ARTICLE 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 ceptor can activate. These modules are cou- specific intracellular sites is an essential facet of cellular regulation. Many signaling pled directly or indirectly to downstream pathways do so by altering the phosphorylation state of tyrosine, serine, or threonine signaling molecules, including enzymes that residues of target proteins. Recently, it has become apparent that regulatory mecha- control phospholipid metabolism, Ras-like nisms exist to influence where and when protein kinases and are activated guanosine triphosphatases (GTPases), pro- in the cell. The role of scaffold, anchoring, and adaptor proteins that contribute to the tein kinases, transcription factors, and specificity of events by recruiting active enzymes into signaling polypeptides that regulate cytoskeletal ar- networks or by placing enzymes close to their substrates is discussed. chitecture and . A genetic test of this concept has been provided in mice by use of the Met tyrosine kinase, which has two closely The speed and precision of signal transduc- through interactions with the targeting sub- spaced autophosphorylation sites within its tion are often taken for granted. Yet, un- units or anchoring proteins that localize COOH-terminal tail that bind a number of derstanding the mechanisms that allow in- these enzymes (3). In addition, kinase bind- signaling proteins with SH2 domains. Con- tracellular signals to be relayed from the cell ing proteins such as 14-3-3 proteins serve as version of these Tyr residues to Phe results membrane to specific intracellular targets adaptor proteins for signaling networks, in the same phenotype as a null mutation, still remains a daunting challenge. Many whereas proteins such as Sterile 5 (Ste 5) despite the fact that the activity of the protein kinases and protein phosphatases and AKAP79 maintain signaling scaffolds of kinase domain is unaltered. In contrast, a have relatively broad substrate specificities several kinases or phosphatases (Fig. 1B) single substitution of an Asn two residues and may be used in varying combinations to (4). Hence, the cell uses related mechanisms COOH-terminal to one of the phosphoty-

achieve distinct biological responses. Thus, for controlling the subcellular organization rosine sites (the ϩ2 position) selectively on February 23, 2009 mechanisms must exist to organize the cor- of tyrosine and Ser-Thr phosphorylation uncouples the receptor from its ability to rect repertoires of enzymes into individual events. In this review, we compare and con- bind the Grb2 SH2/SH3 adaptor protein signaling pathways. One such mechanism trast the protein modules, adaptor mole- and yields a hypomorphic phenotype affect- involves restriction of certain polypeptides cules, targeting subunits, and anchoring pro- ing myoblast proliferation and muscle for- to localized sites of action. This function teins that coordinate signaling networks. mation (6). Thus, the creation of docking can be achieved either by recruitment of sites through autophosphorylation is abso- active signaling molecules into multipro- Mechanisms for Recognition of lutely required for Met function in vivo, tein signaling networks (Fig. 1A) or activa- Phosphotyrosine and Peptide and specific binding to a particular SH2- tion of dormant enzymes already positioned Motifs containing protein is required for one of the www.sciencemag.org close to their substrates (Fig. 1B). Simply receptor’s biological activities. stated, either the enzymes go to the signal SH2 domains are protein modules that rec- The pTyr-binding (PTB) domains of the or the signal goes to the enzymes. ognize short, phosphopeptide motifs com- Shc and insulin receptor substrate-1 (IRS- The assembly of signaling proteins into posed of phosphotyrosine (pTyr) followed 1) proteins (7) recognize phosphopeptide biochemical pathways or networks is typi- by three to five COOH-terminal residues, motifs in which pTyr is preceded by residues fied by the association of autophosphoryl- such as those generated by autophosphoryl- that form a ␤ turn [usually with the con- ated receptor tyrosine kinases with cytoplas- ation of activated receptor tyrosine kinases sensus NPXpY (8)] (9). Specificity is con- mic proteins that contain specialized protein (Fig. 2A) (5). According to this scheme, ferred by hydrophobic amino acids that lie Downloaded from modules that mediate formation of signaling the sequences of the SH2 docking sites on a five to eight residues NH2-terminal to the complexes (Fig. 2) (1). For example, src given dictate pTyr (Fig. 2B) (10) and therefore recognize homology 2 (SH2) domains bind specific which SH2-containing targets associate their ligands in a distinct manner from SH2 phosphotyrosyl residues on activated recep- with the receptor and will therefore help domains (11). PTB domains may serve a tors (Fig. 2A), and src homology 3 (SH3) determine which signaling pathways the re- somewhat different purpose from SH2 do- domains bind to polyproline motifs on a separate set of target proteins (Fig. 2D) (2). Fig. 1. Mechanisms for recruiting or This permits simultaneous association of a localizing signal transduction com- AB single protein containing both SH2 and ponents. (A) Assembly of modular SH3 domains with two or more binding signaling molecules on an activated partners, and hence, the assembly of com- receptor tyrosine kinase. An extra- plexes of signaling proteins around an acti- cellularsignalinputdrivesautophos- vated cell-surface receptor (Fig. 1A). Simi- phorylation of the receptor and larly, subcellular organization of serine- leads to the recruitment of cyto- plasmic proteins that contain vari- threonine kinases and phosphatases occurs ous protein modules. pY, phospho- tyrosine. (B) A localized signaling T. Pawson is at the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Cana- complex of three anchored signal- da. J. D. Scott, Howard Hughes Medical Institute, Vollum ing enzymes. Each enzyme is inactive when bound to the anchoring protein but is released and activated Institute, Portland, OR 97201–3098, USA. by different signals. Enz., enzyme.

