PERSPECTIVE PERSPECTIVE Discovering the first tyrosine kinase Tony Hunter1 Salk Institute for Biological Studies, La Jolla, CA 92037-1002

Edited by Joseph Schlessinger, Yale University School of Medicine, New Haven, CT, and approved May 26, 2015 (received for review April 27, 2015)

In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson’s group that the Rous sarcoma virus (RSV) v-Src–transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src–associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month.

Transmissible RNA and DNA tumor viruses transformation; however, molecular mecha- of the v-Src protein (2), but Joan Brugge and that caused tumors in vertebrate animals nisms of viral transformation remained Ray Erikson, using an antitumor serum, beat were discovered in the early and middle parts shrouded in mystery. The breakthrough us to the post, reporting in September 1977 of the 20th century, leading to the idea that came with the discovery that the v-Src that v-Src is a 60-kDa protein (3), and sub- they might serve as models of human cancer. protein was associated with a protein kinase sequently that a related 60-kDa c-Src protein But not until they were adapted for infection activity in vitro and that the kinase activity is present in normal cells. This finding was and growth in tissue culture cells was it pos- of v-Src from ts mutant RSV was temper- quickly followed by Marc Collett and Ray sible to exploit these viruses for mechanistic ature-sensitive (1). From the outset, the idea Erikson’s remarkable discovery, reported in studies of carcinogenesis. In the 1970s, excite- that protein phosphorylation might be a PNAS in April 1978 (1), that WT v-Src pro- mentmountedasthefirstmolecularanalyses mechanism of viral transformation was very tein isolated in an immunoprecipitate (IP) of both types of tumor virus began to teach attractive, because of the well-known role of made with tumor serum had an associated us about the viral genes involved in cell trans- reversible phosphorylation in regulating pro- protein kinase activity that was lacking when formation. Partial nucleotide sequences of tein function and activity. This discovery ts mutant v-Src was isolated from cells at the the SV40 and polyomavirus (Py) small DNA prompted a flurry of activity in laboratories nonpermissive temperature (we had already tumor viruses were reported, enabling predic- around the world working on other tumor learned from Ray Erikson about this discov- tion of ORFs, and this information, com- viruses, who immediately set out to test ery in January that year). Surprisingly, the – bined with the use of antisera from tumor- whether their favorite viral transforming v-Src–associated kinase activity phosphory- bearing animals, led to the identification of protein had a protein kinase activity in lated the heavy chain of the precipitating an- virally encoded proteins that might be re- the hope that aberrant protein phosphoryla- tibodies. By determining which Ig heavy chain sponsible for malignant transformation. The tion might be a universal mechanism of amino acid was being phosphorylated in the SV40 and Py papovaviruses both encode a malignant transformation. — reaction, they found that the phosphorylated large and a small T (tumor) antigen, but Py Unbeknownst to each other, three groups residue was phosphothreonine (pTh)r; at the was found to encode a third, middle-sized T Alan Smith together with Mike Fried at the time, phosphoserine (pSer) and pThr were antigen from an overlapping reading frame. Imperial Cancer Research Fund (ICRF) in the only known phosphoamino acids present The Py and SV40 T antigen proteins are London, Brian Schaffhausen with Tom in proteins. Independently, the group of expressed early in infection before replication Benjamin at Harvard Medical School, and our Michael Bishop and Harold Varmus reported of viral DNA and trigger proliferation of rest- group, in the Tumor Virology Laboratory in October 1978 that v-Src has an associated ing cells. The ∼60-kDa middle T antigen was (TVL) at the Salk Institute, who had all deduced to be most important for Py trans- played a part in characterizing the Py middle formation, initially because of its absence in T(mT)protein—set out to test whether mT Author contributions: T.H. wrote the paper. cells infected with transformation defective also had an associated protein kinase activity. The author declares no conflict of interest. Py mutants. At about the same time, the At that time in 1978, my group, in collabo- This article is a PNAS Direct Submission. v-Src–transforming protein of the Rous sar- ration with Karen Beemon and Bart Sefton, This article is part of the special series of PNAS 100th Anniver- coma virus (RSV) RNA tumor virus was iden- was already working on RSV. In 1976, we sary articles to commemorate exceptional research published in “ tifiedasa60-kDaprotein,againthroughthe had set out to try to identify the elusive PNAS over the last century. See the companion article, Trans- – forming gene product of Rous sarcoma virus phosphorylates use of tumor sera. v-Src transforming protein, using the newly tyrosine” on page 1311 in issue 3 of volume 77, and see Inner The need for protein products of viral developed mRNA-dependent reticulocyte Workings on page 7886. genomes for tumorigenesis was originally lysate in vitro translation system pro- 1Email: [email protected].

