Signaling Through Scaffold, Anchoring, and adaptor proteins

Signaling Through Scaffold, Anchoring, and adaptor proteins

(Parte 1 de 3)

DOI: 10.1126/science.278.5346.2075 , 2075 (1997); 278Science et al.Tony Pawson, Adaptor Proteins Signaling Through Scaffold, Anchoring, and

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Signaling Through Scaffold, Anchoring, and

Adaptor Proteins Tony Pawson and John D. Scott

The process by which extracellular signals are relayed from the plasma membrane to specific intracellular sites is an essential facet of cellular regulation. Many signaling pathways do so by altering the phosphorylation state of tyrosine, serine, or threonine residues of target proteins. Recently, it has become apparent that regulatory mechanismsexisttoinfluencewhereandwhenproteinkinasesandphosphatasesareactivated in the cell. The role of scaffold, anchoring, and adaptor proteins that contribute to the specificity of signal transduction events by recruiting active enzymes into signaling networks or by placing enzymes close to their substrates is discussed.

The speed and precision of signal transduction are often taken for granted. Yet, understanding the mechanisms that allow intracellular signals to be relayed from the cell membrane to specific intracellular targets still remains a daunting challenge. Many protein kinases and protein phosphatases have relatively broad substrate specificities and may be used in varying combinations to achieve distinct biological responses. Thus, mechanisms must exist to organize the correct repertoires of enzymes into individual signaling pathways. One such mechanism involves restriction of certain polypeptides to localized sites of action. This function can be achieved either by recruitment of active signaling molecules into multiprotein signaling networks (Fig. 1A) or activation of dormant enzymes already positioned close to their substrates (Fig. 1B). Simply stated, either the enzymes go to the signal or the signal goes to the enzymes.

The assembly of signaling proteins into biochemical pathways or networks is typified by the association of autophosphorylated receptor tyrosine kinases with cytoplasmic proteins that contain specializedprotein modules that mediate formation of signaling complexes (Fig. 2) (1). For example, src homology 2 (SH2) domains bind specific phosphotyrosyl residues on activated receptors (Fig. 2A), and src homology 3 (SH3) domains bind to polyproline motifs on a separate set of target proteins (Fig. 2D) (2). This permits simultaneous association of a single protein containing both SH2 and SH3 domains with two or more binding partners, and hence, the assembly of complexes of signaling proteins around an activated cell-surface receptor (Fig. 1A). Similarly, subcellular organization of serinethreonine kinases and phosphatases occurs through interactions with the targeting subunits or anchoring proteins that localize these enzymes (3). In addition, kinase binding proteins such as 14-3-3 proteins serve as adaptor proteins for signaling networks, whereas proteins such as Sterile 5 (Ste 5) and AKAP79 maintain signalingscaffolds of several kinases or phosphatases (Fig. 1B) (4). Hence, the cell uses related mechanisms for controlling the subcellular organization of tyrosine and Ser-Thr phosphorylation events. In this review, we compare and contrast the protein modules, adaptor molecules, targeting subunits, and anchoring proteins that coordinate signaling networks.

Mechanisms for Recognition of Phosphotyrosine and Peptide Motifs

SH2 domains are protein modules that recognize short, phosphopeptide motifs composed of phosphotyrosine (pTyr) followed by three to five COOH-terminal residues, such as those generated by autophosphorylation of activated receptor tyrosine kinases (Fig. 2A) (5). According to this scheme, the sequences of the SH2 docking sites on a given receptor tyrosine kinase dictate which SH2-containing targets associate with the receptor and will therefore help determine which signaling pathways the re- ceptor can activate. These modules are coupled directly or indirectly to downstream signaling molecules, including enzymes that control phospholipid metabolism, Ras-like guanosine triphosphatases (GTPases), protein kinases, transcription factors, and polypeptides that regulate cytoskeletal architecture and cell adhesion.

A genetic test of this concept has been provided in mice by use of the Met receptor tyrosine kinase, which has two closely spaced autophosphorylation sites within its COOH-terminal tail that bind a number of signaling proteins with SH2 domains. Conversion of these Tyr residues to Phe results in the same phenotype as a null mutation, despite the fact that the activity of the kinase domain is unaltered. In contrast, a single substitution of an Asn two residues COOH-terminal to one of the phosphotyrosine sites (the 12 position) selectively uncouples the receptor from its ability to bind the Grb2 SH2/SH3 adaptor protein and yields a hypomorphic phenotype affecting myoblast proliferation and muscle formation (6). Thus, the creation of docking sites through autophosphorylation is absolutely required for Met function in vivo, and specific binding to a particular SH2- containing protein is required for one of the receptor’s biological activities.