www.sciencemag.org ⅐ SCIENCE ⅐ VOL. 278 ⅐ 19 DECEMBER 1997 2075 mains, because they are found primarily as consequences. An individual PDZ-contain- allows efficient activation of the TRP chan- components of docking proteins that recruit ing protein could bind several subunits of a nel by PLC-␤ in response to stimulation of additional signaling proteins to the vicinity particular channel, and thereby induce rhodopsin and deactivation through phos- of an activated receptor. The PTB domains channel aggregation. This could be en- phorylation of TRP by PKC (22). Thus, of proteins such as X11, FE65, and Numb hanced by the ability of a protein such as InaD appears to act as a scaffolding protein can bind nonphosphorylated peptide motifs PSD-95 to form oligomers through NH2- to organize light-activated signaling events (12), indicating that PTB domains are prin- terminal intermolecular disulfide bonds (Fig. 3B). cipally peptide recognition elements, unlike (18). Furthermore, the individual PDZ do- Domains that bind proline-rich motifs. SH3 SH2 domains that appear devoted to the mains of a protein such as PSD-95 can have domains bind proline-rich peptide sequences job of pTyr recognition. distinct binding specificities, leading to the with the consensus PXXP that form a left- PDZ domains have a somewhat similar formation of clusters that contain heteroge- handed polyproline type II helix (Fig. 2D) mechanism of -binding to PTB do- neous groups of proteins. Thus, the ability (23). A principal role of SH3 domains is in mains, in which the peptide binds as an of the third PSD-95 PDZ domain to bind forming functional oligomeric complexes at additional strand to an antiparallel ␤ sheet the cell-adhesion molecule neuroligin may defined subcellular sites, frequently in con- (13). A distinguishing feature of PDZ do- direct the N-methyl-D-aspartate receptor junction with other modular domains (2). mains is their recognition of short peptides NMDA2 and Kϩ channels, which interact There are certain parallels between SH3 and with a COOH-terminal hydrophobic resi- with the first and second PDZ domains, to PDZ domains. Proteins can have multiple due and a free carboxylate group, as exem- specific synaptic sites (19). Two further SH3 domains, potentially allowing cluster- plified by the E(S/T)DV motif at the properties of PDZ domains or proteins that ing of several distinct ligands, and Ser or Thr COOH-terminus of certain contain them may expand their potential phosphorylation adjacent to the proline-rich subunits (Fig. 2C) (14). These interactions for regulating signal transduction. First, ligand may influence SH3 domain interac- can promote clustering of transmembrane some PDZ domains may bind internal pep- tions (24). Serine phosphorylation of a PDZ receptors at specific subcellular sites and tide sequences and, indeed, have a propen- or SH3 recognition site results in uncoupling have an especially important role in the sity to undergo homotypic or heterotypic of signaling proteins, in contrast to the au- spatial organization of voltage- and ligand- interactions with other PDZ domains (20). tophosphorylation of receptor tyrosine ki- gated ion channels at synapses, because Second, proteins with PDZ domains fre- nases, which promotes the assembly of sig- shaker-type Kϩ channels and all three class- quently contain other interaction modules, naling complexes at the SH2 acceptor site. es of glutamate receptors are recognized by including SH3 and LIM domains, and cat- WW domains are very small modules of distinct PDZ domain proteins (Fig. 3A) alytic elements such as tyrosine phospha- 35 to 40 residues that also bind proline-rich on February 23, 2009 (15, 16). Specificity is conferred by ligand tase or nitric oxide synthase domains. PDZ motifs, commonly with the consensus PPXY residues at the –2 to –4 positions relative to interactions may therefore both coordinate or PPLP (Fig. 2E) (25). The WW domains of the COOH-terminus and may be regulated the localization and clustering of receptors the E3 ubiquitin protein ligase Nedd4 bind by phosphorylation, because the –2 residue and channels, and provide a bridge to such proline-rich motifs in an amiloride- of PDZ-binding sites is often a hydroxy- the or intracellular signaling sensitive epithelial Naϩ channel, likely lead- amino acid. In particular, a COOH-termi- pathways. ing to channel degradation (Fig. 3A) (26). nal motif (RRESAI) on the inwardly recti- The sophistication of signaling networks Channel mutations that disrupt this interac- fying Kϩ channel Kir 2.3 binds the second maintained by PDZ interactions is illustrat- tion cause a human hypertensive disorder,