deduced through the isolation and use of grammed with RSV virion RNA. We suc- This article contains supporting information online at www.pnas.org/ viral mutants temperature sensitive (ts) for ceeded in identifying C-terminal fragments lookup/suppl/doi:10.1073/pnas.1508223112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1508223112 PNAS | June 30, 2015 | vol. 112 | no. 26 | 7877–7882 Downloaded by guest on October 4, 2021 protein kinase activity that also phosphorylates the heavy chain of antibodies in their anti-RSV tumor serum, but they did not identify the phosphorylated amino acid (4). August 1978–May 1979 Prompted by the Erikson group’s success in using anti-RSV tumor sera, we set out to raise our own anti-RSV tumor sera by inoculating baby rabbits with a mammotropic strain of RSV, obtaining our first active tumor serum in November 1977. We had used this serum to show that an IP of in vitro-translated v-Src had protein kinase activity that phosphory- lated the Ig heavy chain and that the kinase activity of in vitro-translated ts mutant v-Src was greatly reduced, providing strong sup- port for the conclusion that this activity is intrinsic to v-Src, as we reported in a paper submitted in November 1978 (5). It was our experience with the RSV IP kinase assay that allowed Mary Anne Hutchinson, Walter Eckhart, and me to quickly set up and test whether IPs made with an anti-Py tumor Fig. 1. First experiments demonstrating polyoma middle T and v-Src tyrosine kinase activity. (A) Autoradiogram (4 d serum contained a T antigen-associated with screen) of the products of a partial acid hydrolysate (2 h at 110 °C in 6 M HCl) of the 32P-labeled polyoma (Py) protein kinase activity. The first experiment middle T antigen band separated by cellulose thin layer electrophoresis toward the anode at pH 1.9 (90 min at 1 kV). done in August 1978 showed that a band the The mT band was isolated from an SDS/PAGE gel of the products of an in vitro kinase assay carried out with an size of mT itself was phosphorylated in immunoprecipitate (IP) made with Py antitumor serum from polyomavirus (Py)-infected mouse 3T6 cells (6). The origin, vitro. In subsequent experiments, we found the cathode and anode, the positions of the pSer and pThr markers (circled) visualized by ninhydrin staining, and free orthophosphate (Pi) are indicated. “X” marks the 32P-labeled spot, later shown to be pTyr. (B) Autoradiogram (4 d with that this band was missing when a non- screen) of 32P-labeled acid/pronase hydrolysates separated by thin layer electrophoresis at pH 1.9 as in A. From the transforming Py mutant was used, pro- left: acid hydrolysate of a Py large T antigen (LT) band from 32P-labeled infected cells; acid hydrolysates of Py LT and viding the first clue that the mT-associated middle T antigen (MT) bands from a gel of an in vitro kinase assay carried out on an antitumor serum IP; pronase kinase activity might be important for Py proteolysate of a Py MT band; acid hydrolysate of an Ig heavy chain band from an in vitro kinase assay carried out on a v-Src immunoprecipitate made with RSV antitumor serum. The origin, the cathode and anode, and the positions of the transformation. Subsequently, using strains marker pSer, pThr, pTyr, and free orthophosphate (Pi) are indicated. of Py with different sized mT antigens, we showed that the phosphorylated band was indeed mT itself, and not the Ig heavy chain, about analyzing whether mT was phosphor- our flat-bed TLE set up on July 2, this marker which is about the same size. All three groups ylated on Thr or Ser, which I did by carrying pTyr proved to migrate between pSer and 32 working on Py mT obtained similar evidence out partial acid hydrolysis of a P-labeled mT pThr. As is widely known, it turned out that for an mT-associated protein kinase activity isolated from a band identified by gel separa- the reason that pTyr had resolved from pThr and met up at the 1979 Cold Spring Harbor tion of an IP kinase assay (see Fig. S1 for the wasthatIhadbeentoolazytomakeupfresh (CSH) Symposium on Tumor Viruses, held page from my lab notebook). On June 14, pH 1.9 buffer, which we routinely reused. May 31–June 5, 1979. Each group gave a 1979, I analyzed the hydrolysate using cellu- Upon repeated reuse the pH