Signaling—2000 and Beyond

Ships that pass in the night and speak each other in passing Only a signal shown and distant voice in the darkness —Henry Wadsworth Longfellow, “The Theologian's Tale” (1863) When the history of signal transduction is written in 2100, what will be remembered of the exciting advances that occurred in the latter half of the twentieth century? Everyone will surely have their own list of landmark findings that have contributed to our current picture of signal transduction. Undoubtedly there are seminal earlier events, but from my own perspective this era began with the discovery in the mid-1950s that phosphorylation can reversibly alter the activity of an enzyme through the combined action of a protein kinase and a protein phosphatase (41Krebs E.G Beavo J.A Phosphorylation-dephosphorylation of enzymes.Annu. Rev. Biochem. 1979; 48: 923-959Crossref PubMed Scopus (1388) Google Scholar). At much the same time the hormones adrenalin and glucagon were found to increase the level of intracellular 3′5′ cyclic AMP, and the concept of the second messenger was born. About 10 years later the cAMP-dependent protein-serine kinase (PKA) was isolated as a target for cAMP, and through its pleiotropic substrate specificity PKA was shown to be responsible for many of the effects of cAMP. With these findings the field of signal transduction was born. Since these early days, progress in understanding mechanisms of signal transduction has been astonishingly rapid. In the past 40 years many important themes and principles of signal transduction have emerged. The highly conserved nature of eukaryotic signaling pathways has been revealed, and defects in signaling have been found as the underlying basis of cancer and other human diseases. Progress has benefited tremendously from structural and genetic analysis, the development of analytical techniques and reagents, and the availability of pharmacological modulators of signaling. What are some of the major milestones? The identification of G proteins and their role in activating membrane bound adenylyl cyclase to synthesize cAMP in response to hormonal stimulation in the mid-1970s was another great step forward in understanding transmembrane signal transduction (22Gilman A.G G proteins transducers of receptor-generated signals.Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4616) Google Scholar). The study of G proteins revealed the principle that hydrolysis of protein-bound GTP could act as a signaling switch, and also brought us very near to the membrane receptor itself. The cloning of hormone receptors that are coupled to adenylyl cyclase as well as several neurotransmitter and drug receptors revealed that they all had a close relationship to rhodopsin, the seven transmembrane domain G protein–coupled light receptor. This suggested that serpentine G protein–coupled receptors would be a large family, and, indeed, with the recent addition of odorant receptors, this has become by far the largest receptor family, numbering over a thousand. Analysis with purified components showed that the liganded receptor interacts directly with the heterotrimeric G protein leading to the exchange of GTP for GDP bound to the α subunit, which thereupon dissociates from the β/γ subunits, allowing both the α.GTP and the β/γ complexes to signal to downstream effectors, such as adenylyl cyclase, again by direct interaction. Sustained signaling in response to a stimulus is generally undesirable, and therefore mechanisms for signal termination are required. The study of G proteins revealed that signaling is turned off when the α subunit hydrolyzes the bound GTP, either spontaneously or upon interaction with a GTPase activating protein (GAP), permitting the β/γ complex to rebind. During the 1980s and 1990s several other distinct principles of transmembrane signaling have been uncovered. The discovery of a new class of protein kinases associated with the polyomavirus, v-Src and v-Abl viral transforming proteins that phosphorylate tyrosine immediately suggested that tyrosine phosphorylation plays a role in growth control (30Hunter T Cooper J.A Protein-tyrosine kinases.Annu. Rev. Biochem. 1985; 54: 897-930Crossref PubMed Google Scholar). Tyrosine phosphorylation was quickly revealed as a major mechanism of transmembrane signaling. The demonstration that EGF stimulated tyrosine phosphorylation EGF receptor in membrane preparations led to the identification of a large family of ligand-stimulated receptor protein-tyrosine kinases (PTKs). Receptor PTKs are all type I transmembrane proteins with a cytoplasmic domain that has intrinsic catalytic activity that is activated upon ligand binding. This established that intracellular protein phosphorylation can be used as a direct means of transmembrane signal transduction. Another important step forward in understanding how signals are transmitted across the membrane came with the discovery that receptors lacking intrinsic catalytic activity can be coupled to nonreceptor PTKs via noncovalent association with the cytoplasmic domain of a receptor subunit, thus forming “binary” receptors (59Neet K Hunter T Vertebrate non-receptor protein-tyrosine kinase families.Genes Cells. 