PDZ domain of a 95-kD postsynaptic den- ed by InaD, a polypeptide with five PDZ Liddle’s syndrome (27). WW domains may www.sciencemag.org sity protein called PSD-95. PKA phospho- domains that regulates phototransduction also regulate catalytic function, as suggested rylation at the –2 Ser of the PDZ recogni- in Drosophila melanogaster photoreceptors by a structural analysis of the peptidyl-prolyl tion site uncouples the channel from PSD- (Fig. 3B) (21). InaD associates through dis- cis-trans isomerase Pin1. Pin1, which inter- 95 and results in inhibition of Kϩ conduc- tinct PDZ domains with a calcium channel acts with cell cycle components such as the ␤ tance (Fig. 3A) (17). (TRP); - , the target of NIMA, possesses an NH2- Many proteins have multiple PDZ do- rhodopsin-activated heterotrimeric guanine terminal WW domain that forms one part of mains (up to seven in known proteins), nucleotide–binding protein (Gq␣); and a ligand-binding surface, which includes an which may have at least two important protein kinase C (PKC). InaD organizes ␣ helix from the rotamase domain. The WW Downloaded from these proteins into a signaling complex that domain may thereby contribute to substrate

ABC Fig. 3. Localization of signaling Amiloride-sensitive + AMPA A Kir 2.3 K molecules with ion channels. (A) Na channel channels receptor L-type 2+ Modulation of certain ion chan- Ca channel nels may be coordinated by PKC modular docking proteins or DEF Nedd4 CaN PKA scaffold proteins that tether sig- AKAP 79 naling enzymes in proximity to PSD 95 ion channels. Each ion channel GuK is identified above, and the B Rhodopsin docking or anchoring proteins Fig. 2. Protein modules for the assembly of sig- TRP are indicated below. CaN, cal- Ca2+ channel naling complexes. Several modular domains have cineurin. (B)InDrosophila eye, Eye been identified that recognize specific sequences PKC InaD coordinates the location of on their target acceptor proteins. These sequenc- β PKC and phospholipase ␤ in re- PDZ PDZ es, in single-letter code, are indicated for (A) SH2 CaM PDZ lation to the channel. Calmodu- domains, (B) PTB domains, (C) PDZ domains, (D) PDZ InaD lin (CaM) and InaC also associ- InaC SH3 domains, (E) WW domains, and (F) 14-3-3 ate with the channel. PDZ Drosophila eye proteins. hy indicates hydrophobic residues.

2076 SCIENCE ⅐ VOL. 278 ⅐ 19 DECEMBER 1997 ⅐ www.sciencemag.org ARTICLE recognition (28). An intriguing feature of Docking and Scaffolding Proteins ponents of RTK signaling pathways in Dro- Pin1 is the ability of its catalytic domain to in Receptor Signaling sophila (41). This genetic analysis supports specifically recognize phosphorylated (Ser/ the notion that SH2-docking proteins aug- Thr)Pro motifs, raising the possibility that One way receptors may amplify their signal- ment and regulate tyrosine kinase signaling. Ser-Thr phosphorylation of cell cycle reg- ing is to use adaptor proteins that provide ulators creates a recognition site for Pin1, additional docking sites for modular signal- Signaling Networks for which in turn could modify their confor- ing proteins. Typically, docking proteins Serine-Threonine Phosphorylation mation and functional properties (29). A have an NH2-terminal membrane-targeting conserved domain, EVH1, has recently element, either a PH domain or a myristy- The importance of protein-protein interac- been identified in profilin-binding pro- lation site, and a PTB domain that directs tions is not confined to signaling steps con- teins such as VASP and Mena, and has association with an NPXY autophosphoryl- trolled by tyrosine phosphorylation. Al- been shown to bind proline-rich peptide ation site on a specific receptor (Fig. 4). though some Ser-Thr kinases and phospha- sequences such as the (E/D)FPPPPX(D/E) Once it is associated with the appropriate tases were originally thought to be consti- motif found in the ActA protein of Listeria activated receptor, the docking protein be- tutively attached to intracellular loci monocytogenes. The EVH1 domain may comes phosphorylated at multiple sites that through association with targeting subunits couple cytoskeletal proteins such as zyxin interact with specific SH2 domains of sig- or anchoring proteins, it is now clear that and vinculin, and bacterial ActA, to actin naling proteins. For example, IRS-1 and some are also recruited into signaling net- remodeling (30). IRS-2 (two principal substrates of the insu- works (3). Apparently, protein modules

