Specificity In Signal Transduction
What are the underlying biochemical mechanisms through which specificity is generated during signal transduction, and the means by which signaling molecules may act in combination to generate complex biological responses? Although a rather large fraction of genes within nucleated cells appear to function in the processes of signal transduction and cellular organization (Plowman et al. 1999), it is still remarkable that only a few thousand gene products can control the sophisticated behaviors of many different cell types. This suggests that signaling proteins must act in a combinatorial fashion, since there are insufficient proteins for each to have a single biological role. As an example, there are billions of neurons in the human brain, each of which must project its axon to the appropriate target, let alone undertake the complex biochemical events associated with neurotransmission and synaptic plasticity. Clearly, the signaling molecules that function in the process of axon guidance must act in a combinatorial way to generate the extreme complexity of the human nervous system.

Protein domains and motif interactions display suprisingly flexibility. For instance single amino acid substitutions can alter the binding specificity of SH2 domains (Click on the image to see how this occurs). This apparent flexibility may have an evolutionary advantage, in the sense that SH2 domain binding specificity might change rather rapidly, allowing the formation of new signaling connections as metazoan organisms became more complex.

SH2 domains serve as a proto-type for a large and growing family of flexible, modular protein domains found in intracellular signaling proteins (see SMART).

Image Not Available The surfaces of the PLC-gamma1, Src and Grb2 SH2 domains are shown in blue, with their corresponding peptide ligands (pYIIPLPD, pYEEI, pYVNV respectively) shown in yellow. In each case the pTyr is to the right. For PLC-gamma1, the +1 Ile of the ligand fits into the start of a hydrophobic groove, framed by a Cys (beta-D5) shown in green. In the Src SH2 domain this Cys is replaced by a Tyr (in green) which makes a flat surface that selects for charged residues in the +1 and +2 positions. Src has a pocket that accommodates the hydrophobic side chain of the +3 Ile, which is formed in part by a Thr (EF1) shown in red. In the Grb2 SH2 domain this Thr is replaced by a Trp (in red) which fills up the pocket and forces the phosphpeptide into a beta-turn. Changing the Cys in PLC-gamma1 to Tyr converts the PLC-gamms1 SH2 domain to a Src-like specificity, and conversely changing the Thr in Src to Trp results in a Grb2-like specificity.

Generality of Protein-Protein Interactions in Signaling:

Thus, rather like a RTK that engages a modular protein (Grb2), which in turn recruits a signaling enzyme (Sos1) to activate the Ras pathway, so Fas binds a modular adaptor, FADD, that couples to the enzyme caspase 8 and lights the apoptotic fuse. Thus, receptors involved in signaling pathways that do not use phosphorylation as a primary mechanism for information transfer, nonetheless make use of modular protein-protein interactions to specifically activate their targets.

Image Not Available Modular protein-protein interaction domains convey signals from activated receptors using a variety of recognition motifs. Both the TNF and FGF receptors employ modular protein-protein interaction domains to convey signals. The activated TNF-R1 trimer binds a docking protein, TRADD, through death domain (DD)-DD interactions. TRAD, in turn, binds to a variety of adaptors, including FADD, TRAFs and RIP. FADD recruits pro-caspase 8 which initiates a proteolytic cascade resulting in apoptosis. Recruitment of TRAF-2 via the TRAF-C domain initiates the Jnk pathway, while recruitment of RIP activates NFkB signaling. In an analogous manner, the FGF-R is clustered by FGF and proteoglycan. The myristoylated FRS2 scaffold protein binds to the FGF-R through its PTB domain, becomes phosphorylated on multiple tyrosines, and consequently binds the SH2 proteins Grb2 and Shp2. The Grb2 adaptor recruits Sos1 through its SH3 domain, and Sos1 acts as a GEF for the Ras GTPase. Activated Ras can potentially stimulate multiple pathways to promote cell survival, activate transcription or cause cytoskeletal rearrangement.

Although there are many differences in the details between RTK and TGFbeta receptor signaling, there are also a number of parallels. The receptor targets are modular, and they form complexes with their receptors. In both cases, phosphorylation regulates protein-protein interactions, although in distinct ways. In addition, both RTKs and TGFbeta-receptors can employ docking proteins with phospholipid- and protein-interaction domains, that aid in the recruitment of targets to the receptor. Indeed, the regulation of Smad signaling is somewhat reminiscent of Stats, SH2-containing proteins that function downstream of cytokine receptors to control gene expression (Darnell, 1997) (Click the figure to see a comparison of these pathways).

