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Phosphotyrosine Signaling: Evolving a New Cellular Communication System

Wendell A. Lim1,2,* and Tony Pawson3,4,* 1Howard Hughes Medical Institute 2Department of Cellular and Molecular Pharmacology University of California, San Francisco, San Francisco, CA 94158, USA 3Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada 4Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada *Correspondence: [email protected] (W.A.L.), [email protected] (T.P.) DOI 10.1016/j.cell.2010.08.023

Tyrosine phosphorylation controls many cellular functions. Yet the three-part toolkit that regulates phosphotyrosine signaling—tyrosine kinases, phosphotyrosine phosphatases, and Src Homology 2 (SH2) domains—is a relatively new innovation. Genomic analyses reveal how this revolutionary signaling system may have originated and why it rapidly became critical to metazoans.

Throughout human history, new technolo- (TyrK) phosphorylate specific target with an evolutionary process? Proteins gies and technological platforms have tyrosine residues, phosphotyrosine phos- that bind or remove a posttranslational constantly been invented. Only a small phatases (PTP) remove the phosphates, modification would seem useless without fraction of these technologies go on to and Src Homology 2 (SH2) domains an enzyme to generate the modification be widely adopted, but these can recognize the modifications (Pawson and, in principle, would not provide ultimately have transformational conse- 1995). Together, these three modules a fitness advantage leading to its retention quences. In the evolutionary history of form the ‘‘writer,’’ ‘‘eraser,’’ and ‘‘reader’’ and spread. The pTyr signaling platform living organisms, we know that innovative toolkit that is common to many diverse provides a case study to look for plausible molecular systems have appeared at key cellular information processing platforms stepwise pathways of the evolution of points in time, and these are thought to (Figure 1A). A rich array of diverse and a multipart system. have played a transformative role in major complex regulatory schemes can be Here, we reconstruct a possible history evolutionary transitions in the tree of life. achieved through the dynamic interplay for the evolution of pTyr signaling. This But how do such innovative molecular of these three modular functions (Pawson reconstruction is based on the recent systems emerge, and how and why do et al., 1993; Pawson, 1995; Bhattachar- sequencing of the genomes of a number some proliferate and become stably yya et al., 2006; Kholodenko, 2006). of organisms that originated both before adopted by subsequent lineages? A combination of these modules can and after the emergence of metazoans An example of such an innovative lead to higher-order functions (Figure 1B). from single-celled eukaryotic ancestors molecular system is phosphotyrosine For example, there are several proteins (King et al., 2008). The genome sequence (pTyr)-based . This containing a combination of SH2 and of the choanoflagellate, Monosiga brevi- molecular system for transmitting cellular kinase domains that can generate collis, has been particularly illuminating regulatory information is estimated to positive feedback (phosphorylation of as choanoflagellates are thought to be have appeared relatively recently in the tyrosine sites leads to SH2-mediated one of the closest single-celled relatives history of life—600 million years ago, recruitment of the kinase, and subse- of metazoans. We present a model for just prior to the emergence of multicellular quently, more extensive phosphorylation) how this three-part signaling system (King et al., 2003; Pincus et al., (Pawson, 2004). Similarly SH2-phospha- could have plausibly evolved in a stepwise 2008; Manning et al., 2008). The pTyr sig- tase domain combinations can generate manner. We propose that once the naling system has become an essential negative feedback (Tonks and Neel, complete three-part system was in place, part of metazoan biology. For example, 2001). it may have rapidly taken hold in subse- pTyr signaling plays a central role in many The three-part pTyr signaling toolkit quent lineages because it could generate cell-to-cell communication pathways, thus raises a classic question in evolu- new regulatory behaviors without signifi- including those that regulate proliferation, tionary biology: how do complex, interde- cant cross-interference with existing differentiation, adhesion, hormone res- pendent systems arise? It is clear why regulatory circuits. We also discuss the ponses, and immune defense (Hunter, a new system encompassing a writer, possible role of this new communication 2009). eraser, and reader might be extremely system in facilitating the transformative In modern metazoans, pTyr signaling is useful. But, given their interdependence, evolutionary shift to multicellularity. mediated by a toolkit of three distinct how could these individual components Given the incomplete record, however, functional modules: tyrosine kinases arise in a stepwise fashion consistent such an evolutionary reconstruction is