of the buffer talk at the meeting and reported that the lose thin layer electrophoresis (TLE) at dropped from 1.9 to ∼1.7, and this small pH mT-associated kinase activity had properties pH 1.9. As told elsewhere, the autoradiogram difference was responsible for the resolution 32 consistent with its being involved in Py revealed a P-labeled compound running of pTyr and pThr, which comigrate at pH 1.9. transformation. After discussions at the bar, between the pSer and pThr markers located Indeed, reviewer 1 of our paper did ask what we agreed to cosubmit papers to Cell de- by ninhydrin staining (Fig. 1A). When I re- amino acid was phosphorylated, and, in the scribing these findings as soon as we returned peated this analysis 10 d later and obtained revised paper [submitted to Cell on September from the meeting at Cold Spring Harbor the same result, it was clear that this obser- 21, received on September 25, and accepted Laboratories. The papers were submitted in vation was not an artifact and needed an on September 27 (!)], we included our evi- June 1979. Even before we had submitted our explanation. Luckily, my biochemical train- dence that the mT-associated kinase phos- paper, however, I knew that one likely re- ing at the University of Cambridge had phorylates Tyr as a new figure (figure 7 in ref. viewer question would be the following: taught me that there was a third hydroxy- 6). Our paper was published in Cell in De- What amino acid was being phosphorylated amino acid, namely tyrosine (Tyr), and I cember 1979, back to back with papers from in mT and was this activity a Thr-specific guessed that this novel phosphorylated com- the other two groups, who did not include kinase like v-Src? pound might be phosphotyrosine (pTyr). any data, however, on the amino acid target But I needed some marker pTyr to formally specificity of the mT-associated kinase (6–8). — June 1979 The First Sighting of establish this was the case. By mixing POCl3 At this point, with a major discovery tan- Phosphotyrosine with Tyr in water, I succeeded in extracting a talizingly close, on July 3, 1979, I set off with To preempt this question, the day after our small amount of authentic pTyr from the a group of friends from the Institute for the paper was submitted on June 11, 1979, I set ensuing black tar (!), and, when tested using two-day drive to Idaho with all our rafting

7878 | www.pnas.org/cgi/doi/10.1073/pnas.1508223112 Hunter Downloaded by guest on October 4, 2021 becausewewerenotproteinkinaseexperts, rather due to tight association with the PERSPECTIVE we thought we might have missed a prior c-Src protein (10), providing the first ex- report of protein–tyrosine phosphorylation, ample of a tumor virus-transforming protein and so I decided to ask a few experts to see commandeering and activating a cellular whether anyone had ever found a tyrosine- signaling protein. specific kinase activity. I called Ed Krebs at Although the in vitro data convinced us the University of Washington in Seattle, the that both Py mT antigen and RSV v-Src had acknowledged leader in the protein kinase associated tyrosine kinase activity, they did field, Joli Traugh at the University of Cal- not tell us whether mT and v-Src also acted ifornia, Riverside, and Gordon Gill at the as tyrosine kinases in the cell. To begin to test University of California, San Diego (two this possibility, Bart Sefton and I analyzed other protein kinase mavens), and also 32P-labeled v-Src and the associated 80-kDa George Taborsky at the University of Cal- (later shown to be the Hsp90 chaperone) and ifornia, Santa Barbara, who had written a 50-kDa (later shown to be the Cdc37 cocha- classic review on phosphoproteins. None of perone) proteins, which coprecipitated with them had heard of a tyrosine kinase although Fig. 2. v-Src phosphorylates tyrosine in the Ig heavy chain. This figure shows an autoradiogram of a 2D I did find out that free phosphotyrosine is separation of a partial acid hydrolysate of the Ig heavy used as a tyrosine storage compound in in- chainbandfromaninvitrokinaseassay.First(hori- sect eggs, due to its very high solubility. At zontal) dimension was pH 1.9 thin layer electropho- this stage, it looked like we had discovered a resis with the origin on