The Genesis of Tyrosine Phosphorylation

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Tony Hunter

Salk Institute for Biological Studies, La Jolla, California 92037 Correspondence: [email protected]

Tyrosine phosphorylation of proteins was discovered in 1979, but this posttranslational modification had been “invented” by evolution more than a billion years ago in single- celled eukaryotic organisms that were the antecedents of the first multicellular animals. Because sophisticated cell–cell communication is a sine qua non for the existence of multicellular organisms, the development of cell-surface receptor systems that use tyrosine phosphorylation for transmembrane signal transduction and intracellular signaling seems likely to have been a crucial event in the evolution of metazoans. Like all types of protein phosphorylation, tyrosine phosphorylation serves to regulate proteins in multiple ways, including causing electrostatic repulsion and inducing allosteric transitions, but the most important function of phosphotyrosine (P.Tyr) is to serve as a docking site that promotes a specific interaction between a tyrosine phosphorylated protein and another protein that contains a P.Tyr-binding domain, such as an SH2 or PTB domain. Such docking interactions are essential for signal transduction downstream from receptor tyrosine kinases (RTKs) on the cell surface, which are activated on binding a cognate extracellular ligand, and, as a consequence, elicit specific cellular outcomes.

he first eukaryotic tyrosine kinases (TKs) quenced human genome (Manning et al. 2002), Twere discovered through studies of animal the number of ePK genes stands at 478 (see Fig. tumor virus transforming proteins, such as pol- 1), and the total number of protein kinase genes, yoma virus middle T antigen and the Rous sar- which includes other protein kinases either dis- coma virus v-Src protein (Eckhart et al. 1979; tantly related to the ePKs or unrelated to the Hunter and Sefton 1980). When the v-Src TK ePKs, is 566. Surprisingly, given the scarcity of sequencewas reported in 1983, this immediately P.Tyr in cellular proteins, 90 kinases are classified led to the revelation that despite their unique as TKs (Fig. 1), although a small numberof these amino acid specificity, the TKs are related to lack significant kinase activity, but have con- the Ser/Thr kinases, exemplified by the cAMP- served noncatalytic functions. dependent protein kinase. Bioinformatic analy- The first hint that a growth factor receptor sis and targeted cDNA cloning quickly revealed might have an intrinsic protein kinase activity the existence of a surprisingly large number came from Stanley Cohen’s 1978 report that of related protein kinases, now known as the EGF stimulated protein phosphorylation in a eukaryotic protein kinase (ePK) family. Based membrane preparation from A431 cells (Car- on bioinformatic analysis of the completely se- penter et al. 1978), which have an extraordinari-

Editors: Joseph Schlessinger and Mark A. Lemmon Additional Perspectives on Signaling by Receptor Tyrosine Kinases available at www.cshperspectives.org Copyright # 2014 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a020644 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a020644

