proteins40. The cytokines and have been dependent protein kinase in 1997 (ref. 62). (EGF) receptor was also shown to be a pro- engaged by their receptors. After the JAK tein , switched on when EGF kinases are activated, their substrates, the engages the receptor41. Similar findings ‘signal tranducers and activators of tran- Future trends were made for the insulin receptor a couple scription’ (STATs; reviewed in ref. 48), are Our understanding of protein phosphory- of years later42. phosporylated; modified STATs then medi- lation has now reached the stage where its The discovery that many growth-factor ate directly. The identification importance in almost every physiological receptors were protein tyrosine kinases of a signalling pathway in which the sub- event is recognized. A well-deserved Nobel stimulated the search for their physiologi- strate is phosphorylated at the plasma Prize was awarded to Ed Fischer and Ed cal substrates. But, surprisingly, it was the membrane and then migrates to the nucle- Krebs in 1992 for their pioneering studies receptors themselves that seemed to be the us to regulate transcription without any in this area. But there are, undoubtedly, most prominent cellular substrates, fre- other intervening steps was initially met many surprises still in store, and our quently becoming phosphorylated at mul- with astonishment. But the persuasive understanding of signal integration is still tiple tyrosine residues. These puzzling genetic evidence validating these conclu- in its infancy. In particular, we now realize observations were explained when it was sions led to rapid acceptance by the scien- that regulatory circuits are ‘wired up’ in shown that proteins containing the Src tific community. distinct ways in different cells. Therefore, homology 2 (SH2) domain43 are able to although transformed cell lines have been bind directly to phosphorylated growth- invaluable in helping us to dissect particu- factor receptors because of their ability to Cascading protein kinases lar signalling pathways, I would expect the recognize particular phosphotyrosine-con- Some have dubbed the 1990s as the decade emphasis of research (apart from ) taining sequences44. Receptor ‘autophos- of protein kinase cascades. It is surprising to shift increasingly to the analysis of ‘real’ phorylation’ is therefore critical in induc- that, although the first protein kinase cas- cells and tissues. ing the binding sites for cytoplasmic targets cade was identified in 1968 (ref. 10), it took One of the major gaps in our knowledge with SH2 domains, which then stimulate more than 20 years before further examples concerns the identities of the key substrates ‘downstream’ pathways to mediate the of this phenomenon were identified. An of protein kinases and how their phospho- effects of the signal. insulin-stimulated protein kinase that phos- rylation contributes to the changes in cell The first protein tyrosine phosphatase phorylated -associated protein- physiology evoked in response to particular (PTP1B) was purified in the late 1980s 2 (MAP2) was identified in the late 1980s signals. If a third of the 30,000 proteins (ref. 45) and there was great excitement and termed MAP kinase49 (its name was encoded by the human genome contain when it was found to be homologous to later changed to mitogen-activated protein covalently bound phosphate, an ‘average’ leukocyte common antigen CD45 (ref. 46), kinase — still MAP kinase — to reflect its protein kinase (on the basis of the proba- often found on the surface of haematopoi- activation by many mitogens and growth ble number of protein kinases) would be etic cells. Before this, the function of CD45 factors in different cells). This enzyme was expected to phosphorylate about 20 differ- was unknown. These discoveries generated found to be activated by the phosphoryla- ent proteins in vivo, and an ‘average’ pro- enormous interest in this new family of tion of a threonine and a tyrosine residue50, tein phosphatase would be expected to enzymes and nearly 100 members were catalysed by a ‘dual specificity’ MAP kinase dephosphorylate 60 proteins. These num- subsequently identified, including many kinase51,52, through a Ras-dependent sig- bers are conservative, in part because receptor tyrosine phosphatases. nalling pathway53. This ‘classical’ MAP closely related protein kinase and protein