right (small circle) and the anode on left; second (vertical) dimension was chro- novel type of protein kinase. matography in isobutyric acid/ammonia. Positions of — the pSer, pThr, and pTyr markers detected by ninhydrin September 1979 v-Src Is a Tyrosine staining are shown. Reproduced from ref. 9. Kinase There was still a nagging concern that the appearance of pTyr in mT antigen was gear for a 7-d self-guided rafting trip on the somehow an artifact of using acid hydrolysis. Middle Fork of the Salmon River, which To rule this possibility out, I decided to carry began on July 6. After getting off the river on out total pronase proteolysis of 32P-labeled July 14, I flew directly from Salmon, Idaho by mT to generate free (phospho)amino acids in chartered plane to Salt Lake City, and then another way. Together with the mT proteo- on to England for the DNA Tumor Virus lysate, I decided to spot on the same TLE – meeting being held in Cambridge, July 16 20. plate an acid hydrolysate of 32P-labeled heavy I talked at the meeting about the mT-associ- chain phosphorylated by v-Src to serve as an ated kinase but did not say anything about the authentic pThr control. This plate was run at novel tyrosine kinase activity. After the Cam- pH 1.9 on September 18, and, much to my bridge meeting, I visited my sister in London amazement, the spot from the heavy chain and my parents in Kent and then spent a few comigrated with pTyr and not pThr (Fig. days giving talks in Hamburg and Freiburg, 1B). Separation of the same heavy chain hy- finally getting back to La Jolla on August 8. In drolysate in two dimensions on September retrospect, it is hard to believe that I did not 19 showed that it contained only 32P-labeled rush back to the laboratory sooner! pTyr. On September 23, Bart Sefton and I quickly repeated the experiment and August 1979—Polyoma mT Antigen Is obtained the same result, and carried out a Phosphorylated on Tyrosine in Vitro total pronase digest of the 32P-labeled heavy To prove unequivocally that mT was being chain to confirm the presence of pTyr. This phosphorylated on Tyr, a number of addi- unexpected finding meant that Marc Collett tional experiments were needed. First, we and Ray Erikson had unluckily been misled developed a better method for synthesizing into concluding that v-Src is a Thr kinase pTyr so that we had enough marker for because pThr and pTyr comigrate at pH 1.9 subsequent analyses and also set up a 2D Fig. 3. c-Src has tyrosine kinase activity. This figure on Whatman 3MM paper, which they had 32 separation on cellulose thin-layer plates, us- shows an autoradiogram of P-labeled partial acid hy- used for electrophoresis. On October 3, I drolysates of the Ig heavy chain bands isolated from ing electrophoresis at pH 1.9 in the first analyzed Ig heavy chain that had been phos- immune complex kinase assays, carried out with anti-RSV dimension followed by chromatography in a phorylated in an IP by the c-Src protein, the tumor serum IPs from the indicated sources, separated by foul-smelling mixture of isobutyric acid and cellular homolog of v-Src, and found that it thin layer electrophoresis at pH 1.9 with the anode at the 0.5 M NH OH (5:3) to resolve pSer, pThr, — top. From the left: v-Src isolated from infected chick 4 too phosphorylated the heavy chain on Tyr cells (lane A); v-Src synthesized in vitro in an mRNA- and pTyr better. On August 28, I analyzed the first cellular tyrosine kinase [figure 2 in dependent rabbit reticulocyte cell free system (lane B); 32 the acid hydrolysis product of P-labeled mT Hunter and Sefton (9), reproduced in Fig. endogenous c-Src isolated from human HBL-100 mam- in two dimensions, and the 32Pcompound 3]. It is interesting to note that Sara Court- mary epithelial cells (lane C); and endogenous c-Src isolated comigrated precisely with pTyr (6). At this from chicken embryo cells (lane D). The origin (horizontal neidge showed subsequently that the tyro- arrows on left and right), and the positions of the marker point, we were convinced that authentic pTyr sine kinase activity associated with Py mT pSer, pThr, pTyr, and free orthophosphate (Pi) are indicated. was being generated in this in vitro assay, but, antigen is not an intrinsic activity, but Reproduced from ref. 9.