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T. Hunter

FGFR2 FGFR3 Tr kC EphB2 Tr kB FGFR1 FGFR4

EphB1 Tr kA FLT1/VEGFR1 EphA5 KDR/VEGFR2 MuSK ROR2 ROR1 Fms/CSFR EphB3 Ret Kit EphA3 DDR2 Mer DDR1 Tyro3/ FLT4 EphA4 Axl PDGFRα Sky FLT3 EphA6 IGF-1R IRR PDGFRβ InsR Ye s EphB4 Met EGFR HER2/ErbB2 Src Ron Ros MLK3 EphA7 ALK Tie2 MLK1 Lyn LTK Tie1 HCK Fyn HER4 EphA8 RYK CCK4/PTK7 MLK4 MLK2 Lck Fgr Ack Tnk1 Tyk2 Jak1 HER3 Jak2 EphA2 Jak3 TK BLK DLK TKL Syk Zap70/SRK ANKRD3 SgK288ZAK EphA1 PYK2/FAK2 LZK Lmr1 FAK Lmr2 ALK4 ITK C-Raf/Raf1 β FRK ZAK BRaf TGF R1 TEC EphB6 KSR KSR2 Srm RIPK2 Brk TXK Lmr3 IRAK1 ALK7 BTK IRAK3 LIMK1 ARaf BMPR1B Etk/BMX EphA10 LIMK2 TESK1 ILK TAK1 BMPR1A CTK RIPK3 TSK2 HH498 ALK1 CSK ALK2 AbI2/Arg IRAK2 ActR2 Abl Fes Fer RIPK1 ActR2B β Jak3~b LRRK2 TGF R2 MEKK2/MAP3K2 Jak2~b LRRK1 MISR2 MEKK3/MAP3K3 Tyk2~b suRTK106 BMPR2 ASK/MAP3K5 ANPα/NPR1 IRAK4 MAP3K8 STE ANPβ/NPR2 Jak1~b MAP3K7 KHS1 MOS MST1 YSK1 HPK1 KHS2 MST2 MST3 HSER sgk496 WNK1 MEKK6/MAP3K6 GCK WNK3 MST4 DYRK2 PBK DYRK3 GUCY2D WNK2MAP3K4 HGK/ZC1 DYRK4 NRBP1 NRBP2 DYRK1A GUCY2F MEKK1/MAP3K1 OSR1 MINK/ZC3 DYRK1B WNK4 STLK3 TNIK/ZC2 MLKL NRK/ZC4 STRAD/STLK5 MYO3A PERK/PEK SGK307 STLK6 SLK PKR LOK MYO3B HIPK1 HIPK3 GCN2 SgK424 TAO1 HIPK2 SCYL3SCYL1 Tpl2/COT TAO2 SCYL2 NIK TAO3 PAK1 CLK4 HIPK4 PRP4 HRI PAK3 PAK4 CLK1 CLIK1 PAK2 IRE1 MAP2K5 PAK5/PAK7 CLK2 IRE2 CLIK1L CLK3 TBCK PAK6 MAP2K7 MEK1/MAP2K1 RNAseL GCN2~b MEK2/MAP2K2 MSSK1 TTK SgK071 KIS CMGC SRPK2 MYT1 SEK1/MAP2K4 MKK3/MKK6 SRPK1 α CK2 1 Wee1 δ MAK CK2α2 CDC7 SgK196 Wee1B CK1 β ICK PRPK TTBK1 CK1ε GSK3 Haspin GSK3α MOK TTBK2 CK1α1 CDKL3 CK1α2 CDKL2 PINK1 SgK493 CK1γ2 SgK269 VRK3 CDKL1 SgK396 CDKL5 ERK7 SgK223 γ CDKL4 ERK4 Slob CK1 1 ERK3 SgK110 PIK3R4 CK1γ3 NLK SgK069 Bub1 ERK5 SBK IKKα BubR1 CK1 ERK1/p44MAPK IKKβ VRK1 CDK7 IKKε VRK2 ERK2/p42MAPK PLK4 p38γ PITSLRE TBK1/NAK MPSK1 p38δ JNK1 JNK2 TLK2 JNK3 CDK10 GAK β AAK1 TLK1 PLK3 p38 CDK8 p38α CDK11 CAMKK1 ULK3 PLK1 PLK2 CDK4 CCDK BIKE CAMKK2 BARK1/GRK2 CDK6 ULK1 BARK2/GRK3 RHOK/GRK1 Fused GRK7 GRK5 PFTAIRE2 SgK494 ULK2 ULK4 GRK4 PFTAIRE1 CDK9 GRK6 Nek6 RSKL1 PCTAIRE2 Nek7 Nek10 sgk495 Nek8 PASK PDK1 MSK1 RSKL2 RSK1/p90RSK CDK7 PCTAIRE1 PCTAIRE3 CDK5 CHED Nek9 LKB1 MSK2 RSK4 RSK2 Nek2 chk1 p70S6K RSK3 Akt2/PKBβ AurA/Aur2 p70S6Kβ Akt1/PKBα cdc2/CDK1 Nek11 Akt3/PKBγ CDK3 CDK2 Nek4 AurB/Aur1 SGK1 AurC/Aur3 PKN1/PRK1 SGK2 SGK3 Trb3 Pim1 Pim2 LATS1 PKG2 Nek3 PKG1 PKN2/PRK2 Trb2 Pim3 Tr io LATS2 PKN3 Nek5 Trad NDR1 PRKY PKCδ Trb1 Obscn~b NDR2 PKCθ PRKX η Nek1 SPEG~b YANK1 MAST3 PKC MASTL PKCε Obscn STK33 YANK2 PKCι PKCξ SPEG YANK3 PKAγ PKAα PKCγ MAST2 TTN PKAβ smMLCK TSSK4 ROCK1 PKCα HUNK ROCK2 Atypical protein kinases Chk2/Rad53 PKCβ skmMLCK SSTK SNRK MAST4 MAST1 DMPK ADCK1 DRAK2 CRIK DRAK1 TSSK3 NIM1 ADCK5 PKD2/PKCμ TSSK1 AGC ABC1 ADCK3 sgk085 TSSK2 DMPK2 ADCK4 DCAMKL3 caMLCK DAPK2 PKD1 DCAMKL1 ADCK2 DAPK3 MELK PKD3/PKDν DAPK1 CASK DCAMKL2 MRCKβ ChaK1 VACAMKL MRCKα ChaK2 MNK1 MAPKAPK5 PhKγ1 AlphaK3 AMPKα2 MNK2 PhKγ2 Alpha MAPKAPK2 PSKH1 EEF2K AMPKα1 CaMKIIγ MAPKAPK3 β PSKH2 AlphaK2 CAMK BRSK2 CaMKIIα CaMKII AlphaK1 BRSK1 RSK4~b CaMKIIδ SNARK RSK1~b CaMKIV Brd2 ARK5 MSK2~bMSK1~b Brd3 Brd Brd4 QSK RSK2~b RSK3~b BrdT CaMKIβ PDHK2 SIK PDHK3 QIK PDHK PDHK1 CaMKIγ PDHK4 BCKDK MARK4 CaMKIα δ ATM MARK3 CaMKI ATR MARK1 MARK2 PIKK mTOR/FRAP DNAPK SMG1 TRRAP RIOK3 RIO RIOK1 RIOK2 TIFα TIF1 TIF1γ TIF1β

Figure 1. The human kinome. Based on the catalog of human protein kinases compiled by Manning et al. (2002), an unrooted relatedness tree was constructed using the catalytic domain sequences of the 478 eukaryotic protein kinases (ePKs). The seven major branches of the kinome are indicated: AGC, CAMK, CMGC, TK, TKL, STE, and CK1. The ends of the branches representing individual kinases are labeled with the names of each protein kinase. The TK (tyrosine kinase) branch at the top of the tree has 90 members. The RTKs are present in four major branches: EPH, INSR/TRK/AXL, FGFR/PDGFR/CSF-1R, and EGFR. The atypical protein kinases, shown in the inset at the bottom left, fall into seven small families, which are either distantly related to the ePKs or else unrelated in sequence. (Illustration reproduced courtesy of Cell Signaling Technol- ogy, Inc., www.cellsignal.com.)

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The Genesis of Tyrosine Phosphorylation