Cell 142, September 3, 2010 ª2010 Elsevier Inc. 661 by an upstream Ser/Thr kinase on both a Thr and Tyr residue within its activation loop; Cdk1 is phosphorylated on Tyr14 by the inhibitory kinase Wee1). These tyrosine modifications are clearly not recognized by SH2 domains, but they exert direct allosteric effects within the proteins in which they occur. Thus, PTP domains may have provided a fitness benefit by negatively modulating these rare but functionally important phosphor- ylation events. Consistent with this model, the proteins PTP2 and PTP3 in yeast clearly have a functionally important role in downregulating MAPK-mediated sig- naling in response to pheromones or osmolarity changes, explaining their Figure 1. The Writer, Reader, Eraser pTyr Toolkit fitness benefit (Pincus et al., 2008). In (A) In pTyr signaling, the (TyrK), Src Homology 2 (SH2), and phosphotyrosine phosphatase addition, PTPs may have played a general (PTP) domains form a highly interdependent signaling platform. This platform serves as the writer, reader, role buffering against the occasional and eraser modules, respectively, for processing pTyr marks. (B) Components of pTyr signaling can be used to build complex circuits. For example, recruitment of an harmful stray phosphorylation of function- SH2-TyrK protein to an initiating pTyr site can lead to amplification of through ally important tyrosines. a positive feedback loop. Where did these PTPs come from? PTPs are likely to have arisen from highly speculative. For example, we have tyrosine phosphatase activity. We a common ancestor of the related dual- cannot rule out more complex paths refer to the single putative ‘‘SH2’’ domain specificity phosphatases, which are also involving cycles of evolutionary gain and in yeast, found within the gene Spt6,as found in most single-celled eukaryotes loss of components, nor the possibility a proto-SH2 domain because it does not (Kennelly, 2001; Alonso et al., 2004). that similar components in distinct line- show pTyr binding (the domain has been Dual-specificity phosphatases are cata- ages have independent origins. Nonethe- reported to show phospho-Ser/Thr lytic domains that can dephosphorylate less, this model may provide a useful binding; Dengl et al., 2009). Thus, func- both pSer/Thr and pTyr substrates. The framework for focusing studies of pTyr tionally, it cannot be considered a PTP and dual-specificity phosphatase signaling origins and the origins of analo- ‘‘reader’’ domain that is part of a pTyr catalytic domains are distinct but are gous multicomponent signaling plat- regulatory system. These observations evolutionarily related. They share a forms. suggest a simple model: the first step in common fold and the core catalytic motif

We describe three possible stages in the evolution of the three-part pTyr HC(X)5R, in which a phosphocysteine the emergence of the modern pTyr signaling machinery was likely to have enzyme intermediate is generated during signaling toolkit, each represented by an been the emergence of a functional tyro- catalysis. (Sometimes, both dual-speci- extant model organism (Figure 2). These sine phosphatase. But why would PTPs ficity phosphatases and classical PTPs stages are representative; we do not arise in the pre-tyrosine kinase world? are referred to as PTPs; here, we use claim to define the exact path of evolution, What functional use and fitness advan- this nomenclature only for the classical but rather focus on identifying the domi- tage would this eraser domain provide in PTP domains that act only on pTyr). The nant classes of stable intermediates that organisms lacking a writer domain? domains of dual-specificity phosphatases can exist in the broader evolutionary land- The answer may lie in the fact that some have a shallower active site than classical scape. Ser/Thr kinase domains, which are more PTPs, which may explain why they can ancient than tyrosine kinases (dating dephosphorylate either Tyr or Thr/Ser PTPs in a Pre-Tyrosine back close to the origins of eukaryotes), residues. In some lineages, dual-speci- Kinase World can carry out sporadic but functionally ficity phosphatases have functionally What came first, TyrK, PTP, or SH2 important phosphorylation of tyrosines. diverged further, giving rise to members domains? Sequence analysis suggests Phosphoamino acid analysis of yeast that can act on lipid substrates, such as that it was PTP domains. The genome of reveals a small but significant population the phosphoinositide phosphatases a simple single-celled eukaryote like the of pTyr (Schieven et al., 1986). Moreover, PTEN and the myotubularins (Alonso budding yeast Saccharomyces cerevisiae certain events, such as the activation et al., 2004). Thus, the PTPs appear to shows no TyrK proteins and one proto- of mitogen-activated protein kinases have arisen from a somewhat promis- SH2 domain, but a handful of PTP (MAPKs) and inhibition of the cell-cycle cuous class of multifunctional phospha- proteins (Pincus et al., 2008)(Figure 2, kinase Cdk1, are known to involve phos- tases. Stage 1). Most fungi have no more than phorylation of tyrosine residues (for acti- Despite the presence of PTP proteins in five PTP proteins, and several of these vation, a MAPK must be phosphorylated fungi, there are striking differences