The pTyr-binding (PTB) domains of the

Shc and insulin receptor substrate-1 (IRS- 1) proteins (7) recognize phosphopeptide motifs in which pTyr is preceded by residues that form a b turn [usually with the consensus NPXpY (8)] (9). Specificity is conferred by hydrophobic amino acids that lie five to eight residues NH2-terminal to the pTyr (Fig. 2B) (10) and therefore recognize their ligands in a distinct manner from SH2 domains (1). PTB domains may serve a somewhat different purpose from SH2 do-

T. Pawson is at the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada. J. D. Scott, Howard Hughes Medical Institute, Vollum Institute, Portland, OR 97201–3098, USA.

ABFig. 1. Mechanisms for recruiting or localizing signal transduction com- ponents. (A) Assembly of modular signaling molecules on an activated receptor tyrosine kinase. An extracellularsignalinputdrivesautophosphorylation of the receptor and leads to the recruitment of cytoplasmic proteins that contain various protein modules. pY, phosphotyrosine. (B) A localized signaling complex of three anchored signalingenzymes.Eachenzymeisinactivewhenboundtotheanchoringproteinbutisreleasedandactivated by different signals. Enz., enzyme.

on May 28, 2009 w.sciencemag.org Downloaded from mains, because they are found primarily as components of docking proteins that recruit additional signaling proteins to the vicinity of an activated receptor. The PTB domains of proteins such as X11, FE65, and Numb can bind nonphosphorylated peptide motifs (12), indicating that PTB domains are principally peptide recognition elements, unlike SH2 domains that appear devoted to the job of pTyr recognition.

PDZ domains have a somewhat similar mechanism of ligand-binding to PTB domains, in which the peptide binds as an additional strand to an antiparallel b sheet (13). A distinguishing feature of PDZ domains is their recognition of short peptides with a COOH-terminal hydrophobic residue and a free carboxylate group, as exemplified by the E(S/T)DV motif at the COOH-terminus of certain ion channel subunits (Fig. 2C) (14). These interactions can promote clustering of transmembrane receptors at specific subcellular sites and have an especially important role in the spatial organization of voltage- and ligandgated ion channels at synapses, because shaker-type K1 channels and all three classes of glutamate receptors are recognized by distinct PDZ domain proteins (Fig. 3A) (15, 16). Specificity is conferred by ligand residues at the –2 to –4 positions relative to the COOH-terminus and may be regulated by phosphorylation, because the –2 residue of PDZ-binding sites is often a hydroxyamino acid. In particular, a COOH-terminal motif (RRESAI) on the inwardly rectifying K1 channel Kir 2.3 binds the second PDZ domain of a 95-kD postsynaptic density protein called PSD-95. PKA phosphorylation at the –2 Ser of the PDZ recognition site uncouples the channel from PSD- 95 and results in inhibition of K1 conductance (Fig. 3A) (17).

Many proteins have multiple PDZ domains (up to seven in known proteins), which may have at least two important consequences. An individual PDZ-containing protein could bind several subunits of a particular channel, and thereby induce channel aggregation. This could be enhanced by the ability of a protein such as

PSD-95 to form oligomers through NH2- terminal intermolecular disulfide bonds

(18). Furthermore, the individual PDZ domains of a protein such as PSD-95 can have distinct binding specificities, leading to the formation of clusters that contain heterogeneous groups of proteins. Thus, the ability of the third PSD-95 PDZ domain to bind the cell-adhesion molecule neuroligin may direct the N-methyl-D-aspartate receptor NMDA2 and K1 channels, which interact with the first and second PDZ domains, to specific synaptic sites (19). Two further properties of PDZ domains or proteins that contain them may expand their potential for regulating signal transduction. First, some PDZ domains may bind internal peptide sequences and, indeed, have a propensity to undergo homotypic or heterotypic interactions with other PDZ domains (20). Second, proteins with PDZ domains frequently contain other interaction modules, including SH3 and LIM domains, and catalytic elements such as tyrosine phosphatase or nitric oxide synthase domains. PDZ interactions may therefore both coordinate the localization and clustering of receptors and channels, and provide a bridge to the cytoskeleton or intracellular signaling pathways.

The sophistication of signaling networks maintained by PDZ interactions is illustrated by InaD, a polypeptide with five PDZ domains that regulates phototransduction in Drosophila melanogaster photoreceptors (Fig. 3B) (21). InaD associates through distinct PDZ domains with a calcium channel (TRP); phospholipase C-b, the target of rhodopsin-activated heterotrimeric guanine nucleotide–binding protein (Gqa); and protein kinase C (PKC). InaD organizes these proteins into a signaling complex that allows efficient activation of the TRP channel by PLC-b in response to stimulation of rhodopsin and deactivation through phosphorylation of TRP by PKC (2). Thus, InaD appears to act as a scaffolding protein to organize light-activated signaling events (Fig. 3B).