Hunter PNAS | June 30, 2015 | vol. 112 | no. 26 | 7879 Downloaded by guest on October 4, 2021 v-Src from 32P-labeled RSV-infected chicken embryo fibroblasts [figure 3 in Hunter and Sefton (9)]. Two-dimensional separation of the partial acid hydrolysates of the 32P- labeledv-Src,p80,andp50samples,carried out on October 1, showed that p80/Hsp90 contained exclusively pSer whereas v-Src contained equal amounts of pSer and pTyr [figure 4 D–F in Hunter and Sefton (9)]. p50/ Cdc37 also contained pSer and pTyr, be- coming the first cellular protein identified as a target for a tyrosine kinase. Two-dimen- sional analysis of tryptic digests of the same 32P-labeled v-Src band [figure 4A in Hunter and Sefton (9)] revealed that v-Src had two main phosphopeptides: Partial acid hydroly- sis of the isolated peptides revealed that the Fig. 4. v-Src contains pSer and pTyr in different tryptic peptides. Tryptic peptides were eluted from a 2D cellulose peptide derived from the C-terminal half of thin layer separation of a tryptic digest of v-Src obtained from 32P-labeled chicken embryo cells [shown in figure 4A in v-Src contained pTyr whereas the peptide Hunter and Sefton (9)]. The eluted α (ALPHA) and β (BETA) peptides were subjected to partial acid hydrolysis, and the hydrolysates were resolved in two dimensions exactly as described in Fig. 2. Autoradiograms of the TLE plates are from the N-terminal half contained pSer (Fig. shown. (A) α peptide. (B) β peptide. Subsequent work showed that the α peptide is derived from the N-terminal half of 4; this figure was in the original version of the v-Src and contains pSer17 and that the β peptide is derived from the C-terminal half of v-Src, which comprises the paper submitted to PNAS but was omitted catalytic domain, and contains pTyr416, the major autophosphorylation site. Positions of the pSer, pThr, and pTyr from the revised version to save space). These markers detected by ninhydrin staining are shown, and the origins are indicated by small circles at the bottom right. results convinced us that pTyr was a bona fide and stable in vivo posttranslational To try to obtain better separation of pThr infected with a ts v-Src mutant RSV was modification and suggested that v-Src might andpTyr,ItestedwhetherTLEatpH3.5 rapidly reduced to the level in normal cells autophosphorylate and also phosphorylate would work as a second dimension, based on after shifting the cells from the permissive the cellular p50/Cdc37 protein. a suggestion from Jack Rose, a fellow faculty growth temperature to the restrictive temper- October 1979—v-Src Phosphorylates member at the Salk, who knew that this ature when the cells are no longer trans- ’ Tyrosine in Cellular Proteins methodhadbeenusedinDavidBaltimores formed. Together, these experiments provided ’ If RSV transforms cells by expressing an ac- and Eckhard Wimmer s groups to resolve the compelling evidence that v-Src itself is the tive tyrosine kinase, then one might expect to pTyr generated upon hydrolysis of the po- kinase responsible for phosphorylating cel- find increased levels of pTyr-containing liovirus protein linked to genomic RNA from lular proteins on tyrosine in RSV-trans- proteins in RSV-infected cells. To test pSer and pThr. Electrophoresis at pH 3.5 formed cells and also implied that there had whether v-Src tyrosine kinase activity causes worked extremely well, giving a large sepa- to be cellular protein phosphatases that could increased tyrosine phosphorylation, I took a ration of pTyr from pThr, and it allowed us remove phosphate from pTyr. Even though 32P-labeled RSV-infected chick cell lysate to establish conditions for a 2D separation of wehadobtainedthesetemperatureshiftdata prepared by Bart Sefton and extracted the total pSer: pThr and pTyr, using TLE at pH 1.9, before we submitted the paper to PNAS, and protein into phenol. Protein was TCA- followed by TLE at pH 3.5 at right angles to one of the reviewers even asked for this ex- precipitated from the phenol layer, and the first dimension, instead of having to use a periment, we did not include this result in 32P-labeled lipids were then removed from nauseating isobutyric acid containing buffer the final version due to space constraints. the protein pellet by chloroform:methanol in the second dimension. The new pH The temperature shift data were included in (2:1) extraction, which was then subjected to 1.9/pH 3.5 2D TLE separation proved essential a subsequent paper published in Cell in partial acid hydrolysis. This rather uncon- for resolving the much less abundant pTyr September 1980 (see figure 1 in ref. 11). ventional extraction seemed to