662 Cell 142, September 3, 2010 ª2010 Elsevier Inc. Figure 2. Evolution of pTyr Signaling Shown is a possible path for the emergence of phosphotyrosine (pTyr) signaling. We postulate three successive stages, each represented by what is observed in a modern organism. The thickness of the tree reflects the approximate degree of usage of pTyr signaling (thicker lines mean more usage). Stage 1 (exemplified by the budding yeast Saccharomyces cerevisiae) reflects the situation in early eukaryotes, in which PTPs emerged but were limited in number and complexity. They were most likely used to reverse or process sporadic cross-phosphorylation of tyrosine residues by Ser/Thr kinases. S. cerevisiae has fewer than five PTP proteins and no functional SH2 or TyrK domains. Stage 2 reflects systems in which functional SH2 domains emerged that were able to bind to pTyr motifs. Together with Ser/Thr kinases with increased cross-reactivity for Tyr (such as tyrosine kinase-like or dual specificity Ser/Thr kinases), these systems may reflect the most primitive of pTyr writer/reader/eraser systems. However, the lack of a dedicated Tyr kinase may have limited the utility and expansion of this toolkit. This stage is potentially represented by the slime mold, Dictyostelium discoideum. Stage 3 reflects systems that evolved after the emergence of the modern TyrK domain. We postulate that the full writer/reader/eraser system was of so much greater utility that its use expanded dramatically. This likely resulted in many more proteins in these families, as well as much more complex, multidomain architectures than those seen in the earlier stages. This stage is represented by both the multicellular metazoan and unicellular choanoflagellate lineages. between these proteins and those proteins in which the PTP module has transfer enzymes; Bordo and Bork, found in metazoans (Figure 2). For been functionally recombined with multi- 2002). Thus, fungal PTP proteins are example, there are far fewer PTPs in ple other signaling modules. In contrast, very simple (one to two domains) and fungi (5/genome versus 40/genome in fungi, the PTP domains are all either lack the combinatorial complexity of in metazoans) and they are considerably in simple single-domain proteins or in metazoan PTP proteins. The simplicity less complex in domain architecture combination with a single rhodanase-like and low number of PTPs in yeast sug- (Pincus et al., 2008). Metazoan PTP domain (a putative regulatory domain gests that in early single-celled eukary- proteins tend to be large multidomain that is homologous to a class of sulfur otes, PTP domains had fairly limited

Cell 142, September 3, 2010 ª2010 Elsevier Inc. 663 functional utility, especially when com- pTyr-binding SH2 domains and may kinase domain followed by an SH2 pared to their broad and complex usage therefore provide a living representative domain, a domain combination that is in metazoans. of this second evolutionary stage (Fig- somewhat similar to metazoan cyto- Unlike PTPs, there are no known pTyr- ure 2, Stage 2). Dictyostelium is a soil- plasmic tyrosine kinases like Src (Monia- binding SH2 domains in fungi, although living amoeba that has a unicellular kis et al., 2001). The Shk catalytic domain, there is one clearly homologous domain lifestyle in the presence of bacterial however, lacks motifs characteristic of found in the yeast protein SPT6. This food. However, when food is depleted, bona fide tyrosine kinases and biochemi- protein, which has a domain with an individual cells aggregate in response to cally displays dual specificity toward SH2-like sequence and fold, is involved the chemoattractant cAMP to form serine/threonine and tyrosine residues. in the regulation of transcription elonga- a multicellular structure, which then Indeed, Dictyostelium differs from tion, and the SH2 domain binds to the develops into a fruiting body through the metazoans and choanoflagellates in that Ser/Thr phosphorylated C-terminal tail of differentiation of stalk and spore cells. its genome does not encode any modern RNA polymerase II. The domain does The rudimentary pTyr-SH2 system in tyrosine-specific protein kinases. For not