ly high number of surface EGF receptors. This duced dimerization and intermolecular phos- group’s subsequent July 1979 paper concluded phorylation was originally proposed as an RTK that EGF stimulated threonine phosphorylation activation mechanism by Yarden and Schles- of proteins in membranes (Carpenter et al. singer (1987), and based on much subsequent 1979). However, with the realization that phos- work, this is now accepted as the general mech- phothreonine (P.Thr) and P.Tyr comigrate on anism of RTK activation by ligands, although electrophoresis at pH 1.9, they reevaluated the specific details differ between different this conclusion, and in September 1980 the Co- RTK subfamilies. Ligand binding either alters hen group published that EGF actually stimu- the conformation of a preexisting RTK dimer lated Tyr phosphorylation (Ushiro and Cohen or induces RTK dimer formation, which results 1980). By June 1981, purified EGF receptor in juxtaposition of the catalytic domains and preparations had been shown to have TK activ- activation in trans, either through an induced ity (Chinkers and Cohen 1981), and EGF had conformational change or via transphosphory- been shown to stimulate Tyr phosphorylation in lation of activating residues in the activation the cell, resulting in Tyr phosphorylation of spe- loop or the cytoplasmic juxtamembrane do- cific proteins within minutes of EGF treatment main. Once activated, RTKs autophosphorylate (Hunter and Cooper 1981). The 1984 cloning of at additional sites, enabling recruitment of SH2 the EGF receptor revealed that it has a catalytic and PTB domain P.Tyr-binding proteins, and domain related to that of the c-Src TK (Down- also directly phosphorylate substrates to propa- ward et al. 1984; Lin et al. 1984), confirming the gate downstream signals. In this regard, al- intrinsic nature of the TK activity. In quick though we have structures of dimerized ligand- succession, several additional growth factor re- bound RTK extracellular domains, and cyto- ceptors were shown to have TK activity, starting plasmic domains, structures of an intact li- with the PDGF receptor in 1982. Further RTKs ganded RTK dimer are still needed to under- were added through directed cloning and se- stand exactly how ligand-induced dimerization quence analysis combined with biochemical of the extracellular domain results in catalytic testing, and by the end of the decade .10 activation through juxtaposition of the cyto- RTKs had been reported. By this time, it was plasmic kinase domain (Arkhipov et al. 2013; clear that ligand-induced Tyr phosphorylation Endres et al. 2013). was a major mechanism for the transmission of signals across the plasma membrane. This con- TYROSINE PHOSPHORYLATION clusion was reinforced by the discovery of two AND DISEASE component receptors, like the antigen receptors and the cytokine receptors, in which the ligand- The discovery that transforming proteins of binding subunit of the receptor complexes with tumor viruses had TK activity immediately a cytoplasmic TK, which is activated upon li- suggested that unbridled Tyr phosphorylation gand binding. We now know that the human might be a potent transforming mechanism. genome encodes 58 RTKs grouped in 20 distinct Analysis of temperature-sensitive transforming families (Lemmon and Schlessinger 2010). mutants of Rous sarcoma virus showed that In general, RTKs are type 1 transmembrane v-Src TK activity correlated precisely with trans- proteins with an extracellular ligand-binding forming potential, providing direct evidence domain linked by a transmembrane domain to that Tyr phosphorylation was required for trans- an intracellular domain that includes a TK cat- formation (Sefton et al. 1980). A search for hu- alytic domain, and, usually,an unstructured car- man tumor oncogenes quickly revealed that boxy-terminal tail that possesses autophosphor- chronic myelogenous leukemia (CML) results ylation sites (Lemmon and Schlessinger 2010). from the fusion of the BCR gene with the RTK kinase activity is increased in response to c-ABL TK gene, yielding the BCR-ABL fusion binding of a cognate ligand, such as a growth protein, a constitutively activated TK that is en- factor, to the extracellular domain. Ligand-in- coded by the t22:9 Philadelphia chromosomal

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T. Hunter

fusion (Hunter 2007). BCR-ABL TK activity is agnosed with an ALK mutation (Hallberg and necessary for transformation of myeloid cells in Palmer 2010). As of August 2013, 19 TKIs have culture and leukemia in animals. Subsequently, been approved for cancer therapy, and, notably, many additional oncogenicallyactivated human 12 of these target activated RTKs; several more TK mutants have been reported in cancer. Sev- TKIs are in phase III trials, and are likely to be eral of these are mutant forms of RTKs, includ- approved in the near term. In addition, five ap- ing many instances in which a chimeric protein proved protein antibody drugs are directed is made as a result of the fusion of a dimerization against RTK extracellular domains or their li- domain from one protein with the cytoplasmic gands, with more in the pipeline. catalytic domain of an RTK, resulting in a constitutively activated TK. WHY WAS TYROSINE PHOSPHORYLATION The finding that an activated TK was causal MISSED FOR SO LONG? in human disease spurred efforts to develop protein kinase inhibitors as cancer therapeutics. Ser/Thr kinase activities were first identified in The first effort to develop selective protein ki- 1954 (Burnett and Kennedy 1954), but, even nase inhibitors began in the 1980s with the goal though as Phoebus Levene, who reported the of using these to study protein kinase function synthesis of P.Tyr in 1933 (Levene and Shcor- in the cell (Hidaka et al. 1984), and ultimately to muller 1933) had realized that phosphorylation develop them as therapeutics. The importance of the tyrosine hydroxyl is theoretically possible, of elevated Tyr phosphorylation in cancer trig- phosphorylation of Tyr in proteins was not de- gered efforts to develop selective inhibitors scribed until nearly 25 years later (Eckhart et al. against individual TKs known to be activated 1979; Hunter and Sefton 1980). This long gap by mutation or overexpression in different types was despite the fact that, as we now know, a large of cancer. In 1998, the first drug antagonizing a number of TKs exists (Manning et al. 2002). TK was approved for cancer therapy; trastuzu- Why was Tyr phosphorylation missed for so mab (Herceptin), an inhibitory monoclonal an- long? Unlike many Ser and Thr phosphoryla- tibody directed against the extracellular domain tions, most Tyr phosphorylations are very short- of the HER2 RTK, is used for therapy of HER2- lived owing to the presence of extremely active positive breast cancer. This was quickly followed P.Tyr-specific phosphatases (PTPs) that rapidly in 2001 by the approval of a small-molecule TK dephosphorylate any P.Tyr residue that is not inhibitor (TKI), imatinib (Gleevec), an inhibi- protected through binding to an SH2 or PTP tor of the activated BCR-ABL TK responsible domain or via an intramolecular interaction. A for CML (Hunter 2007). Imatinib has proved good example of how rapidly phosphate on Tyr to be remarkably successful in treating CML, turns over is the finding that the EGF-induced and most patients who are put on treatment P.Tyr residues on the EGF receptor have half- during the chronic indolent phase of the disease lives of only a few seconds, and are turned over go into long-term remission, provided they con- .100 times during the early phase of the cellular tinue taking the drug. Since 2001, several addi- EGF response when the EGF receptor is maxi- tional small-molecule TKIs and protein drugs mally phosphorylated (Kleiman et al. 2011). As have been approved forcancer therapy, targeting a result, P.Tyrconstitutes ,1% of the total phos- a wide range of mutationally activated or over- phohydroxy-amino acids in proteins in a typical expressed TKs characteristic of different human mammalian cell, even when stimulated with cancers. A striking recent example is crizotinib, growth factors to activate RTKs or other types which is a selective inhibitor of the ALK RTK of receptor systems that signal through TKs. To that is activated by chromosomal translocation compound this scarcity, for many years the in about 4% of non-small-cell lung cancers. routine method for identifying phosphoamino Within 4 years of the discovery of ALK fusion acids in proteins involved electrophoretic sepa- genes in NSCLC in 2007, crizotinib had been ration of partial acid hydrolysates of 32P-labeled approved for treatment of NSCLC patients di- proteins at pH 1.9, and, at this pH, P.Thr and