Image Not Available Stats and Smads: Linking receptors to transcription. IFNg receptor activation induces protein-tyrosine kinases of the Jak family. These act on the cytoplasmic tail of the receptor, creating SH2 docking sites. STAT proteins are recruited to the membrane via their SH2 domain, and are themselves phosphorylated by the Jak kinases. Phosphorylated STATs dimerize and translocate to the nucleus where they activate transcription by binding directly to specific DNA sequences. In a somewhat anlogous manner, R-SMADs are recruited to activated TGFb receptors from their membrane anchor protein SARA. Following phosphoylation, the R-SMAD protein forms heterodimers with SMAD4 and translocates to the nucleus

Serine/Threonine Phosphorylation:

Phosphorylation of serine and threonine residues also play critical roles in regulating a variety of signalling events. For instance, pSer/Thr-dependent recognition of proteins by members of the F-box family appears to be a common and critical mechanism for the selective destruction of signaling and cell cycle proteins by ubiquitin-mediated proteolysis (Craig and Tyers, 1999; Tyers and Willems, 1999). Cell cycle checkpoint controls initiated by DNA damage or other mechanisms also relies heavily upon pSer/pThr switches. (See below for more details).

Image Not Available A simplified scheme indicating the role of serine/threonine phosphorylation events in regulating DNA-damage- induced cell cycle checkpoint control: DNA damage results in phosphorylation mediated stabilization of p53 by a variety of kinases (ATM, ATR, DNA-PK, Jnk, and CKI). This blocks the interaction of p53 with Mdm2 that normally results in efficient ubiquitin-targeted degradation of p53 by the proteasome. Activated p53 induces the transcription of genes such as p21cip/waf, a cyclin dependent kinase inhibitor that blocks the action of CyclinE/Cdk2 and thereby prevents the phosphorylation of targets such as Rb, an event that is required for G1 to S transition. DNA damage stabilization of p53 thereby blocks cell cycle progression at the G1 to S checkpoint. Cyclin E1 is itself degraded at the G1 to S boundary following phosphorylation on Thr-380. Phosphorylation at this site is sufficient to target Cyclin E1 to the SCF E3 ubiquitin ligase complex composed of cdc53/CUL-1, Skp1, Rbx-1, cdc34 (E2), and a putative F-box protein. DNA damage also results in the activation of Chk1 kinase that phosphorylates the Cdc25C phosphatase on Ser-216. 14-3-3 protein binds to phosphorylated Cdc25C and sequesters this complex in the cytoplasm. Active Cdc25C is required to activate CyclinB/Cdk2 to allow G2 to M transition. Thus, DNA damage also blocks cell cycle progression at the G2 to M checkpoint.

Scaffold Molecules:

Many of the signaling pathways that control serine/threonine phosphorylation are composed of a succession of protein kinases (MAPK cascades for example), raising the issue as to how specificity is preserved under such circumstances. Clearly, protein-serine/threonine kinases preferentially phosphorylate specific motifs in their substrates, but the experience with tyrosine kinases suggests that they might have more extensive interactions with their targets. Protein kinases are often anchored to a scaffolding protein that may either facilitate the flow of information from one kinase to another, or hold the kinase in a latent state close to the receptor that will induce its activation (Whitmarsh and Davis, 1998; Pawson and Scott, 1997). Similarly, MAPKs, and possibly many other serine/threonine kinases, select their substrates first through a non-catalytic docking interaction, which determines the substrate to be phosphorylated. While the ability of scaffolding proteins to organize the protein kinases and phosphatases that regulate serine/threonine phosphorylation is typified by the A kinase anchoring proteins (AKAPs) (Colledge and Scott, 1999). (See thumbnail below for examples of scaffold interactions that specify signalling cascades).

Image Not Available Scaffold mediated assembly of signaling pathways. A; Jip1 acts as a scaffold for the mammalian Jnk MAPK cascade. Jip1 has separate binding sites for Jnk, and the upstream kinases MKK7 (MAPKK), MLK3 (MAPK), and HPK1. Jip1 also has SH3 and PTB domains that may tether the complex to additional proteins involved in upstream activation or localization. B; The Erk MAPK docks to target proteins. Once tethered in this manner, Erk phosphorylates the substrate, Rsk1. C; The regulatory cyclin A subunit of Cdk2 binds substrates with a conserved RXL motif, such as p107. D; The AKAP protein Yotiao binds to the NMDA receptor, PKA in the inactive form, and PP1 in the active form. In doing so, Yotiao creates close physical association of the components that repress resting NMDA receptor and enhance channel activation.

Signaling to the Cytoskeleton:

It is common when considering signaling pathways to dwell on events that culminate in the nucleus. However, signaling pathways that control the cytoskeleton and adhesion of cells to the extracellular matrix are essential for guided cell migration, including processes such as axon guidance and topographic map formation in the brain. Among the guidance receptors are members of the Eph family of RTKs that interact with ligands, termed ephrins, which are themselves anchored to the cell surface, either through a GPI linkage (A type ephrins) or a transmembrane sequence joined to a conserved cytoplasmic tail (B type ephrins).The binding of ephrins to neuronal cells expressing Eph receptors induces remodeling of the actin cytoskeleton and growth cone collapse, in a fashion that is dependent on receptor kinase activity and the juxtamembrane pTyr sites (Drescher et al. 1995; Binns et al. 2000). These data suggest that activated Eph receptors can communicate with signaling proteins that regulate the cytoskeleton. There are a number of candidates that might fulfil this role, including the Nck adaptor. Nck binds the protein-serine/threonine kinase Pak that in turn potentiates the signal through Rac and PIX.