bind to pTyr (Dengl et al., 2009). Inter- Dictyostelium is important for aspects of example, metazoan STAT proteins are estingly, a single SPT6 ortholog, with the this differentiation process, including usually phosphorylated by Janus tyrosine same overall domain architecture, is intracellular responses to both cAMP kinases (JAKs), but there are no JAKs in found in all eukaryotes, including all fungi and the morphogen differentiation in- Dictyostelium (Kay, 1997). This suggests and metazoans (but not prokaryotes). This ducing factor or DIF (which induces the the possibility that signaling proteins con- finding suggests that in early eukaryotes, differentiation of prestalk cells), as well taining SH2 domains such as STATs a proto-SH2 domain emerged to perform as for transcriptional regulation in res- evolved before the modern tyrosine a highly specialized function—one that ponse to hyperosmotic stress. These kinases with which they are associated was unrelated to the flexible modular observations are consistent with early in metazoans. The identity of the kinase pTyr recognition function of the modern pTyr-SH2 signaling playing a role in responsible for STAT tyrosine phosphory- SH2 domain. This proto-SH2 domain cellular responses to changing environ- lation, and the consequent formation of most likely did not ‘‘read’’ pTyr modifica- mental conditions. SH2-binding sites, remains mysterious. tions, but instead recognized a special- The Dictyostelium genome specifies 13 How, then, is tyrosine phosphorylation ized related modification. Thus, although proteins with SH2 domains (as well as of Dictyostelium proteins such as the SPT6 is likely to represent an early a single Spt6 homolog). These 13 proteins STATs controlled? Thus far, genetic anal- ancestor or relative that eventually gave cluster into five basic domain architec- ysis has not identified a specific relevant rise to modern SH2 domains, it cannot tures, two of which are homologous to kinase, and it has been proposed that, in be considered a functional part of a pTyr metazoan SH2 proteins. Notably, Dic- contrast to mammalian STATs, there regulatory toolkit. We therefore postulate tyostelium has four STAT (signal trans- may be basal constitutive phosphoryla- that early eukaryotes had only a pTyr ducers and activators of transcription) tion of Dictyostelium STAT tyrosine sites, eraser function (mediated by PTPs) with proteins that are very similar to metazoan which is regulated by changes in PTP no specialized complementary reader or STAT transcription factors (Kay, 1997; activity in response to extracellular writer functions. Kawata et al., 1997). For example, they signals (Langenick et al., 2008). One of In summary, the PTP domain and all have an SH2 domain juxtaposed to the PTPs in Dictyostelium, PTP3, binds a structural ancestor of the SH2 domain a DNA-binding region; they are inducibly and dephosphorylates STATc, thereby appear to have arisen in early single- phosphorylated on tyrosine residues in blocking SH2-mediated dimerization and celled eukaryotes, but are likely to have response to stress or the extracellular STATc accumulation in the nucleus. functional origins that are not directly signaling molecule DIF; they undergo Signaling induced by the DIF morphogen related to their later function in modern pTyr/SH2-mediated dimerization and appears to transmit signals by inhibiting pTyr regulatory systems. These compo- then translocate to the nucleus to regulate PTP3 activity and consequently boosting nents may have provided a limited but the expression of specific genes. Dictyos- STATc tyrosine phosphorylation and incremental fitness advantage, even in telium also has an ortholog of the STATc-dependent gene expression. the absence of a specialized tyrosine mammalian E3 Cbl, which Although Dictyostelium lacks true tyro- kinase domain. uses SH2 and Ring domains to couple sine kinases, it is noteworthy that its pTyr signals to the ubiquitination ma- genome has a significant expansion in Toward a Write/Read/Erase System chinery (Langenick et al., 2008). The re- the number of putative dual-specificity In the early days of a more sophisticated maining three domain architectures of protein kinases (there are 70, also known pTyr-signaling system, we suggest that Dictyostelium SH2 proteins are distinct as tyrosine kinase-like or TKL kinases) a proto-SH2 domain (mostly likely a from those found in other sequenced (Manning et al., 2008). This set includes homolog of the yeast Spt6 protein) in organisms. The LrrB protein has an SH2 the Shk catalytic domain, described a single-celled organism acquired the domain linked to a leucine-rich repeat above. It is unlikely that any of these new and functionally beneficial ability to domain (Sugden et al., 2010), whereas kinases are precursors of modern tyrosine bind to pTyr-containing peptide motifs. the FbxB protein has an F-box followed kinases. However, it is plausible that these The slime mold Dictyostelium discoideum by an SH2 domain and ankyrin repeats. represent the first evolutionary form of has the simplest repertoire of bona fide In addition, the Shk proteins have a protein the ‘‘writer’’ function in a prototype pTyr