Phospholipid recognition and membrane tar- lin receptor) have an NH2-terminal PH control the location and assembly of these geting. Not all localization of signaling mod- domain followed by a PTB domain and 18 Ser-Thr kinase signaling networks, provid- ules is directed by interaction with other potential tyrosine phosphorylation sites ing an intriguing parallel with tyrosine ki- proteins. Pleckstrin homology (PH) do- (36). Under physiological circumstances, nase signaling events. mains bind the charged headgroups of spe- both the IRS-1 PH and PTB domains are and protein kinase C an- cific polyphosphoinositides and may there- required for insulin-induced tyrosine phos- choring. In its inactive form, the adenosine by regulate the subcellular targeting of sig- phorylation of IRS-1 and mitogenic signal- 3Ј,5Ј-monophosphate (cAMP)–dependent naling proteins to specific regions of the ing, suggesting that membrane targeting of protein kinase (PKA) is composed of two plasma membrane (Fig. 4) (31). This posi- IRS-1 and physical binding to its receptor catalytic (C) subunits held in an inactive tions such proteins for interactions with tyrosine kinase facilitate insulin-mediated conformation by association with two reg- regulators or targets. In this way, PH do- signaling (Fig. 4) (37). Another membrane- ulatory (R) subunits. Binding of cAMP to on February 23, 2009 mains can couple the actions of phosphati- associated docking protein, Shc, has an the R subunits causes dissocation of the C dyl inositol (PI) kinases, inositol phospha- NH2-terminal PTB domain that binds both subunits, which act to control a wide range tases, and phospholipases to the regulation pTyr sites and phosphoinositides, as well as of biological processes. Although cAMP is of intracellular signaling (32). This is illus- a COOH-terminal SH2 domain, and con- the sole activator of PKA, other regulatory trated by the finding that the product of PI sequently is phosphorylated by a broad proteins control where and when the kinase Ј 3 -kinase, PI-3,4,5-P3, binds specifically to range of receptor tyrosine kinases (11). In is turned on in response to specific stimuli. the PH domain of a protein, Grp1, which contrast, mammalian Gab-1, which has an The concerted actions of adenylyl cyclases can potentially function as an exchange NH2-terminal PH domain, may play a more and phosphodiesterases create gradients and factor for the small GTPase Arf (33). Be- specific role downstream of the Met ty- compartmentalized pools of cAMP, where- www.sciencemag.org cause the p85 regulatory subunit of PI 3Ј- rosine kinase, and p62dok is a prominent as A-kinase anchoring proteins (AKAPs) kinase possesses SH2 domains that control substrate of the Eph receptors that control maintain the PKA holoenzyme at precise enzyme activation by tyrosine phosphoryl- axon guidance (38). Similarly, FRS2, which intracellular sites. Anchoring ensures that ation, it seems that the SH2 and PH do- is myristylated and has a potential PTB PKA is exposed to localized changes in mains of distinct proteins can act sequen- domain, is specifically phosphorylated by cAMP and is compartmentalized with sub- tially in a common pathway to link receptor receptors for fibroblast (FGF) 39 tyrosine kinase signaling to the control of and nerve growth factor (NGF) ( ). Insulin receptor vesicle trafficking. The signaling properties of these docking β subunit Downloaded from

PH domains are also found covalently proteins likely depend on the sequences of Phospholipid linked to other modules, such as SH2, their SH2-binding motifs. Commonly, a spe- N IRS-1 P SH3, and PTB domains, with which they cific Tyr-based motif is reiterated several E may synergize in controlling the activa- times within an individual docking protein. YÐP tion of specific signaling proteins. For ex- Thus, Shc has two YXNX motifs, which can SH2 proteins ample, the Btk cytoplasmic tyrosine kinase couple to Grb2 and the Ras pathway (40); PI 3'-kinase dok Insulin contains an NH2-terminal PH domain p62 has six YXXP motifs, which may bind receptor Grb-2 that binds PI-3,4,5-P and is covalently SH2-containing proteins that influence the kinase 3 Shp-2 linked to SH3, SH2, and kinase domains cytoskeleton (38); and IRS-1 has nine Nck (34). The production of PI-3,4,5-P3 may YXXM motifs, which can bind and activate therefore induce association of Btk with PI 3Ј-kinase. This phenomenon may allow the membrane, facilitating the interaction selective amplification of specific signaling Fig. 4. The IRS-1 docking protein. The IRS-1 docking protein contains an NH -terminal PH do- of the SH2, SH3, and catalytic domains pathways. The Drosophila docking protein 2 with activators and targets. Mutations in Dos, which has the potential to bind several main that potentially mediates interactions with the membrane and a PTB domain that binds a the PH domain that inhibit phospholipid distinct signaling proteins with SH2 do- specific juxtamembrane Tyr autophosphorylation recognition, or of Cys residues in an adja- mains, including Drk (the Drosophila or- site in the insulin receptor. The kinase domain of cent structural element that binds Zn, lead tholog of Grb-2) and corkscrew (an SH2- the activated insulin receptor phosphorylates Tyr to an inherited human B cell defect, X- containing tyrosine ), was origi- residues in IRS-1 that act as docking sites for linked agammaglobulinemia (35). nally identified in a genetic screen for com- multiple SH2-domain signaling proteins.