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The Genesis of Tyrosine Phosphorylation

P.Tyr comigrate (Ushiro and Cohen 1980), the greater distance of the O4 hydroxyl from the meaning that the weak P.Tyr signal is masked peptide backbone allowed evolution of Tyr-spe- by the much more abundant P.Thr signal (5% cific kinases, and also P.Tyr-specific phosphatas- of total cellular phosphoamino acids). This es. Fortuitously, the distinct chemical properties meant that evenwhen kinase activity was assayed of P.Tyr also allow the immune system to gener- in vitro, a protein kinase that phosphorylated ate antibodies that selectively recognize P.Tyr Tyr would have been mistakenly identified as a over P.Ser and P.Thr, and such anti-P.Tyr anti- Thr kinase, as was the case with the EGF receptor bodies have been extraordinarily useful in RTK (Carpenter et al. 1979) and v-Src TK (Col- studying Tyr phosphorylation. lett et al. 1979). Once two-dimensional electrophoretic methods had been developed to sepa- WHERE DID TKs AND PHOSPHATASES rate P.Tyr from P.Thr and P.Ser (Hunter and COME FROM? Sefton 1980), it was possible to show that P.Tyr was present in proteins isolated from cells, and Prokaryotes made early use of the reactive tyro- that its levels increased significantly when an sine hydroxyl group in proteins for regulatory activated TK, such as v-Src (Hunter and Sefton purposes. For example, adenylylation of a spe- 1980), was expressed, or when the cells were cific Tyr residue in glutamine synthetase de- stimulated with EGF to activate the EGF recep- creases its activity (Shapiro and Stadtman tor RTK (Cooper and Hunter 1981). Moreover, 1968). True Tyr phosphorylation is also used individual cellular proteins containing an in- in prokaryotes, and was apparently “invented” creased level of P.Tyr were identified in such separately from the process in eukaryotes, be- cells by two-dimensional gel electrophoresis cause the small family of bacterial receptor-like (Radke et al. 1980; Cooper and Hunter 1981), TKs, known as BY kinases, is unrelated in se- and through physical association with the TKs quence to the large family of Tyr TKs in eukary- themselves (Hunter and Sefton 1980). otes. BY-catalyzed Tyr autophosphorylation is used in bacteria to regulate biosynthesis and export of extracellular polysaccharide (Gran- WHAT IS SPECIAL ABOUT TYROSINE geasse et al. 2012). P.Tyr-specific phosphatases PHOSPHORYLATION? counteract the phosphorylation of BY kinases, From a chemical perspective there is nothing but no specific P.Tyr-binding proteins have been particularly unusual about the chemical prop- found in bacteria, and the consequences of Tyr erties of the O4-phenolic phosphate ester bond phosphorylation are exerted primarily through of P.Tyr, which, like those of phosphoserine allosteric/electrostatic effects. (P.Ser) and P.Thr, is a relatively high-energy Based on the sequence similarities between bond (8–10 kcal). However, because the phos- their catalytic domains, it is likely that conven- phate on Tyr is linked to the O4 position of the tional TKs evolved from Ser/Thr kinases, which phenolic ring, it lies much further away from the in turn appear to have been derived from bacte- peptide backbone than the phosphate on the b- rial small-molecule kinases, like the eukaryotic- OH groups of Ser and Thr, and, in consequence, like kinases (ELKs), such as the aminoglycoside this in itself provides an element of binding spe- kinases (Kannan et al. 2007), which have a dis- cificity. In addition, the phenolic ring of P.Tyr is tantly related catalytic domain that has a very unique in providing significant additional bind- similar three-dimensional fold. Dual-specificity ing energy for phosphospecific-binding do- kinases that could phosphorylate Ser/Thr and mains that are mediated by hydrophobic or p Tyr may have been an intermediate step in the bond-ring interactions, which P.Ser/Thr cannot evolution of the first Tyr-specific kinase. For ex- make. These properties allowed the evolution of ample, the MAP2K family dual-specificity ki- selective P.Tyr-binding domains, which have nases, which phosphorylate both a Thr and a much deeper binding pockets than those for Tyr in target MAP kinases are of ancient origin, P.Ser and P.Thr-binding domains. In addition, and are found in all eukaryotes. The main re-