664 Cell 142, September 3, 2010 ª2010 Elsevier Inc. three-part regulatory system. The union of sine kinases (Lee and Jia, 2009). It is functions, thus leading to the subsequent an SH2 domain and a dual specificity therefore probable that BY kinases expansion of the complete set. Although kinase domain, as found in the Shk evolved separately from metazoan tyro- the PTP and SH2 domains had utility in proteins, may be an early example of link- sine kinases and operate in a different simpler organisms, their much larger ing ‘‘reader’’ and ‘‘writer’’ modules to fashion. functional potential was not unleashed achieve more complex functions such as The new eukaryotic tyrosine kinase until the emergence of the TyrK domain. positive feedback. Nonetheless, the domain appears to have been a game The rapid expansion of the pTyr limited functionality of the dual specificity changing innovation (Figure 2, Stage 3). signaling machinery in the ancestors of kinases in carrying out tyrosine phosphor- The total number of tyrosine kinase choanoflagellates and animals is reminis- ylation may have limited the capabilities of proteins in both choanoflagellate and cent of how technology expands in this early system. This may explain the metazoan species is in the range of 30– quantum jumps, especially in situations very modest expansion of pTyr signaling 150 per genome (Pincus et al., 2008; involving codependent technologies. For in organisms such as Dictyostelium. Manning et al., 2008). Among sequenced example, the value of the laser expanded These observations paint the following genomes, there is a striking absence of dramatically after the later invention of picture of Dictyostelium pTyr signaling species with only a small number of TyrK the complementary technology of fiber and, by extension, of an early phase in proteins. This all-or-none sudden jump in optics. This codependent technology al- the evolution of pTyr communication. the number of TyrK proteins suggests lowed lasers to be repurposed to rapidly SH2 domains have acquired pTyr-binding their importance as they appear to have displace electrical transmission via activity and are found in several distinct undergone rapid expansion and subse- copper wires as the backbone of global combinations with other types of signaling quent retention. communication (Alwayn, 2004). Thus, domains. Among these, the STAT and Cbl What is perhaps more striking is the although lasers had standalone utility, proteins are shared with metazoans, observation that the emergence of the their major application had to await the whereas the LrrB, FbxB, and Shk proteins TyrK domain and its rapid expansion introduction of complementary tech- are unique to Dictyostelium. But no dedi- correlates with an equally rapid expansion nology. The expansion of molecular com- cated modern tyrosine kinases have of PTP and SH2 domains within the same ponents in biology is likely to be similar. been found, and the dynamic control of genomes (Pincus et al., 2008). Although A toolkit of writer, reader, and eraser tyrosine phosphorylation may be primarily fungi and Dictyostelium have 5 PTP functions may be of full use only when all regulated by PTPs. Although functionally proteins, metazoans, and choanoflagel- components are present. Thus, it may important for aggregation and differentia- lates have 30–40 per genome. Similarly, be common for any system of this type tion, the pTyr signaling system has not Dictyostelium has approximately ten to show a quantum ‘‘all-or-none’’ expan- acquired the pervasive influence evident SH2 domain-containing proteins (fungi sion only when the final piece of the toolkit in M. brevicollis and metazoans, perhaps have none), whereas metazoans and emerges. because of the lack of an efficient tyrosine choanoflagellates have 100 each. kinase. Put another way, Dicytostelium Thus, both PTP and SH2 proteins Applying the New pTyr Toolkit has effective pTyr readers and erasers, undergo a roughly 10-fold increase in to Different Functions but the writer is poorly developed. number per genome after the emergence Although both choanoflagellate and of the TyrK domain. Moreover, the metazoan lineages show a large expan- Invention of TyrK and Expansion proteins containing SH2 and PTP sion of the three-part pTyr regulatory of the pTyr Toolkit domains become far more complex and machinery, the way in which these Current analysis suggests that the varied (Jin et al., 2009). For example, in components are used appears to be quite modern tyrosine kinases arose just prior yeast and