1996; 1: 147-169Crossref PubMed Scopus (121) Google Scholar). For instance, cytokine receptors use members of the JAK PTK family. The discovery of transmembrane protein-serine kinases, first in plants and then as receptors for TGFβ family cytokines in vertebrates, provided another example of the use of ligand-induced intracellular protein phosphorylation as means of transmembrane signal transduction. The fact that receptor PTKs proved to be type I transmembrane proteins raised the conundrum of how a theoretically flexible protein with a single transmembrane domain could propagate a signal across the membrane in response to ligand binding. The seminal finding was that activation of receptor PTK cytoplasmic catalytic domains requires ligand-induced dimerization (71Schlessinger J Signal transduction by allosteric receptor oligomerization.Trends Biochem. Sci. 1988; 13: 443-447Abstract Full Text PDF PubMed Scopus (291) Google Scholar). This juxtaposes the two catalytic domains allowing mutual transphosphorylation of residues in the activation loop of the catalytic domain, leading to enzymatic activation, and autophosphorylation of tyrosines outside the catalytic domain, which are the key to downstream signaling. Receptor protein-serine kinases are also activated by ligand-induced dimerization. Subsequently, ligand activation of many types of surface receptor, including those that do not activate protein kinases directly, has been found to involve oligomerization. The discovery of the SH2 domain as a means of recognizing specific phosphorylated tyrosines in activated receptor PTKs and receptor PTK targets was a breakthrough in understanding how activated PTKs propagate signals, since it revealed how the association of two proteins could be induced by phosphorylation thus propagating a signal (63Pawson T Gish G.D SH2 and SH3 domains from structure to function.Cell. 1992; 71: 359-362Abstract Full Text PDF PubMed Scopus (776) Google Scholar). Indeed, this discovery illustrated a totally new function for protein phosphorylation, namely the regulation of protein–protein association. Other types of phosphotyrosine (P.Tyr)-binding domain have subsequently been found (e.g., PTB domains), but SH2 domains are the most prevalent type of P.Tyr-binding domain involved in signaling downstream of activated receptor PTKs. SH2 domains bind in a sequence-specific fashion, recognizing one or more residues in positions 1–6 C-terminal to the P.Tyr. PTB domains recognize residues up to 5 away on the N-terminal side of P.Tyr, but only a subset of PTB domains (e.g., the Shc and IRS-1 PTBs) bind to their target proteins in a phosphorylation-dependent manner (85van der Geer P Pawson T The PTB domain a new protein module implicated in signal transduction.Trends Biochem. Sci. 1995; 20: 277-280Abstract Full Text PDF PubMed Scopus (230) Google Scholar). SH2 and PTB domain interactions are used as a means of recruiting target proteins to activated PTKs, thus permitting their phosphorylation, and also for translocation to the plasma membrane, where many effector proteins activated by receptor PTKs, such as phospholipase Cγ and PI-3′ kinase, have their substrates (73Schlessinger J SH2/SH3 signaling proteins.Curr. Opin. Genet. Dev. 1994; 4: 25-30Crossref PubMed Scopus (394) Google Scholar). SH2/PTB domains are present not only in proteins with intrinsic enzymatic activity that can be regulated by tyrosine phosphorylation, such as phospholipase Cγ, but also in so-called adaptor proteins, such as Grb2, that bind to and thereby bring effector enzymes to the plasma membrane. Receptor-induced recruitment of proteins to the plasma membrane as a mechanism of activating signaling pathways has emerged as a major theme in signaling. With the identification of the SH2 domain came the realization that most protein–protein interaction domains involved in signal transduction are modular in nature (Figure 1). The study of signaling initiated by receptor PTKs and other protein kinases has uncovered a plethora of different protein interaction domains, ranging in size from 40 to 150 residues, used for inducible or constitutive interactions involved in signaling (62Pawson T Protein modules and signaling networks.Nature. 1995; 373: 573-580Crossref PubMed Scopus (2182) Google Scholar). Examples are SH2, PTB, SH3, WW, FHA, SAM, LIM, PX, EH, EVH1, and PDZ domains. Like the SH2 domain, most of these domains recognize short linear sequences from 4 to 10 amino acids in length, in some cases requiring phosphorylation of a specific Ser/Thr or Tyr within the recognition sequence, thus providing inducible association. These domains fold independently and their N and C termini protrude from one side of the domain with the interaction surface on the opposite side, allowing them to be assembled almost like beads on a string (42Kuriyan J Cowburn D Modular peptide recognition domains in eukaryotic signaling.Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 259-288Crossref PubMed Scopus (455) Google Scholar). Another important step forward was the discovery that domains involved in membrane signaling can recognize molecules other than proteins. For instance, pleckstrin homology domains (PHD), found in many signaling proteins, recognize specific phospholipids and thus allow inducible membrane association dependent on the formation of lipid second messengers (17Ferguson K.M Lemmon M.A Sigler P.B Schlessinger J Scratching the surface with the PH domain.Nat. Struct. Biol. 1995; 2: 715-718Crossref PubMed Scopus (59) Google Scholar). For example, the PHDs of the PDK1 and Akt/PKB protein-serine kinases bind PIP3 and this promotes their membrane recruitment in response to PI-3′ kinase activation and leads to activation of Akt/PKB by PDK1. Ca2+ elevation can regulate membrane association of proteins via Ca2+-dependent interaction of C2/CalB domains with phospholipids. Recently, phosphoserine (P.Ser)-binding domains have also been identified, and these too play roles in signal propagation, by facilitating phosphorylation-dependent protein–protein interactions (91Yaffe M.B Cantley L.C Signal transduction. Grabbing phosphoproteins.Nature. 1999; 402: 30-31Crossref PubMed Scopus (70) Google Scholar). The first example was the phosphorylation-dependent binding of the CBP coactivator protein to the CREB transcription factor when phosphorylated at Ser133. Subsequently, other families of P.Ser/P.Thr-binding domains have been found. Following the identification of the transmembrane receptors, and their proximal targets, intracellular signaling pathways were the next to come on stage. The emergence of a large family of small monomeric G proteins, heralded by Ras, was an important step in understanding transmembrane signaling. Ras is anchored via C-terminal lipid modifications to the inner face of the plasma membrane, and, like heterotrimeric G proteins, is activated by GTP-GDP exchange catalyzed by GTP exchange factors. Ras.GTP levels are increased upon activation of many types of receptor. A key to understanding how Ras is activated was the finding that the Cdc25-related Sos protein has Ras.GTP exchange factor activity, and that the Grb2 SH2/SH3 adaptor protein is constitutively associated with Sos via its SH3 domains (72Schlessinger J How receptor tyrosine kinases activate Ras.Trends Biochem. Sci. 1993; 18: 273-275Abstract Full Text PDF PubMed Scopus (335) Google Scholar). The Grb2/Sos complex is recruited to an activated receptor PTK upon binding of the Grb2 SH2 domain to an autophosphorylated tyrosine in the appropriate sequence context, thus bringing the Sos catalytic domain into proximity with Ras at the plasma membrane and stimulating GTP exchange. Once activated, Ras.GTP interacts with a series of effector proteins, including the Raf protein-serine kinase and PI-3′ kinase, which initiate downstream signaling (88Wittinghofer A Nassar N How Ras-related proteins talk to their effectors.Trends Biochem. Sci. 1996; 21: 488-491Abstract Full Text PDF PubMed Scopus (137) Google Scholar). Analysis of Ras function also led to the discovery of the first GAP, which stimulates GTP hydrolysis and thus acts to terminate Ras signaling (52McCormick F Ras GTPase activating protein signal transmitter and signal terminator.Cell. 1989; 56: 5-8Abstract Full Text PDF PubMed Scopus (406) Google Scholar). Other small G proteins also function as signaling switches that are activated by receptor-induced GTP exchange. For instance, the Rho family small G proteins, Rho, Cdc42, and Rac, are all activated via receptor signaling pathways, and, like Ras, induce divers signaling pathways, which lead to gene expression and, perhaps more importantly, cause dramatic rearrangements in the actin cytoskeleton. Ras was the first signaling protein whose function was shown to be conserved from yeast to vertebrates, and this presaged the identification of many highly conserved signaling pathways throughout the eukaryotic kingdom. A prime example is the receptor PTK-Ras-MAP kinase pathway, where genetic and biochemical analysis has revealed that this pathway exists in essentially identical form in species ranging from nematodes to vertebrates, with most of the components being functionally interchangeable between organisms. Upon binding ligand, many receptors invoke gene expression responses. Indeed, the discovery that growth factors induce de novo expression of a specific set of genes independent of new protein synthesis led to the search for transcytoplasmic signaling pathways that regulate transcription (37Karin M Hunter T Transcriptional control by protein phosphorylation signal transmission from the cell surface to the nucleus.Curr. Biol. 1995; 5: 747-757Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar). In the past 10 years, there has been spectacular progress in understanding how plasma membrane signals are transmitted to the nucleus. The first and simplest transcytoplasmic nuclear signaling mechanism to be identified was the PKA/CREB system (57Montminy M.R Gonzalez G.A Yamamoto K.K Regulation of cAMP-inducible genes by CREB.Trends Neurosci. 