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T. Hunter

quirement for converting a Ser/Thr kinase, Interestingly, no additional RTK families are which phosphorylates the b-OH group of Ser/ shared between Monosiga and Capsaspora, sug- Thr that lies close to the peptide backbone, into gesting that the large numbers of RTKs in both a TK is to redesign the active site so that it can clades evolved independently. Not only is the accommodate a Tyr residue, whose phenolic overall number of TKs in choanoflagellates OH group is 6A˚ further away from the pep- greater than that of a typical vertebrate, but tide backbone than the b-OH group of Ser/Thr. they also possess all the machinery needed for The conventional TKs are characterized by a dif- Tyr phosphorylation-based signaling, including ference in the conserved catalytic domain motif an abundance of PTPs and SH2 domain pro- on the amino-terminal side of the activation teins. These include homologs of several specific loop (HRDLAARN in TKs versus HRDLKPEN metazoan PTP and SH2 proteins, as well as in Ser/Thr kinases; in both cases the Asp residue many novel proteins with unique domain ar- serves as the catalytic base). Both the Arg resi- rangements. Overall, the analysis of TKs in hol- dues in this motif hydrogen bond to the target ozoa suggests that an early holozoan had a ma- Tyr OH group, and the Tyr phenolic ring makes ture set of 6–8 nonreceptor TKs (CTKs), van der Waals interactions with a conserved Pro including SRC, CSK, ABL, TEC, FER, and in the P þ 1 loop, which adopts a different con- FAK, several fast-evolving RTKs, and an exten- figuration in the TKsthan in the Ser/Thr kinases sive network of SH2 and PTB P.Tyr-binding do- and appears to be a major determinant in con- mains, and PTP domain proteins to reverse the ferring specificity for Tyr versus Ser/Thr (Hub- action of TKs, and presumably downstream TK bard et al. 1994). target substrates. In all unicellular and multicel- One can never know with certainty in which lular organisms that have TKs, they constitute species the first TK appeared, but almost cer- 10%–20% PKs, underscoring their fundamen- tainly it was a unicellular organism, because ex- tal importance to cellular physiology. tant unicellular choanoflagellate species, which At what point in the emergence of Tyr phos- lie at the base of the metazoan branch of the phorylation-based signaling did RTKs arise? Be- evolutionary tree, have a well-developed Tyr cause of their key role in intercellular commu- phosphorylation system, with many TK genes nication, RTKs were originally proposed to have (Manning et al. 2008; Pincus et al. 2008). In- evolved in parallel with multicellularity in ani- deed, an unexpectedly complex repertoire of mals, and it was suggested that the development TKs has been found in the genomes of two uni- of Tyr phosphorylation-based signaling may cellular organisms that are close relatives of have played a vital part in the emergence of metazoans, namely, the choanoflagellate Mono- metazoans by providing the essential means of siga brevicollis and the filasterian Capsaspora coordinating function between different cells in owczarzaki. These two holozoan genomes each a multicellular organism. The lack of Tyr phos- encode .100 TKs, more than the total human phorylation in single-celled organisms, such as count, although both organisms are predomi- the yeasts, reinforced this idea. Nevertheless, be- nantly unicellular in lifestyle. Almost nothing is cause of the unexpectedly complex repertoire of known about the functions of these TKs, but the holozoan RTKs, it seems likely that RTKs their sequences do shed light on TK evolution did indeed evolve in single-celled organisms, and the diversity of domain contexts in which and that they were used as a means of sensing TKs can operate. The majority of TKs in both extracellular stimuli, perhaps nutrients or toxic species is predicted to be membrane-spanning compounds. Subsequently, they adapted to a cell-surface receptors (RTKs) (88/128 in Mono- new function in signaling cell–cell interactions siga and 92/103 in Capsaspora), but for the most either directly or through paracrine factors. part these RTKs are not orthologous to metazo- Whether RTKs arose through the fusion of a an RTKs. Possible homologs of EPH and IGF-1R gene encoding a receptor-like protein and a may be present in choanoflagellates, but no oth- gene for a TK catalytic gene, or perhaps from er examples of human RTK families are evident. a receptor-serine kinase, and whether all RTKs

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The Genesis of Tyrosine Phosphorylation

arose from a single progenitor RTK or whether ing networks. Areas in which key methodolog- RTKs arose multiple times are unanswered ical advances were made include detection of questions. P.Tyr, three-dimensional structural analysis, de- The existence of a large family of P.Tyr-spe- generate library methods for defining P.Tyr- cific protein phosphatases (PTPs), which op- binding and TK phosphorylation consensus se- pose the actions of the TKs, underscores the quences, development of sensitive nonradioac- importance of Tyr phosphorylation as an intra- tive kinase assays, generation of specific TKIs cellular signaling system. Like the TKs, PTPs and analog-sensitive protein kinase mutants, developed early in eukaryotic evolution, appar- MS-based P.Tyr proteomics, the use of kinase ently being derived from a family of dual-spe- and domain arrays, functional analysis of TK cificity phosphatases (DSPs), whose evolution- function by RNAi, genetic analysis in model ary origins are obscure, but which are found in organisms through knockout and mutant all extant eukaryotes, serving to dephosphory- knock-in analysis, and live cell imaging tech- late MAP kinases at the activating P.Tyr and niques for spatiotemporal localization of TK P.Thr sites. Unlike the protein kinases, which signaling events using fluorescently tagged pro- are predominantly in a single family, there are teins and biosensors. I will touch on a few of several distinct and unrelated protein phospha- these key advances. tase catalytic domain families. Among these, there are two main P.Tyr phosphatase families: Detection of P.Tyr the classical PTPs, which are selective for P.Tyr, and the DSPs, many of which can hydrolyze Initially, the detection of Tyr phosphorylation P.Tyr but also P.Serand P.Thr. In humans, there and the identification of the first TK substrates are 38 classical PTPs, split into 21 receptor-like required labeling cells with massive doses of 32P- PTPs and 17 nonreceptor PTPs, 61 DSPs (al- orthophosphate, and in vitro kinase reactions though all DSPs hydrolyze phosphate ester link- with g-32P-ATP. However, within 2 years of ages, not all of them act on protein substrates), the discovery of Tyr phosphorylation, antibod- four Asp-based PTPs, three Cdc25 DSPs that act ies that specifically recognize P.Tyr in proteins primarily on the inhibitory P.Thrand P.Tyr res- were developed (Ross et al. 1981; Frackelton idues in the CDKs, and the LMW PTP,for a total et al. 1983), and the use of anti-P.Tyr polyclonal of 107 (Alonso et al. 2004). Taken together, this and then monoclonal antibodies quickly sup- means that nearly as many P.Tyr phosphatases planted in vivo 32P labeling, and greatly in- exist as TK catalytic entities. However, although creased the number of identified TK substrates. there are genetic hints that specific RTK/PTP Ironically, as it turned out, these were not the pairs exist, in general there do not appear to be first man-made anti-P.Tyr antibodies. Immu- one-to-one relationships. nologists had been using phenylarsonate as a hapten in antibody induction experiments since the 1940s, without realizing, as later analysis NEW METHODS HAVE BEEN ESSENTIAL showed, that they were generating antibodies FOR UNDERSTANDING TYROSINE that cross-reacted with P.Tyr, albeit weakly! PHOSPHORYLATION Anti-P.Tyr antibodies have been valuable for It is often said that progress in biology is depen- studying patterns of Tyr phosphorylation in re- dent on advances in technology, and nowhere is sponse to specific stimuli by immunoblotting, this truer than in the field of Tyr phosphoryla- and also for immunostaining cells to identify tion. Historically, many of the advances in un- structures enriched in P.Tyr proteins (Marchisio derstanding Tyr phosphorylation have depend- et al. 1984; Maher et al. 1985). In addition to ed on the development of new methods and sequence-independent P.Tyr antibodies, site- reagents that played and continue to play a vital specific P.Tyr antibodies raised against synthetic part in our progress toward a complete under- P.Tyr-containing peptides have been particular- standing of Tyr phosphorylation-based signal- ly useful for assessing TK activation states in the