Domains that bind proline-rich motifs. SH3 domains bind proline-rich peptide sequences with the consensus PXXP that form a lefthanded polyproline type I helix (Fig. 2D) (23). A principal role of SH3 domains is in forming functional oligomeric complexes at defined subcellular sites, frequently in conjunction with other modular domains (2). There are certainparallelsbetweenSH3 and PDZ domains. Proteins can have multiple SH3 domains, potentially allowing clustering of severaldistinctligands,and Ser or Thr phosphorylationadjacent to the proline-rich ligand may influence SH3 domain interactions (24). Serine phosphorylationof a PDZ or SH3 recognitionsite resultsin uncoupling of signaling proteins, in contrast to the autophosphorylation of receptor tyrosine kinases, which promotes the assembly of signaling complexes at the SH2 acceptor site.

W domains are very small modules of 35 to 40 residues that also bind proline-rich motifs, commonly with the consensus PPXY or PPLP(Fig.2E)(25). TheWW domainsof the E3 ubiquitin protein ligase Nedd4 bind such proline-rich motifs in an amiloridesensitiveepithelialNa1 channel,likely leading to channel degradation (Fig. 3A) (26). Channel mutations that disrupt this interaction cause a human hypertensive disorder, Liddle’s syndrome (27). W domains may also regulate catalytic function, as suggested by a structural analysis of the peptidyl-prolyl cis-trans isomerase Pin1. Pin1, which interacts with cell cycle components such as the protein kinase NIMA, possesses an NH2- terminalWW domainthat forms one part of a ligand-binding surface, which includes an a helixfromtherotamasedomain.TheWW domain may thereby contribute to substrate

Fig. 2. Protein modules for the assembly of signaling complexes. Several modular domains have been identified that recognize specific sequences on their target acceptor proteins. These sequences, in single-letter code, are indicated for (A) SH2 domains, (B) PTB domains, (C) PDZ domains, (D) SH3 domains, (E) W domains, and (F) 14-3-3 proteins. hy indicates hydrophobic residues. Drosophila eye

TRP Ca2+ channel

InaC InaD

GuK

A Amiloride-sensitiveNa channel

Kir 2.3 K+channelsAMPA receptor L-type

Ca2+ channel

Rhodopsin

Nedd4 PSD 95AKAP 79

Eye PKC

Fig. 3. Localization of signaling molecules with ion channels. (A) Modulation of certain ion channels may be coordinated by modular docking proteins or scaffold proteins that tether signaling enzymes in proximity to ion channels. Each ion channel is identified above, and the docking or anchoring proteins are indicated below. CaN, calcineurin. (B)I n Drosophila eye, InaD coordinates the location of PKC and phospholipase b in relation to the channel. Calmodulin (CaM) and InaC also associate with the channel.

on May 28, 2009 w.sciencemag.org Downloaded from recognition (28). An intriguing feature of Pin1 is the ability of its catalytic domain to specifically recognize phosphorylated (Ser/ Thr)Pro motifs, raising the possibility that Ser-Thr phosphorylation of cell cycle regulators creates a recognition site for Pin1, which in turn could modify their conformation and functional properties (29). A conserved domain, EVH1, has recently been identified in profilin-binding proteins such as VASP and Mena, and has been shown to bind proline-rich peptide sequences such as the (E/D)FPPPPX(D/E) motif found in the ActA protein of Listeria monocytogenes. The EVH1 domain may couple cytoskeletal proteins such as zyxin and vinculin, and bacterial ActA, to actin remodeling (30).

Phospholipid recognition and membrane targeting. Not all localization of signaling modules is directed by interaction with other proteins. Pleckstrin homology (PH) domains bind the charged headgroups of specific polyphosphoinositides and may thereby regulate the subcellular targeting of signaling proteins to specific regions of the plasma membrane (Fig. 4) (31). This positions such proteins for interactions with regulators or targets. In this way, PH domains can couple the actions of phosphatidyl inositol (PI) kinases, inositol phosphatases, and phospholipases to the regulation of intracellular signaling (32). This is illustrated by the finding that the product of PI

39-kinase, PI-3,4,5-P3, binds specifically to the PH domain of a protein, Grp1, which can potentially function as an exchange factor for the small GTPase Arf (3). Because the p85 regulatory subunit of PI 39- kinase possesses SH2 domains that control enzyme activation by tyrosine phosphorylation, it seems that the SH2 and PH domains of distinct proteins can act sequentially in a common pathway to link receptor tyrosine kinase signaling to the control of vesicle trafficking.