work and got cleanly from pThr (9%) and pSer (90%) in 32 32 November 1979—The Paper Is rid of the P-labeled RNA and phospholipid hydrolysates of total P-labeled proteins contaminants. Separation of this acid hydro- from RSV-infected chick cells. In an experi- Submitted to PNAS lysate on October 2 by TLE at pH 1.9 fol- ment analyzed between October 14 and 17 With the final piece of evidence establishing lowed by chromatography showed a trace using this new 2D separation, we found that that v-Src acts as a tyrosine kinase in vivo, we of pTyr. However, the resolution of pTyr normal chick cells or chick cells infected by were able to write up our findings for sub- from the massive pThr spot was marginal transformation-defective RSV had low levels mission to PNAS. In those days, PNAS pa- and was not going to persuade the un- of pTyr in proteins (0.05%) and that this level pers had to be communicated by a National initiated that this minor spot was not a was increased up to 10-fold in WT RSV- Academy member, and, luckily, Bob Holley, pThr satellite spot. Nevertheless, when we transformed cells [table 1 and figure 5 in a Nobel prizewinner who had switched to repeated this experiment (on October 10) Hunter and Sefton (9), reproduced here as studying factors that negatively regulate cell and compared 32P-labeled samples from Fig. 5], consistent with v-Src’sactingasa growth after his prizewinning research on uninfected and RSV-infected chick cells, the tyrosine kinase in cells to phosphorylate tRNA structure and move to the Salk In- pTyr spot was visible only in the RSV- cellular proteins. This result was the final stitute, had his laboratory on our floor and infected sample, suggesting that RSV infection piece of data we needed for the paper. We agreed to communicate our paper. We gave indeed stimulated tyrosine phosphorylation of also found that the high level of pTyr in four copies of our paper to Bob Holley on cellular proteins. cellular proteins in chicken embryo fibroblasts November 12, and he sent these copies out to

7880 | www.pnas.org/cgi/doi/10.1073/pnas.1508223112 Hunter Downloaded by guest on October 4, 2021 on tyrosine phosphorylation with v-Src PERSPECTIVE and polyoma mT, respectively, having heard about our work through the grapevine, mostly through telephone calls. The younger generation may be surprised to learn that important scientific news spread quickly even before the Internet and email! I had taken a copy of our accepted PNAS paper to the meeting, and the FMI Xerox machines were kept busy making multiple copies to hand out to everyone that was interested. We also Fig. 5. v-Src increases pTyr levels in whole cell proteins. This figure shows autoradiograms of a 2D separation of sent out over 20 preprints by mail in De- A partial acid hydrolysates of total protein isolated from normal uninfected ( ) and Schmidt-Ruppin A RSV-transformed cember. By the end of 1979, a large number (B) chicken embryo cells labeled for 16 h with 32P-orthophosphate. As shown in the schematic of the separation on the right (C), the first (horizontal) dimension was thin layer electrophoresis at pH 1.9 with the origin (circled) on the of people knew that v-Src was a tyrosine right and the anode on the left; the second (vertical) dimension was thin layer electrophoresis at pH 3.5, with the kinase, and many groups had started work- anode at the top. The positions of the marker pSer, pThr, and pTyr are indicated. On panels A and B the origin is ing on tyrosine phosphorylation in their indicated with a small vertical arrow, and the position of pTyr with a large arrow. Partially hydrolyzed peptides are seen own systems. Indeed, over the next few at the bottom right. Reproduced from ref. 9. months, we received many requests for phosphotyrosine because the compound three (local) reviewers. Remarkably, all of the had collaborated when we were graduate was not commercially available. reviews were received within two weeks (see students together in the mid 1960s in Asher Independently, Owen Witte, Asim Das- Fig. 6 for reviewer 3′s handwritten and Korner’s group in the Department of Bio- gupta, and David Baltimore at MIT had been positive comment and Figs. S2 and S3 for the chemistry in Cambridge, about the phos- working on the Abelson murine leukemia- other two reviews). We addressed the issues photyrosine story. Tim was going to be a transforming protein, v-Abl, and found that they raised by rewriting and also significantly speaker at the meeting and had persuaded it too had an associated kinase activity that shortening the manuscript to fit the 5-page the organizers to ask me—I was squeezed