1990; 13: 184-188Abstract Full Text PDF PubMed Scopus (373) Google Scholar). In this pathway, cAMP, elevated in response to receptor stimulation, activates the R2C2 PKA holoenzyme localized in the cytoplasm by binding to the regulatory (R) subunits, thus releasing the catalytic (C) subunit which translocates to the nucleus. An important step in understanding the cAMP pathway was the identification of the CREB transcription factor, which binds to cAMP response elements in inducible genes, and the demonstration that it is a nuclear target for PKA. Once the C subunit enters the nucleus it phosphorylates CREB at Ser133, triggering binding of the CBP/p300 coactivator and transcription of cAMP-responsive genes. This finding reinforced the notion that transcytoplasmic signaling pathways could use transcription factor phosphorylation as a regulatory mechanism. Many transcription factors are now known to be directly regulated by phosphorylation, through positive or negative control of nuclear import or export, DNA binding, or transactivation activity (37Karin M Hunter T Transcriptional control by protein phosphorylation signal transmission from the cell surface to the nucleus.Curr. Biol. 1995; 5: 747-757Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar). Two separate lines of inquiry, namely analysis of protein-serine kinases activated by receptor PTKs and the study of transcription factor phosphorylation, converged to establish that the MAP kinase pathway is a major mechanism for controlling transcription in eukaryotes (74Seger R Krebs E.G The MAPK signaling cascade.FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3099) Google Scholar). MAP kinase was originally discovered as an insulin-activated protein-serine kinase, and biochemical studies, reinforced by genetic analysis of the pheromone response in budding yeast, showed that this pathway consists of a cascade of three protein kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAP kinase (MAPK) (86Waskiewicz A.J Cooper J.A Mitogen and stress response pathways MAP kinase cascades and phosphatase regulation in mammals and yeast.Curr. Opin. Cell. Biol. 1995; 7: 798-805Crossref PubMed Scopus (525) Google Scholar). These protein kinases are activated in series, such that the MAPKKK phosphorylates the MAPKK at serines in its activation loop, which is thereby activated and phosphorylates the MAPK at a threonine and tyrosine in its activation loop, leading to its activation. Every eukaryotic organism has multiple MAPK pathways, which are largely separate from one another. The elucidation of the modular MAPK cascade with its three consecutive protein kinases had immediate implications for understanding signal amplification and switching, and most importantly for nuclear signaling, because the terminal MAPK, once activated, can migrate into the nucleus, and there phosphorylate and activate transcription factors. Receptor-induced nuclear translocation of a latent cytoplasmic transcription factor into the nucleus emerged as a different transcytoplasmic signaling principle. The NF-κB activation pathway provided the first example of a cytoplasmically sequestered transcription factor (37Karin M Hunter T Transcriptional control by protein phosphorylation signal transmission from the cell surface to the nucleus.Curr. Biol. 1995; 5: 747-757Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar). In this case, NF-κB is held in the cytoplasm through binding to an inhibitor protein, IκB, which masks the nuclear localization signal in the NF-κB heterodimer. Activating stimuli induce phosphorylation of IκB, at specific sites, which targets it for ubiquitin-mediated degradation, thus releasing active NF-κB to migrate into the nucleus. The simplest pathway of this sort is the JAK/STAT system, in which ligand binding to a cytokine receptor activates associated JAK family PTKs, which first phosphorylate receptor subunits and then specific STAT transcription factors, whereupon the STATs dimerize, migrate to the nucleus and activate transcription (37Karin M Hunter T Transcriptional control by protein phosphorylation signal transmission from the cell surface to the nucleus.Curr. Biol. 1995; 5: 747-757Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar). Other examples are activation of the Wnt/Frizzled pathway, which results in translocation of β-catenin/LEF1 transcription factor complexes into the nucleus, and activation of TGFβ family receptors, which leads to phosphorylation of receptor-specific Smad transcription factors, which then assemble with the common Smad4 subunit, translocate into the nucleus, and induce transcription of target genes. Signaling via Notch family receptors utilizes the novel principle of ligand-induced proteolytic cleavage releasing the Notch cytoplasmic domain, which acts as a protein second messenger that migrates to the nucleus and regulates gene expression by binding into and converting the suppressor-of-hairless transcriptional repressor into a transcriptional activator (2Artavanis-Tsakonas S Rand M.D Lake R.J Notch signaling cell fate control and signal integration in development.Science. 