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cell, and both types of P.Tyr antibody have been regulated, both positively and negatively. A nice used for assaying TK activity in vitro. recent example emerging from structural anal- In the last 10 years, phosphoproteomic analysis is the unexpected stimulatory role of specif- ysis using high-throughput, high-sensitivity ic contacts made in cis between the SH2 do- mass spectrometry (MS) instruments has revo- mains of Fes and Abl and the amino lobes of lutionized the identification of Tyr phosphory- their catalytic domains (Filippakopoulos et al. lation sites. Such global analysis of P.Tyr-con- 2008), a mode of regulation that contrasts with taining proteins, using anti-P.Tyr monoclonal the inhibitory role of the combined SH3-SH2 antibodies to enrich for P.Tyr-containing tryptic domain unit in autoinhibition of Src and Abl peptides from digests of cellular proteins fol- family kinase activity, which is exerted through lowed by MS analysis, has revealed an unexpect- a distinct set of SH3 and SH2 contacts with the edly complex repertoire of proteins and sites amino and carboxy lobes. Recently, nuclear that can be phosphorylated on Tyr in metazoans magnetic resonance (NMR) solution analysis (Rush et al. 2005; Rikova et al. 2007). Thousands of kinase catalytic domains and P.Tyr interac- of P.Tyr sites have been reported, and, in the vast tion domains has begun to emphasize the im- majority of cases, the function of these Tyr portance of dynamic motions within the cata- phosphorylation events has not been investigat- lytic domain. This concept has been extended ed. In this regard, it is possible that a significant by the use of microsecond-long molecular dy- fraction of these sites do not have a functional namic simulations of catalytic domain struc- output, and could be construed as “noise” Tyr tures to define transitions between active and phosphorylation. inactive conformations.

Three-Dimensional Structural Analysis Degenerate Peptide Libraries and Target Identification Advances in the use of protein crystallography to define the structures of isolated protein do- Twenty years ago, Cantley and Songyang’s de- mains, intact proteins, and protein complexes velopment of degenerate, position-oriented pep- has played a key role in affording major insights tide libraries to delineate the sequence specificity into how Tyr phosphorylation and dephos- of SH2 domain binding to P.Tyr sites (Zhou phorylation is catalyzed, and how P.Tyr residues et al. 1993), and subsequently to define the pri- are recognized by P.Tyr-binding domains in a mary sequence selectivity of TK catalytic do- sequence-dependent fashion to promote pro- mains (Songyang et al. 1994) revolutionized tein–protein interactions and propagate signal- the field. Degenerate peptide library technology ing initiated by RTK activation at the cell surface has subsequently been widely used to define upon binding a growth factor. The first crystal protein kinase specificity in general, and such structure of a TK, namely, that of the insulin primary sequence preferences have been incor- receptor catalytic domain, yielded the secrets porated into programs such as Scansite; this al- underlying kinase specificity for Tyr versus Ser gorithm enables the user to input a primary se- and Thr (Hubbard et al. 1994; Taylor et al. 1995) quence, and obtain predictions as to which Likewise, structures of the SH2 domain bound protein kinase(s) might phosphorylate a residue to a synthetic P.Tyr peptide revealed that the of interest, which can then be tested experimen- P.Tyr side chain binds into a pocket, such that tally (Obenauer et al. 2003). Such prediction its O4-phosphate interacts with an Arg at the algorithms are continually being improved by base of the pocket, which is too deep for the combining sequence preference with other rele- phosphate on b-OH of Ser or Thr to reach vant contextual information, such as known (Waksman et al. 1992). Higher-order multido- protein–protein interactions obtained from main structures obtained by crystallography systematic interactome analysis, subcellular lo- and cryo-EM tomography analysis have begun calization, genetic epistasis relationships, and to reveal how receptor and nonreceptor TKs are other functional connections (e.g., NetworKIN

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The Genesis of Tyrosine Phosphorylation