in resulted in autophosphorylation of the limit for PNAS. The final version was re- as a speaker in the last session of the meeting v-Abl protein. They determined that the ceived by PNAS on December 3, 1979 (see on the topic of IFN and tumor viruses. As an in vitro phosphorylated v-Abl contained Figs. S4 and S5), but the paper was not indication of how fast word had spread, both pTyr and submitted a paper at the end of published until the March issue in 1980 (9). Ray Erikson and Alan Smith talked in this October 1979, concluding that this phos- session about their very recent experiments phorylation might represent a phosphoenzyme December 1979—First Public Presentations AftertalkingaboutourresultsattheTVL floor meeting at the beginning of October (with the current Editor-in-Chief of PNAS, Inder Verma, at the time an Assistant Pro- fessor on our floor, in attendance), my first public presentation of our work on tyrosine phosphorylation of polyoma mT and v-Src was at a joint meeting between the groups working on tumor viruses at the Salk In- stitute and the Fred Hutchinson Cancer Re- search Center held in Seattle on October 25, 1979. Subsequently, I talked about our work ataHammerCancerWorkshopattheSalk Institute on November 13, 1979, and then again at the University of California, San Diego, Biology Department Noon Seminar on November 20, 1979. The word about our discovery spread through the community very fast and had even reached the other side of the Atlantic, because, at the end of Octo- ber, I received an invitation to speak about our work at the “Protein Phosphorylation and Bio-Regulation” meeting that Julian Gordon, Ernesto Podesta, and George Thomas were coorganizing at the Friedrich- Miescher Institute (FMI) in Basel, December 10–12, 1979. The invitation had come about becauseIhadtoldTimHunt,withwhomI Fig. 6. Reviewer 3′s comments. The handwritten review on the PNAS review form is shown.

Hunter PNAS | June 30, 2015 | vol. 112 | no. 26 | 7881 Downloaded by guest on October 4, 2021 intermediate in phosphate transfer to an- to carry out a series of experiments with evidence was simply too compelling. Of other (protein) substrate. Their Nature pa- v-Src in short order between September 18 course, whether this novel mechanism of per came out at the end of February 1980 and October 17. Thus, amazingly, all of the cell transformation by tumor viruses would (12), shortly before our PNAS paper on experiments demonstrating that v-Src is a be relevant to human cancer we did not the v-Src tyrosine kinase activity. tyrosine kinase and phosphorylates tyro- know at the time, but it did not take long for In keeping with my general philosophy sine in cellular proteins that were included the first human oncogenes encoding mutant about science communication, we had not as figures in the PNAS paper were done in tyrosine kinases to be reported. This re- attempted to keep our discovery of tyrosine a span of just one month—science could alization that activated tyrosine kinases could phosphorylation a secret. Indeed, we had move fast even before the days of molecular play a role in human cancer was the stimulus presented our work in public and told people biology. In retrospect, the key to discover- for the development of tyrosine kinase in- in person or on the phone what we had ing that v-Src is a tyrosine kinase was that hibitor drugs. Starting in 2001, over 20 small foundwellbeforewehadsubmittedourpa- we were working on both types of tumor molecule tyrosine kinase inhibitor drugs have pers on polyoma mT tyrosine phosphoryla- virus simultaneously. now been approved for cancer therapy, with “ tion and v-Src as a tyrosine kinase. Indeed, The PNAS paper Transforming gene more on the horizon. A gratifying outcome Marc Collett, Tony Purchio, and Ray Erikson product of Rous sarcoma virus phosphory- of this set of experiments on a simple chicken ” were able to submit a paper describing their lates tyrosine by me and Bart Sefton was tumor virus. evidence that v-Src is a tyrosine kinase to published in March 1980 three months after Nature in December 1979, based on what we acceptance (immediate online publication ACKNOWLEDGMENTS. Part of this paper was written hadtoldthem,anditwaspublishedinMay was not an option in 1979!). There was no on my trek up Mount Whitney in July 2014, another high point in my career! I want to thank all my colleagues at 1980 (13). In addition, Stanley Cohen, who in press release and no news and views, and we the Salk Institute, including Karen Beemon, Walter Eck- 1978 was the first to show that the EGF re- did not even think about trying to patent the hart, Mary