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.