1999; 284: 770-776Crossref PubMed Scopus (4670) Google Scholar). The finding that lipid-soluble ligands, such as retinoic acid, estrogen, and other hormones, can traverse the plasma membrane without utilizing surface receptors and induce cellular responses by binding to and activating members of a family of zinc finger transcription factors revealed yet another nuclear signaling mechanism (50Mangelsdorf D.J Thummel C Beato M Herrlich P Schutz G Umesono K Blumberg B Kastner P Mark M Chambon P Evans R.M The nuclear receptor superfamily the second decade.Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (5841) Google Scholar). These so-called nuclear receptors prove to be a huge family that transduce transcriptional responses to an astonishing variety of chemicals. In principle, the nuclear receptors represent the simplest possible mechanism for nuclear signaling, although analysis of how ligand binding activates receptor-mediated transcription of target genes reveals a surprisingly complex machinery in which the unliganded receptor heterodimer acts as repressor as a result of its association with histone deacetylases, and ligand binding converts the receptor into an activator by triggering dissociation of histone deacetylases and recruitment of histone acetylases. The discovery in the 1960s of the phosphatidylinositol (PI) cycle, which results in stimulus-induced turnover of PIP2, was the harbinger of the finding of a series of phospholipid-derived second messengers, including diacylglycerol (DAG), IP3, and PI3,4,5P3, and more recently IP4 and IP6 (45Liscovitch M Cantley L.C Lipid second messengers.Cell. 1994; 77: 329-334Abstract Full Text PDF PubMed Scopus (304) Google Scholar). The identification of protein kinase C as a DAG-regulated enzyme provided another example of a second messenger-regulated protein kinase. Phospholipase action on PIP2 not only generates DAG but also IP3, which acts as a second messenger to release Ca2+ from intracellular stores, thus triggering a program of Ca2+-activated events. The unexpected discovery of PI-3′ kinases and the family of 3′ phosphoinositides they generate led to the elucidation of a new phospholipid-based signaling system in which proteins are recruited via PIP3-binding PH domains to the membrane where they are activated (e.g., the Akt/PKB protein-serine kinase) (36Kapeller R Cantley L.C Phosphatidylinositol 3-kinase.Bioessays. 1994; 16: 565-576Crossref PubMed Scopus (547) Google Scholar). The regulated entry and exit of ions across the plasma membrane, through ligand-gated, voltage-sensitive and stretch-activated ion channels and via ion pumps, has important signaling functions, particularly in impulse propagation in the nervous system and in muscle contractility. Receptor-induced changes in cytoplasmic and nuclear Ca2+ levels as a result of release of membrane-bound Ca2+ stores through IP3 or entry of extracellular Ca2+ through plasma membrane ion channels constitute an important signaling mechanism (4Berridge M.J Irvine R.F Inositol phosphates and cell signaling.Nature. 1989; 341: 197-205Crossref PubMed Scopus (3240) Google Scholar). The discovery of calmodulin as a major Ca2+-sensing protein in the cells, and identification of protein targets for Ca2+/calmodulin complexes, including a family of Ca2+/calmodulin activated protein kinases, has provided us with an explanation for how Ca2+ release is translated into molecular consequences. Moreover, the development of cell-permeant Ca2+ sensor fluorophores has afforded us with the most detailed spatiotemporal picture of signaling events occurring in the cell. The intricate and dynamic real-time patterns of local and global Ca2+ release and resorption and the propagation of Ca2+ waves and oscillations are surely the harbinger of how other signaling systems work at the subcellular level. With cAMP, cGMP, and IP3 the concept of the second messenger was well established. However, the discovery that NO is the endothelial cell–derived relaxing factor for vascular smooth muscle and that NO activates soluble cytoplasmic guanylyl cyclase to elevate cGMP revealed, somewhat unexpectedly, that a gas could act as a second messenger (58Murad F Regulation of cytosolic guanylyl cyclase by nitric oxide the NO-cyclic GMP signal transduction system.Adv. Pharmacol. 