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Engstrom, L. Biochem. Biophys. Res. Commun. 70, 696–703 phosphatase isoforms would be expected to questions will be a major challenge for (1976). have overlapping specificities, and many many years to come. 31. Kemp, B. E., Benjamini, E. & Krebs, E. G. Proc. Natl. Acad. Sci. proteins are regulated by multisite phos- Finally, I should mention that recent USA 73, 1038–1042 (1976). phorylation. Moreover, although some pro- events have propelled the development of 32. Shoji, S. et al. Proc. Natl Acad. Sci. USA 78, 848–851 (1981). 33. Nurse, P. Trends Genet. 1, 51–55 (1985). tein kinases are dedicated to the phospho- specific protein-kinase inhibitors for treat- 34. Cohen, P. Trends Biochem. Sci. 1, 38–40 (1976). rylation of one, or just a few substrates (e.g. ing disease right to the top of the pharma- 35. Nakielny, S., Campbell, D. G. & Cohen, P. Eur. J. Biochem. phosphorylase kinase and MAP kinase ceutical agenda. There is going to be a huge 199, 713–722 (1991). kinases), others must have several hundred push over the next 20 or 30 years to devel- 36. Stralfors, P., Hiraga, A. & Cohen, P. Eur. J. Biochem. 149, 295–303 (1985). substrates. Furthermore, the substrates of op drugs that modulate the activities of 37. Bregman, D. B., Bhattacharyya, N, and Rubin, C. S. J. Biol. protein kinases are frequently cell-specific, specific protein kinases and phosphatases. Chem. 266, 4648–4656 (1989). explaining the distinctive effects of different But the remarkable surge of interest in this 38. Colledge, M. & Scott, M. D. Trends Cell Biol. 9, 216–221 (1999). signals in different tissues. The identifica- area over the past few years is another story, 39. Collett, M. S. & Erikson, R. L. Proc. Natl Acad. Sci. USA 75, 2021–2024 (1978). 68 tion of the major substrates of each protein which I cover in another article . 40. Hunter, T. & Sefton, B. M. Proc. Natl Acad. Sci. USA kinase and phosphatase is a massive under- Philip Cohen is at the MRC Protein 77, 1311–1315 (1980). taking, which is going to take at least sever- Phosphorylation Unit, School of Life Sciences, 41. Ushiro, H. & Cohen, S. J. Biol. Chem. 255, 8363–8365 (1980). al decades to solve. But it is at the heart of University of Dundee, Scotland, UK. 42. Kasuga, M., Karlsson, F. A. & Kahn, C. R. Science 215, 185–187 (1982). much that we want to know in the twenty- e-mail: [email protected] 43. Sadowski, I., Stone, J. C. & Pawson, T. Mol. Cell. Biol. first century, including the molecular basis 1. MacKintosh, C., Beattie, K. A., Klumpp, S., Cohen, P. & 6, 4396–4408 (1986). of embryogenesis and the functioning of Codd, G. A. FEBS Lett 264, 187–192 (1990). 44. Anderson, A., Koch, C. A., Grey, L., Ellis, C., Moran, M. F. & the brain. More powerful methods are 2. Cori, G. T. & Green, A. A. J. Biol. Chem. 151, 31–38 (1943). Pawson, T. Science 250, 979–982 (1990). 3. Burnett, G. & Kennedy, E. P. J. Biol. Chem. 211, 969–980 45. Tonks, N. K., Diltz, C. D. & Fischer, E. H. J. Biol. Chem. 263, needed to identify the substrates of particu- (1954). 6722–6730 (1988). lar protein kinases and phosphatases, cou- 4. Fischer, E. H. & Krebs, E. G. J. Biol. Chem. 216, 121–132 46. Tonks, N. K., Charbonneau, H., Diltz, C. D., Fischer, E. H. & pled with further exploitation of important (1955). Walsh, K. A. 27, 8695–8701 (1988). methodological and technical advances, 5. Krebs, E. G. & Fischer, E. H. Biochim. Biophys. Acta 20, 150–157 47. Wilkes, A. F., Harpur, A. G., Kurban, R. R., Ralph, S. J., (1956). Zürcher, G. & Ziemiecki, A. Mol. Cell. Biol. 11, 2057–2065(1991). such as the development of phospho-spe- 6. Sutherland, E. W. & Wosilait, W. D. Nature 175, 169–170 48. Wilks, A. F. & Harpur, A. G. Bioessays 16, 313–320 (1994). cific , specific cell-permeant (1955). 49. Ray, L. B. & Sturgill, T. W. Proc. Natl Acad. Sci. USA inhibitors of protein kinases and phos- 7. Fischer, E. H., Graves, D. J., Crittenden, E. R. S. & Krebs, E. G. 84, 1502–1506 (1987). J. Biol. Chem. 234, 1698–1704 (1959). 50. Anderson, N., Maller, J. L., Tonks, N. K. & Sturgill, T. W. Nature phatases, and cell lines that do not express a 8. Ingebritsen, T. S. & Cohen, P. Eur. J. Biochem. 132, 255–261 343, 651–653 (1990). particular protein kinase or phosphatase. (1983). 51. Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K. In a review article I wrote for Nature 9. Robison, G. A., Butcher, R. W. & Sutherland, E. W. Cyclic AMP & Krebs, E. G. J. Biol. Chem. 266, 4220–4227 (1991). 20 years ago63, I used the term ‘silent’ to Academic Press, London (1971). 52. Gomez, N. & Cohen, P. Nature 353, 170–173 (1991). 10. Walsh, D. A., Perkins, J. P. & Krebs E. G. J. Biol. Chem. 53. Leevers, S. J. & Marshall, C. J. EMBO J. 11, 569–574 (1992). describe phosphorylation sites that did not 243, 3763–3765 (1968). 