Anne Hutchinson, and David Shannahoff, and, ceptor has an associated kinase activity that idea that targeting oncogenic tyrosine kinases particularly, my coauthor Bart Sefton, who helped with — all the experiments in the paper, as well as all the scien- he reported was specific for threonine (14), might be useful therapeutically it was a tists working on DNA and RNA tumor viruses throughout quickly reevaluated this conclusion based on different time in the biological sciences. The the world who shared information and ideas so freely, whatwehaddone,andreportedthatthe concept that v-Src transforms cells through and made the exciting events of 1978/1979 possible. EGF receptor is also a tyrosine kinase in a phosphorylation of cellular target proteins on I also acknowledge the National Cancer Institute for fund- ing the work described here and all the people who have JournalofBiologicalChemistrypaper sub- tyrosine was immediately accepted as true by cited our paper over the past 35 years (over 2,100 as of mitted in June 1980 and published in Sep- the community without any naysayers—the April 2015). tember 1980 (15). In fact, by the end of 1980, we knew that there were at least four distinct tyrosine kinases: v-Src/c-Src (11, 13), v-Abl 1 Collett MS, Erikson RL (1978) Protein kinase activity associated 10 Courtneidge SA, Smith AE (1983) Polyoma virus transforming (12), v-Fps/v-Fes (16, 17), and the EGF re- with the avian sarcoma virus src gene product. Proc Natl Acad Sci protein associates with the product of the c-src cellular gene. Nature USA 75(4):2021–2024. 303(5916):435–439. ceptor (15). 2 Beemon K, Hunter T (1977) In vitro translation yields a possible Rous 11 Sefton BM, Hunter T, Beemon K, Eckhart W (1980) Evidence that sarcoma virus src gene product. Proc Natl Acad Sci USA 74(8):3302–3306. the phosphorylation of tyrosine is essential for cellular transformation Coda 3 Brugge JS, Erikson RL (1977) Identification of a transformation- by Rous sarcoma virus. Cell 20(3):807–816. specific antigen induced by an avian sarcoma virus. Nature 12 Witte ON, Dasgupta A, Baltimore D (1980) Abelson murine The totally unexpected functional common- 269(5626):346–348. leukaemia virus protein is phosphorylated in vitro to form ality between the transforming proteins of an 4 Levinson AD, Oppermann H, Levintow L, Varmus HE, Bishop JM phosphotyrosine. Nature 283(5750):826–831. RNAtumorvirusandaDNAtumorvirus (1978) Evidence that the transforming gene of avian sarcoma virus 13 Collett MS, Purchio AF, Erikson RL (1980) Avian sarcoma virus- encodes a protein kinase associated with a phosphoprotein. Cell transforming protein, pp60src shows protein kinase activity specific that our work had uncovered immediately 15(2):561–572. for tyrosine. Nature 285(5761):167–169. suggested that tyrosine phosphorylation of 5 Sefton BM, Hunter T, Beemon K (1979) Product of in vitro 14 Carpenter G, King L, Jr, Cohen S (1979) Rapid enhancement of cellular proteins might be a universal mech- translation of the Rous sarcoma virus src gene has protein kinase protein phosphorylation in A-431 cell membrane preparations by activity. JVirol30(1):311–318. epidermal growth factor. J Biol Chem 254(11):4884–4891. anism of viral transformation. At the time, 6 Eckhart W, Hutchinson MA, Hunter T (1979) An activity phosphorylating 15 Ushiro H, Cohen S (1980) Identification of phosphotyrosine as a we did not know whether the Py mT-asso- tyrosine in polyoma T antigen immunoprecipitates. Cell 18(4):925–933. product of epidermal growth factor-activated protein kinase in A-431 7 Smith AE, Smith R, Griffin B, Fried M (1979) Protein kinase activity cell membranes. J Biol Chem 255(18):8363–8365. ciated activity was intrinsic or due to an as- associated with polyoma virus middle T antigen in vitro. Cell 18(4): 16 Barbacid M, Beemon K, Devare SG (1980) Origin and functional sociated enzyme, but, for v-Src we had strong 915–924. properties of the major gene product of the Snyder-Theilen strain of evidence that the tyrosine kinase activity 8 Schaffhausen BS, Benjamin TL (1979) Phosphorylation of polyoma feline sarcoma virus. Proc Natl Acad Sci USA 77(9):5158–5162. T antigens. Cell 18(4):935–946. 17 Pawson T, et al. (1980) A strain of Fujinami sarcoma virus which was intrinsic and essential for RSV trans- 9 Hunter T, Sefton BM (1980) Transforming gene product of Rous sarcoma is temperature-sensitive in protein phosphorylation and formation, and this insight had enabled us virus phosphorylates tyrosine. Proc Natl Acad Sci USA 77(3):1311–1315. cellular transformation. Cell 22(3):767–775.

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