1994; 26: 19-33Crossref PubMed Scopus (212) Google Scholar). This finding rekindled interest in cGMP-mediated signaling, which is mainly effected by the cGMP-dependent protein kinase, although there are other targets for cGMP. Although much of the emphasis in signal transduction has been on eukaryotes, prokaryotes should not be neglected in this litany of progress. For instance, an important new signaling principle was established with the discovery of the two component histidyl-aspartyl phosphorelay systems, comprised of a sensor “histidine” protein kinase that phosphorylates a response regulator on an aspartate residue (80Stock J.B Stock A.M Mottonen J.M Signal transduction in bacteria.Nature. 1990; 344: 395-400Crossref PubMed Scopus (469) Google Scholar). These systems allow prokaryotes to respond to a wide variety of extracellular stimuli. However, somewhat surprisingly, two component systems are rare in eukaryotes, and for the most part seem to have been superseded by conventional protein kinases. Likewise, although animals and fungi have received the lion's share of attention, plants have also taught us new principles in signal transduction. For instance, the first receptor protein-serine kinases were found in plants, and one type of leucine-rich receptor kinase may be directly regulated by plant steroid family ligands. Moreover, ethylene, an important plant hormone, was the first gas shown to induce cellular responses, and significant progress has recently been made in understanding how ethylene signals. What does the future hold? There are many areas in signaling where it is safe to predict that rapid progress will be made, but because of its preeminence as a mechanism of signal transduction, most attention will be paid to protein phosphorylation and its role in intracellular signaling. A central theme in receptor-initiated signaling is the use of modular protein–protein interaction domains, either for inducible or constitutive interactions (62Pawson T Protein modules and signaling networks.Nature. 1995; 373: 573-580Crossref PubMed Scopus (2182) Google Scholar). Well over a hundred putative protein domains have been defined by comparative sequence analysis (http://smart.EMBL-Heidelberg.de). Although the function of many of these domains is known, many remain uncharacterized and a majority of them could prove to be new protein–protein interaction domains utilized in signaling pathways. Partners for these domains can be identified by two-hybrid screens and affinity methods combined with the use of degenerate peptide libraries to establish a binding consensus sequence. Several phospholipid interaction domains, including PH and FYVE domains, are already known that provide inducible membrane association in response to generation of lipid second messengers (e.g., PIP3) or stabilize membrane association of signaling proteins via binding to constitutive membrane phospholipids. Given the importance of membrane recruitment in signaling, additional lipid recognition domains seem certain to be discovered. Identification of nonprotein targets for such domains should be aided by affinity chromatography combined with mass spectrometric analysis. The finding that short linear motifs were sufficient for recognition by protein interaction domains came as something of a surprise, given the complex nature of the subunit interaction surfaces found in multisubunit proteins. However, the fact that recognition only requires such short sequences combined with the functional independence of these domains presumably facilitated the rapid genesis of new protein–protein interactions during evolution. Indeed, how easily these domains could be duplicated and used to evolve new signaling proteins is illustrated by the experimental portability of these domains. For example, replacement of the C terminus of the Sos Ras activator with the Grb2 SH2 domain yields a fusion protein that largely rescues the defects in Grb2−/− ES cell differentiation (8Cheng A.M Saxton T.M Sakai R Kulkarni S Mbamalu G Vogel W Tortorice C.G Cardiff R.D Cross J.C Muller W.J Pawson T Mammalian Grb2 regulates multiple steps in embryonic development and malignant transformation.Cell. 1998; 95: 793-803Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). The ability to make chimeric signaling proteins of this sort to test hypotheses about specific signaling connections will be increasingly useful as an analytical tool. In just 10 years it has become apparent how widely used sequence-dependent P.Tyr recognition is for tyrosine phosphorylation-mediated signaling. To add to the well-established SH2 and PTB domains, novel P.Tyr-binding domains have recently been identified in c-Cbl, IRS-2, and Gab1. Interestingly, structural analysis indicates that the P.Tyr-binding domain in c-Cbl is a variant SH2 domain (53Meng W Sawasdikosol S Burakoff S.J Eck M.J Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase.Nature. 1999; 398: 84-90Crossref PubMed Scopus (243) Google Scholar), whereas the Gab1 P.Tyr-binding domain appears to have a novel