Dictyostelium, SH2 and PTP different. When one examines the domain to the evolution of the metazoans. Aside proteins normally are very simple one or types that co-occur with TyrK, SH2, or from metazoans, canonical tyrosine two domain proteins. However, in line- PTP domains, one finds many distinct kinases have thus far only been observed ages that have modern TyrK proteins, combinations that are unique to each in the choanoflagellates, which appear to SH2 and PTP proteins almost always lineage (Pincus et al., 2008; Manning be the closest known single-celled rela- comprise three to ten domains. et al., 2008). These differences in domain tives of metazoans (King et al., 2008). These observations are consistent with combinations imply distinct functions for The absence of significant numbers of the following model. When a far more proteins containing these domains in the such tyrosine kinases in any other branch efficient TyrK domain—or ‘‘writer’’ func- choanoflagellate and metazoan lineages of life suggests that this new catalytic tion—emerged, this dramatically increased (Li et al., 2009). Assuming that the evolu- domain evolved in a recent common the functional utility of the pre-existing PTP tion of new TyrK, SH2, and PTP proteins ancestor of choanoflagellates and meta- (eraser) and SH2 (reader) domains. As occurred by recombination with new zoans, most likely as a branch of the older a three-part toolkit—a catalytic domain to accessory domains (Jin et al., 2009; Pei- Ser/Thr kinases. Some bacteria do have generate pTyr, an interaction domain to sajovich et al., 2010), this observation specialized tyrosine kinases (BY kinases), bind to these pTyr sites, and an enzyme also implies that the complete signaling but these resemble P loop NTPases to dephosphorylate them—this domain toolkit emerged only shortly before the (nucleotide triphosphatases) and are set could be used to encode and execute divergence of metazoans and choanofla- structurally unrelated to eukaryotic tyro- a far wider and diverse range of regulatory gellates (i.e., shortly before the evolution

Cell 142, September 3, 2010 ª2010 Elsevier Inc. 665 of metazoan multicellularity) and that communication. Because of this valuable gous new molecular information curren- much of the divergent expansion of these high encoding potential, there is extreme cies could have, in principle, been able domain families occurred after the lineage pressure to quickly fill this region of the to serve as the substrate for dramatic split. spectrum. Moreover, the exact type of phenotypic innovation. In this context, Thus, earlier assumptions that pTyr information carried by each region of the plants make extensive use of protein signaling is only used in metazoan cell- spectrum is flexible—for example, the phosphorylation and have numerous cell communication are clearly incorrect. same region of the spectrum can be as- transmembrane receptor Ser/Thr kinases, Choanoflagellates do not form the com- signed to different functions in different but they lack conventional tyrosine plex and permanent cell-cell organization countries. We hypothesize that the new kinases, indicating that pTyr-based that metazoans do, yet surprisingly they pTyr signaling system that emerged prior signaling is not the only mechanism of have a comparable (if not greater) number to metazoans presented similar new information transfer through which organ- of pTyr signaling proteins (Manning et al., opportunities to transmit more informa- isms can achieve multicellularity. 2008). Sequencing of other organisms tion. This virgin system was rapidly that arose near the origins of metazoans exploited, though the way it has been Is pTyr Signaling Saturated? is ongoing. Preliminary data also suggest used appears to be different in the two How close is the pTyr signaling system to a large number of pTyr signaling proteins branches (metazoans and choanoflagel- being saturated? Is there still available en- in other single-celled relatives of meta- lates) that emerged after the complete coding potential that could be tapped for zoans. Thus, it may be more reasonable toolkit was established. the evolution of new pathways and to view the pTyr signaling system as an It is tempting to speculate that the behaviors? It is difficult to answer these innovative but generic information pro- emergence of a new signaling system questions. However, the fact that new cessing system that could potentially be with high encoding potential may have pTyr signaling proteins appear to be asso- used for transmitting many different types played a key role in the emergence of ciated with advanced processes like of information. a new, complex biological function such adaptive immunity suggests that there as metazoan multicellularity. Such large- was still some remaining encoding poten- Orthogonal Signaling: A Platform scale phenotypic