54. Whitman, M., Downes, C. P., Keeler, M., Keller, T. & Cantley, L. seem to influence the activities of enzymes 11. Bishop, J. S. & Larner, J. Biochim. Biophys. Acta 171, 374–377 C. Nature 332, 644–646 (1988). directly. Many such sites have been identi- (1969). 55. Traynor-Kaplan, A. E., Harris, A. L., Thompson, B. L., Taylor, P. fied. Do silent have 12. Soderling, T. R. et al. J. Biol. Chem. 245, 6617–6628 (1970). & Sklar, L. A. Nature 334, 353–356 (1988). 13. Heilmeyer, L. M. G.Jr, Meyer, F., Haschke, R. H. & Fischer, E. H. 56. Toker, A. & Cantley, L. C. Nature 387, 673–676 (1997). important physiological functions that we J. Biol. Chem. 245, 6649–6656 (1970). 57. Vlahos, C. J., Matter, W. F., Hui, K. Y. & Brown, R. F. J. Biol. do not yet understand, or is there, as Ed 14. Brostrom, C. O., Hunkeler, F. L. & Krebs, E. G. J.Biol. Chem. Chem. 269, 5241–5248 (1994). Krebs has suggested, a considerable amount 246, 1961–1967 (1971). 58. Ui, M., Okada, T., Hazeki, K. & Hazeki, O. Trends Biochem. Sci. of noise in the system? 15. Cohen, P., Burchell, A., Foulkes, J. G. & Cohen, P. T. W. FEBS 20, 303–307 (1995). Lett. 92, 287–292 (1978). 59. Franke, T. F. et al. Cell 81, 727–736 (1995). There was evidence many years ago that 16. Linn, T. C., Pettit, F. H. & Reed, L. J. Proc. Natl Acad. Sci. USA 60. Burgering, B. M.Th and Coffer, P. J. Nature 376, 599–602 (1995). there are protein kinases that phosphorylate 62, 234–241 (1969). 61. Cross, D. A. E., Alessi, D. R., Cohen, P., Anjelkovic, M. & and residues64; it was also 17. Corbin, J. D., Reimann, E. M., Walsh, D. A. & Krebs, E. G. Hemmings, B. A. Nature 378, 785–788 (1995). pointed out at that time that the acid labil- J. Biol. Chem. 245, 4849–4851 (1970). 62. Alessi, D. R. et al. Curr. Biol. 7, 261–269 (1997). 18. Ljungstrom, O., Hjelmqvist, G. & Engstrom, L. Biochim. 63. Cohen, P. Nature 296, 613–620 (1982). ity of these phosphorylated residues make Biophys. Acta 358, 289–298 (1974). 64. Smith, D. L., Chen, C-C, Bruegger, B. B., Holtz, S. L., Halpern, them difficult to study with conventional 19. Kuo, J. F. & Greengard, P. Proc. Natl Acad. Sci. USA R. M. & Smith, R. A. Biochemistry 13, 3780–3785 (1974). methods. Although protein kinases that 64, 1349–1355 (1969). 65. Swanson, R. V., Alex, L.A. & Simon, M. I. Trends Biochem. Sci. phosphorylate histidine residues are now 20. England, P. J. FEBS Lett. 50, 57–60 (1975). 19, 439–518 (1994). 21. Tada, M., Kirschberger, M. A. & Katz, A. M. J. Biol. Chem. 66. Levy-Favatier, F., Delpech, M. & Kruh, J. Eur. J. Biochem. well established as integral components of 250, 2640–2647 (1975). 166, 617–621 (1987). 65 sensing mechanisms in bacteria , it is still 22. Langan, T. A. Proc. Natl Acad. Sci. USA 64, 1276–1283 (1969). 67. Khandelwahl, R. M., Mattoo, R. L. & Waygood, E. B. FEBS Lett. unclear whether the phosphorylation of 23. Dabrowska, R., Sherry, J. M. F., Aromatorio, D. K. & 162, 127–132 (1983). Hartshorne, D. J. Biochemistry 17, 253–258 (1978). 68. Cohen, P. Nature Rev. Drug Disc. 1, 309–315 (2002). residues other than serine, threonine and 24. Schulman, H. & Greengard, P. Nature 271, 478–479 (1978). tyrosine is widespread or significant in 25. McGuinness, T. L., Lai, Y., Greengard, P., Woodgett, J. R. & mammalian cells. Are protein lysine kinas- Cohen, P. FEBS Lett. 163, 329–334 (1983). Acknowledgements es, protein kinases66,or even pro- 26. Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U. & Nisdhizuka, Y. This perspective is a personal account of some of the important J. Biol. Chem. 255, 2273–2276 (1980). events in protein phosphorylation research that have taken place tein kinases that use phosphoryl donors 27. Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B. & over the past 50 years or more. I apologize to the scientists whose other than ATP (e.g. phosphoenolpyru- Cohen, P. FEBS Lett. 137, 80–84 (1982). many important discoveries that could not be included or refer- vate67), represented among the many pro- 28. Liu, J., Farmer, J. D., Lane, W. S., Friedman, J., Weissman, I. & enced in this article because of space restrictions. The work carried Schreiber, S. L. Cell 66, 807–815 (1991). out in my laboratory is supported by the UK Medical Research teins in the human genome for which a 29. Daile, P. & Carnegie, P. R. & Young, J. D. Nature 257, 416–418 Council, The Royal Society of London, Diabetes U.K., The Louis function has yet to be ascribed? Providing (1975). Jeantet Foundation, AstraZeneca, Boehringer Ingelheim, the answers to these and other intriguing 30. Zetterqvist, O., Ragnarsson, U., Humble, E., Berglund, L. & GlaxoSmithKline, NovoNordisk and Pfizer.

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