and NetPhorest) (Linding et al. 2008). In the to define phosphorylation sites that are con- case of SH2 domain specificity, this approach served through evolution (Beltrao et al. 2012). has been extended by using arrays of P.Tyr-con- Sites that are conserved are most likely to have taining peptides derived from physiological Tyr important physiological functions. In addition, phosphorylation sites and recombinant SH2 knowledge of the primary sequence preference domains (Machida et al. 2007; Tinti et al. 2013). for a PK, combined with its interaction part- In an attempt to solve the thorny problem of ners, subcellular localization, signaling pathway which PK phosphorylates a particular site in the information, and functional genetic analysis cell, a number of new methods for identifying can be used to define potential new PK targets targets of individual PKs have been developed. (Linding et al. 2008). Information obtained One such method involves cross-linking of a from biochemical analysis, si/shRNA screens, catalytic domain to substrate proteins bound phosphoproteomic data sets, protein interac- to it in the cell; this is achieved by making a tomes, and other sources, can be used to build Cys substitution mutation at the substrate phos- phosphorylation networks. Such networks can phorylation site, and then using a cross-linker then be subjected to computer simulation to designed to covalently couple the Cys to the Lys predict input/output responses, and modula- in the ATP-binding site, followed by MS identi- tion by cross talk and feedback mechanisms. fication of the kinase bound to the tagged sub- This is particularly important in diseases of ab- strate (Statsuk et al. 2008). In another method, errant signaling, such as cancer, because it may an analog-sensitive PK mutant is used to thio- ultimately be possible to predict the conse- phosphorylate substrates in vitro using g-S-ATP, quences of using a specific inhibitor on signal- and then thiophosphorylated residues are mod- ing outcome based on the genotype of a tumor, ified with an adduct that can be recognized by an and thereby determine which combinations of antibody, allowing the modified peptides to be signal transduction inhibitor drugs might be immunoaffinity enriched and identified by MS most effective. (Allen et al. 2007). In another approach, spotted arrays of complete proteomes can be phosphor- Protein Kinase Assays, Inhibitors, and ylated by a purified TK to identify potential Kinase Engineering substrates, and arrays of isolated catalytic domains can be tested for phosphorylation of a Traditionally, PK assays involved using a protein specific protein of interest. Many nonreceptor substrate or a short synthetic peptide corre- TKs interact with targets via their SH2 or SH3 sponding to a known high-affinity phosphory- domains, and a new method for identifying SH2 lation site for the PK in question, combined and SH3 domain binding partners in the cell has with g-32P-ATP to monitor phosphorylation. recently been developed. This method takes ad- However, short Tyr-containing peptides are vantage of “unnatural amino acid” (UAA) tech- generally poor substrates for TKs, although nology, in which a UV photoactivatable UAA is the synthetic polymer poly(Glu.Tyr) has proved incorporated into a protein domain at specific useful as a generic TK substrate. The general sites on its ligand-interacting surface in appro- trend toward nonradioactive assays has led to priately engineered cells, allowing cross-linking the use of the sequence-independent anti- to bound proteins when cells are irradiated with P.Tyr MAbs or sequence-specific anti-P.Tyr an- UV,and recovery of the tagged domains for MS tibodies to assay phosphorylation, using FRET, analysis (Okada et al. 2011; Uezu et al. 2012). often in a high-throughput format for inhibitor screening. More recently, MS-based methods using mass-encoded substrate peptides have Bioinformatic Analysis of Tyrosine been developed, and these can be multiplexed Phosphorylation to simultaneously assay multiple PKs in a mix- The wealth of global phosphoproteomics data ture, such as a cell lysate, with high sensitivity now available allows cross-species comparisons and speed. Assays in which His-tagged TKs are

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T. Hunter

bound to NTA-modified lipid vesicles have came out of the same series as a compound proved very useful in recapitulating Tyr phos- that was ultimately taken forward into the clinic. phorylation on cell membranes, because of With the use of any inhibitor, however selective, the large increase in local protein concentration the caveat is that the cellular response may be afforded by using a two-dimensional surface due to an off-target effect, and the expression of (Zhang et al. 2006). Another important ad- mutant forms of the target PK designed to be vance has been the development of FRET-based resistant to the inhibitor can provide a crucial biosensors to measure PK activity in living cells. control for inhibitor specificity, including allo- These genetically encoded reporters, such as steric inhibitors (Holt et al. 2009). AKAR (Zhang et al. 2001), have a short peptide A major advance in studying PK function substrate sequence, coupled by a linker to an was Shokat’s development of mutant PKs in appropriate phosphobinding domain flanked which mutation of the “gatekeeper” residue at by YFP and CFP, such that phosphorylation of the base of the catalytic cleft to a residue with a the substrate site on the biosensor results in its smaller side chain allows the binding of base- interaction with the phosphobinding domain modified forms of ATPwith bulky groups at the intramolecularly, leading to a change in the N6 position of the purine ring, which are ex- YFP-CFP FRET signal that can be measured in cluded by the WT PK. This method was origi- living cells to report local activity of the PK in nally developed to identify PK substrates in cell question (Ting et al. 2001). One of the first PKs lysates using g-32P-labeled N6-modifed ATPan- this method was applied to was the EGF recep- alogs (Shah et al. 1997). Subsequently, Shokat tor RTK, in which the EKAR biosensor was used extended this concept by developing cell-per- to assess activation of the EGF receptor in re- meant, purine-based inhibitors with similar sponse to addition of EGF. Another fluores- bulky groups at the N6 position that can be cence-based method for assaying substrate Tyr used to specifically inhibit these “analog-sensi- phosphorylation in cells makes use of a modi- tive” PKs, without affecting other PKs in the cell fied SH2 domain in which a Trp lying adjacent (Bishop et al. 2000). This strategy has been very to the bound phosphate is replaced with a cou- powerful, particularly in organisms in which marin derivative using UAA technology; P.Tyr gene replacement technology can be used to binding is read out as a change in fluorescence replace the resident PK gene with the as mutant output (Lacey et al. 2011). gene. Treatment of the as mutant PK-expressing The development of selective TK inhibitors cells with such analog inhibitors has allowed has been a primary focus of the pharmaceutical identification of substrate proteins of the PK industry because of the importance of TKIs in of interest, characterized by a rapid decrease in cancer therapy. Methods for developing truly their phosphorylation (Holt et al. 2009). selective inhibitors are continually improving, and fragment/scaffold-based approaches, com- Genetic Analysis of Tyrosine bined with structure-based refinement are gen- Phosphorylation erating inhibitors with exquisite selectivity, although there are some arguments for designing Genetic screens in Drosophila and Caenorhabdi- multitargeted TKIs for cancer therapy. Selective tis elegans afforded some of the first glimpses inhibitors are an important research tool, be- into the functions of RTKs when mutants de- cause they allow rapid and reversible inhibition fective in differentiation and development were of the target kinase in cells, which can avoid shown to harbor mutations in RTK genes. Tar- compensatory mechanisms often observed geted knockout and knock-in studies in mice with knockout or knockdown studies. In this have also provided a wealth of information regard, it would undoubtedly benefit the re- about RTK function in vivo. More recently, the search community enormously if pharma could advent of RNAi technology has enabled si/ be persuaded to make available without strings shRNA depletion of individual TKs and PTPs selective inhibitors for research purposes that revealing which Tyr phosphorylation events are