evolutionary innova- tial in the system as late as the evolution of for Biological Innovation tions may require and coincide with mammals. The evolutionary history re- When the three-part pTyr system first innovations in basic molecular compo- constructed here begs many questions. emerged, it presented a new platform nents (King, 2004; Rokas, 2008). Are there new regulatory toolkits evolving with which to transmit information that Indeed, we speculate that pTyr now or in the future? Will these new tool- was orthogonal to pre-existing signaling signaling may provide a more general kits be the substrate required for the systems. Because it was based on a model for the generation of multicompo- next big evolutionary innovation? distinct covalent modification, new regu- nent biological systems, involving first a The importance of new molecular tool- latory circuits could be assembled with limited stepwise development of elements kits is conversely also very relevant to these components without significant that together have a rudimentary biolog- the emerging field of synthetic biology, in cross-interference with pre-existing net- ical utility, followed by an explosive which the goal is to engineer cellular works. Thus, this brand new signaling spread, once all of the components of systems with new functions. A major apparatus probably had a high encoding the mature system are in place. Explora- potential limitation is how to build such potential for evolving dramatically new tion of this concept, and further analysis new functions in a reliable fashion that functions, such as those involved in multi- of the evolution of pTyr signaling, will be does not cross-interfere in unanticipated cellularity. One possible problem that assisted by the increasing sequence infor- ways with existing systems (Lim, 2010). could be caused by the expansion of the mation being gathered for both unicellular Can we develop new synthetic molecular new pTyr signaling enzymes might be and multicellular eukaryotes (Srivastava signaling currencies that are orthogonal excessive general phosphorylation of et al., 2010), which will no doubt yield to existing natural ones, and would these tyrosine residues throughout the pro- surprises akin to the discovery of exten- systems dramatically facilitate our ability teome. Interestingly, however, organisms sive pTyr signaling in M. brevicollis. More- to reliably and predictably endow cells using pTyr signaling may have developed over, as genomic information bracketing with innovative new functions? a simple solution to deal with this other major evolutionary transitions problem—a decrease in the tyrosine becomes available, it will be interesting Conclusions content of proteins across the proteome to see whether these innovations are Current data suggest that PTP and SH2 is observed to correlate with tyrosine also associated with the explosive expan- domains evolved before modern TyrK kinase expansion (Tan et al., 2009). sion of new molecular toolkits. domains, most likely to process pTyr A new orthogonal signaling system like A key point here is that the specific modifications sporadically catalyzed by the pTyr signaling platform can be viewed emergence of the pTyr toolkit may not Ser/Thr kinases. However, the PTP and as analogous to a newly opened region in have been essential for the evolution of SH2 domain protein families did not the telecommunications spectrum. New multicellularity, but rather, any number of expand dramatically until the emergence frequencies provide the opportunity for new orthogonal signaling toolkits with of an efficient TyrK. We postulate that transmitting large amounts of information the same high encoding potential could only with the complete toolkit of writer as there is little interference from existing have served a similar role. Other analo- (TyrK), reader (SH2), and eraser (PTP)

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Ernberg Li, W., Scarlata, S., and Miller, W.T. (2009). functions. for helpful comments. This work was supported by Biochemistry 48, 5180–5186. Thus, we are able to reconstruct a plau- the Howard Hughes Medical Institute (W.L.), the Lim, W.A. (2010). Nat. Rev. Mol. Cell Biol. 11, sible model by which the pTyr signaling National Institutes of health (GM55040, GM62583, 393–403. machinery could have evolved in a rela- GM081879, and EY016546—W.L.), the Packard Manning, G., Young, S.L., Miller, W.T., and Zhai, Y. Foundation (W.L.), the National Science Foundation (2008). Proc. Natl. Acad. Sci. USA 105, 9674–9679. tively simple stepwise manner into what Synthetic Biology Engineering Research Center Moniakis, J., Funamoto, S., Fukuzawa, M., today is a complex and highly interdepen- (W.L.), the Canadian Institutes for Health Research Meisenhelder, J., Araki, T., Abe, T., Meili, R., dent system. 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