Image Not Available A signaling pathway to the cytoskeleton. Axon guidance receptors potentially bind the Nck adaptor, through both SH2-and SH3-mediated interactions. The second SH3 domain of Nck recruits the Pak protein-serine/threonine kinase to the membrane. Pak kinase activity is induced by binding of GTP-Cdc42 to a CRIB motif. Pak can also generate activated Cdc42 through the associated PIX GEF. (Pro = proline-rich motif).

Two signaling proteins appear central to controlling the cytoskeleton, and may serve as convergence points downstream from a variety of guidance receptors (Figure 7). The Drosophila Enabled (Ena) protein, and its mammalian homolog Mena, are stongly implicated in regulating actin dynamics (Lanier and Gertler, 2000). Another multi-domain protein that acts in neurons to control axon guidance is UNC-73 (Steven et al. 1998), the C. elegans counterpart of mammalian Trio (Debant et al. 1996). Unc-73 appears to couple upstream signals to the regulation of Rho family GTPases, and thus to reorganization of the cytoskeleton.

Image Not Available Modular proteins that may integrate cytoskeletal signaling. Mena interacts with receptors, focal adhesion components and bacterial pathogens through its EVH1 domain, and has multiple motifs that potentially couple to the actin cytoskeleton. UNC-73/Trio has an N-terminal domain related to yeast Sec 14, multiple spectrin repeats, and two DH-PH domains that activate Rac and Rho respectively. The signaling pathways that lie upstream of UNC-73/Trio are not well established.

Signaling to the Cytoskeleton:

In the body, a cell is simultaneously exposed to multiple, potentially contradictory signals, in the form of soluble hormones and ligands anchored to adjacent cells or the ECM. The cell must have mechanisms for converting these various signals into a defined response. Furthermore, the cell must monitor its internal state, so that it does not attempt to divide before achieving the appropriate mass, for example. There are some clues as to how these types of regulation may be achieved. In one mode of integration, activation of a key signaling protein requires input from two or more distinct biochemical pathways. The Rsk1 protein-serine/threonine kinases exhibits such behaviour. Rsk1 has two catalytic domains, of which the N-terminal domain phosphorylates downstream targets. The N-terminal kinase domain is controlled by multiple inputs, including the C-terminal domain. The Erk MAPK binds a basic docking site at the extreme C-terminus of Rsk1, and phosphorylates sites in the linker region between the two kinase domains and in the C-terminal domain, which are essential for activation. Full activation, however, also requires phosphorylation of the N-terminal kinase domain by the PIP3-responsive protein kinase PDK1 (Richards et al. 1999; Nebreda and Gavin, 1999). Rsk1 activation, therefore, requires inputs from both the Erk MAPK pathway and the PI3K pathway (Click on the thumbnail sketch for a full view).

Clearly much of the integration of signaling pathways occurs at the level of transcriptional promoters. Thus the transcription factors regulated by the TGF-b and Wnt signaling pathways (Smads and beta-catenin complexed with Lef1/Tcf, respectively) can physically interact with one another and synergistically activate gene expression during embryonic development (Nishita et al. 2000).

An alternative scenario is that two different pathways have opposing effects, and the cell may therefore want to prevent their simultaneous activation. A process of this sort is seen for signaling with the interferon-gamma receptor which, through activation of the Stat3 transcription factor, induces expression of Smad7 (Ulloa et al. 1999)

Image Not Available Integration of signaling pathways. A; Activation of Rsk1 requires phosphorylation by both PDK1 and ERK MAPK. Thus the PI3K and Ras pathways synergize to stimulate the Rsk1 protein-serine/threonine kinase. B; Signaling through the interferon (IFN) gamma receptor/Stat3 pathway induces the expression of the inhibitory Smad7, which blocks the activity of the TGF-beta receptor. Thus one cytokine pathway inhibits the activation of a distinct pathway. C; Both the Wnt receptor (DFz2) and the related Frizzled signal through the multi-domain protein Dishevelled. Wnt signaling involves the DIX and PDZ domains of Dishevelled, which activate the beta-Catenin pathway. Frizzled signals through the C-terminal DEP domain to activate the JNK MAPK pathway.


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Additional Reading (Reviews):

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Pawson, T. (1995) Protein modules and signalling networks. Nature 373, 573-580.

Pawson, T. and Scott, J.D. (1997) Signaling through scaffold, anchoring, and adaptor proteins. Science 278, 2075-2080.

Starr, R. and Hilton, D.J. (1999) Negative regulation of the JAK/STAT pathway. BioEssays 21, 47-52.

Tessier-Lavigne, M. and Goodman, C.S. (1996) The molecular biology of axon guidance. Science 274, 1123-1133.

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Wrana, J. (2000) Regulation of Smad activity. Cell 100, 189-192.

Adapted from Tony Pawson and Piers Nash (2000)
Protein-protein interactions define specificity in signal transduction.
Genes & Development 14 (9): 1027-1047.