PH domains are also found covalently linked to other modules, such as SH2, SH3, and PTB domains, with which they may synergize in controlling the activation of specific signaling proteins. For example, the Btk cytoplasmic tyrosine kinase

contains an NH2-terminal PH domain that binds PI-3,4,5-P3 and is covalently linked to SH3, SH2, and kinase domains

(34). The production of PI-3,4,5-P3 may therefore induce association of Btk with the membrane, facilitating the interaction of the SH2, SH3, and catalytic domains with activators and targets. Mutations in the PH domain that inhibit phospholipid recognition, or of Cys residues in an adjacent structural element that binds Zn, lead to an inherited human B cell defect, X- linked agammaglobulinemia (35).

Docking and Scaffolding Proteins in Receptor Signaling

One way receptors may amplify their signaling is to use adaptor proteins that provide additional docking sites for modular signaling proteins. Typically, docking proteins have an NH2-terminal membrane-targeting element, either a PH domain or a myristy- lation site, and a PTB domain that directs association with an NPXY autophosphorylation site on a specific receptor (Fig. 4). Once it is associated with the appropriate activated receptor, the docking protein becomes phosphorylated at multiple sites that interact with specific SH2 domains of signaling proteins. For example, IRS-1 and IRS-2 (two principal substrates of the insu- lin receptor) have an NH2-terminal PH domain followed by a PTB domain and 18 potential tyrosine phosphorylation sites (36). Under physiological circumstances, both the IRS-1 PH and PTB domains are required for insulin-induced tyrosine phosphorylation of IRS-1 and mitogenic signaling, suggesting that membrane targeting of IRS-1 and physical binding to its receptor tyrosine kinase facilitate insulin-mediated signaling (Fig. 4) (37). Another membraneassociated docking protein, Shc, has an

NH2-terminal PTB domain that binds both pTyr sites and phosphoinositides, as well as a COOH-terminal SH2 domain, and consequently is phosphorylated by a broad range of receptor tyrosine kinases (1). In contrast, mammalian Gab-1, which has an

NH2-terminal PH domain, may play a more specific role downstream of the Met ty- rosine kinase, and p62dok is a prominent substrate of the Eph receptors that control axon guidance (38). Similarly, FRS2, which is myristylated and has a potential PTB domain, is specifically phosphorylated by receptors for fibroblast growth factor (FGF) and nerve growth factor (NGF) (39).

The signaling properties of these docking proteins likely depend on the sequences of their SH2-bindingmotifs.Commonly,a specific Tyr-based motif is reiterated several times within an individual docking protein. Thus, Shc has two YXNX motifs, which can couple to Grb2 and the Ras pathway (40); p62dok has six YXXP motifs,which may bind SH2-containing proteins that influence the cytoskeleton (38); and IRS-1 has nine YXXM motifs, which can bind and activate PI 39-kinase. This phenomenon may allow selective amplification of specific signaling pathways. The Drosophila docking protein Dos, which has the potential to bind several distinct signaling proteins with SH2 domains, including Drk (the Drosophila ortholog of Grb-2) and corkscrew (an SH2- containing tyrosine phosphatase), was originally identified in a genetic screen for com- ponents of RTK signaling pathways in Drosophila (41). This genetic analysis supports the notion that SH2-docking proteins augment and regulate tyrosine kinase signaling.

Signaling Networks for Serine-Threonine Phosphorylation

The importance of protein-protein interactions is not confined to signaling steps controlled by tyrosine phosphorylation. Although some Ser-Thr kinases and phosphatases were originally thought to be constitutively attached to intracellular loci through association with targeting subunits or anchoring proteins, it is now clear that some are also recruited into signaling networks (3). Apparently, protein modules control the location and assembly of these Ser-Thr kinase signaling networks, providing an intriguing parallel with tyrosine kinase signaling events.

Protein kinase A and protein kinase C anchoring. In its inactive form, the adenosine 39,59-monophosphate (cAMP)–dependent protein kinase (PKA) is composed of two catalytic (C) subunits held in an inactive conformation by association with two regulatory (R) subunits. Binding of cAMP to the R subunits causes dissocation of the C subunits, which act to control a wide range of biological processes. Although cAMP is the sole activator of PKA, other regulatory proteins control where and when the kinase is turned on in response to specific stimuli. The concerted actions of adenylyl cyclases and phosphodiesterases create gradients and compartmentalized pools of cAMP, whereas A-kinase anchoring proteins (AKAPs) maintain the PKA holoenzyme at precise intracellular sites. Anchoring ensures that PKA is exposed to localized changes in cAMP and is compartmentalized with sub-

Insulin receptor β subunit

Insulin receptor kinase

(Parte 1 de 3)

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