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The Genesis of Tyrosine Phosphorylation

affected by the loss of individual TKs and PTPs, (Mikhailik et al. 2007). Over the past few years, as well as kinome-wide functional screens new P.Tyr-binding domains have been identified (MacKeigan et al. 2005), which have given us (e.g., the Hakai HYB domain and the PLC-d important insights into Tyr phosphorylation- variant C2 domain), and there may be other based signaling networks. examples, but, in contrast to the SH2 domain, these will not be found in large families. A high priority should be efforts to determine the func- Subcellular Localization of Tyrosine tions of the thousands of reported Tyr phos- Phosphorylation Events in Fixed and phorylation sites, bearing in mind the possibil- Living Cells ity that some fraction of them may be silent. Major advances in light microscopy, such as An important concept to consider is that confocal and superresolution microscopy, have P.Tyr not only signals directly, but can also made it possible to study signaling processes in couple with other posttranslational modifica- real time in living cells (and organisms) at in- tions (PTMs) in the same protein to provide a creasing resolution, using genetically encoded unique signaling output dependent on both GFP or RFP reporters or microinjected fluores- PTMs being present simultaneously, thus creat- cently labeled antibodies. This has made it pos- ing an AND logic gate. In this regard, the sible to visualize protein movements in the cell phenolic hydroxyl group of Tyr itself is subject in response to an external signal, and also use to additional PTMs, namely, sulfation and ni- FRET-based reporters to determine where a tration, and also adenylylation (AMPylation) protein kinase is active in the cell or where a (Worby et al. 2009). Such PTMs might compete protein–protein interaction occurs in the cell. with phosphorylation of specific Tyr under de- Superresolution microscopy, which breaks the fined circumstances, although this would de- diffraction limit of light, now makes it possible pend on stoichiometry. to resolve the position of single signaling pro- From a technology perspective, more sensi- tein molecules to within a few nm, and this un- tive and selective genetically encoded TK bio- precedented advance affords insights into local- sensors are needed. These will be particularly ized signaling structures, such as nanoclusters, useful in studying nuclear signaling by TKs, which can act as signaling depots (Lillemeier where biosensors localized to the nucleus could et al. 2010). help address the somewhat controversial issue of whether RTKs actively phosphorylate targets in the nucleus following their activation, either FUTURE through intramembrane cleavage and traffick- What does the future of tyrosine phosphoryla- ing of the released cytoplasmic domain into the tion hold? Given the unabated rate of progress nucleus, or through translocation of the intact since its discovery more than 30 years ago, we RTK into the nucleus. TK biosensors used in are certainly in for more surprises and insights, conjunction with superresolution microscopy and undoubtedly, this will require the develop- will also be helpful in studying membrane ment of new technologies. It seems unlikely that nanoclusters as signaling nodes. additional dedicated TKs will be identified, but Although structural analysis has already the recent report that PKM2 can phosphorylate taught us a great deal about Tyr phosphoryla- STAT3 on Tyr705 (Gao et al. 2012) means that tion, we need to define the structures of higher- other enzymes that use ATP, or another sub- order signaling complexes and nanoclusters in strate with an energy-rich phosphate, might the membrane. Single molecule analysis in T also moonlight as TKs under special circum- cells has indicated the importance of signaling stances. Likewise, additional P.Tyr phosphatase protein clusters that preexist before activation activities may emerge; a recent example is STS- (Lillemeier et al. 2010; Sherman et al. 2011), 1, a member of the histidine phosphatase family and this principle seems likely to hold true that has been reported to be a P.Tyr phosphatase for other systems. We can expect to learn that

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The Genesis of Tyrosine Phosphorylation

Tony Hunter

Cold Spring Harb Perspect Biol 2014; doi: 10.1101/cshperspect.a020644

Subject Collection Signaling by Receptor Tyrosine Kinases

CSF-1 Receptor Signaling in Myeloid Cells The Genesis of Tyrosine Phosphorylation E. Richard Stanley and Violeta Chitu Tony Hunter The EGFR Family: Not So Prototypical Receptor Structure-Function Relationships of ErbB RTKs in Tyrosine Kinases the Plasma Membrane of Living Cells Mark A. Lemmon, Joseph Schlessinger and Donna J. Arndt-Jovin, Michelle G. Botelho and Kathryn M. Ferguson Thomas M. Jovin Tie2 and Eph Receptor Tyrosine Kinase Activation Receptor Tyrosine Kinases: Legacy of the First and Signaling Two Decades William A. Barton, Annamarie C. Dalton, Tom C.M. Joseph Schlessinger Seegar, et al. The Spatiotemporal Organization of ErbB The Role of Ryk and Ror Receptor Tyrosine Receptors: Insights from Microscopy Kinases in Wnt Signal Transduction Christopher C. Valley, Keith A. Lidke and Diane S. Jennifer Green, Roel Nusse and Renée van Lidke Amerongen Insulin Receptor Signaling in Normal and Regulation of Receptor Tyrosine Kinase Ligand Insulin-Resistant States Processing Jérémie Boucher, André Kleinridders and C. Colin Adrain and Matthew Freeman Ronald Kahn Central Role of RET in Thyroid Cancer Molecular Mechanisms of SH2- and Massimo Santoro and Francesca Carlomagno PTB-Domain-Containing Proteins in Receptor Tyrosine Kinase Signaling Melany J. Wagner, Melissa M. Stacey, Bernard A. Liu, et al. Receptor Tyrosine Kinase-Mediated Angiogenesis Eph Receptor Signaling and Ephrins Michael Jeltsch, Veli-Matti Leppänen, Pipsa Erika M. Lisabeth, Giulia Falivelli and Elena B. Saharinen, et al. Pasquale Biology of the TAM Receptors Effects of Membrane Trafficking on Signaling by Greg Lemke Receptor Tyrosine Kinases Marta Miaczynska For additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/

For additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/