www.sciencemag.org ⅐ SCIENCE ⅐ VOL. 278 ⅐ 19 DECEMBER 1997 2077 strates. The AKAPs have two types of pro- AKAPs were thought to exclusively tar- (Fig. 3). Hence, these proteins may repre- tein sequence that direct PKA function: a get the type II PKA. However, a family of sent an emerging class of mammalian tar- conserved amphipathic helix binds the R dual function AKAPs has now been discov- geting proteins that organize signal trans- subunit dimer of the PKA holoenzyme and ered that bind RI or RII (46). Although in duction events by bringing kinases together. a specialized targeting region tethers indi- vitro studies indicate that RI binds several Phosphatase targeting. The dephosphoryl- vidual PKA-AKAP complexes to specific AKAPs, with affinity one one-hundredth ation of proteins is of equal importance to subcellular structures (Fig. 5A) (42). As a that of RII, the micromolar binding-con- protein phosphorylation for the regulation consequence, PKA is held by the AKAP in stant interaction is within the physiological of cellular behavior, and the functions of an inactive state at a defined intracellular concentration range of RI and AKAPs in- protein phosphatases seem to be controlled location, where it is poised to respond to side cells. Thus, type I PKA anchoring may by targeting interactions like those de- cAMP by the local release of active C sub- be relevant under certain conditions where scribed for protein kinases. Indeed, a com- unit. Thus, a pleiotropic protein kinase can RII concentrations are limiting (47). Cer- mon feature of many intracellular tyrosine rapidly phosphorylate specific targets in re- tain AKAPs bind multiple signaling en- phosphatases is the presence of noncatalytic sponse to a defined signal. zymes. AKAP79 functions as a signaling domains that direct the phosphatase to spe- Information on the AKAP motifs that scaffold for PKA, PKC, and protein phos- cific compartments. For example, two mam- direct R subunit–binding and subcellular phatase 2B at the postsynaptic densities of malian tyrosine phosphatases, Shp-1 and localization has been exploited to alter the neurons, whereas AKAP250 (gravin) tar- Shp-2, contain tandem NH2-terminal SH2 distribution of PKA inside cells. Heterolo- gets both PKA and PKC to the membrane domains that both regulate phosphatase ac- gous expression of AKAP75, or its human cytoskeleton and filopodia of cells (48). tivity and allow these enzymes to be recruit- homolog AKAP79, redirects PKA to the Anchored signaling scaffolds may permit ed into complexes of specific pTyr-contain- periphery of HEK 293 cells. This submem- the integration of signals from distinct sec- ing proteins (51). brane targeting of PKA enhances cAMP- ond messengers to preferentially control se- Among the Ser-Thr phosphatases, PP-1, stimulated phosphorylation of the ␣1 sub- lected phosphorylation events. PP-2A, and PP-2B interact with distinct unit of the cardiac L-type Ca2ϩ channel The Ca2ϩ phospholipid–dependent pro- targeting proteins or subunits (3, 4). PP-1 and increases ion flow (43). Conversely, tein kinase, PKC, exerts a wide range of associates with glycogen particles in liver microinjection of “anchoring inhibitor pep- biological effects. Various isoforms of PKC through a “glycogen-targeting subunit” tides,” which compete for the RII-AKAP are differentially localized inside cells. (GL), whereas the skeletal muscle form of interaction, displaces the kinase from an- Whereas the attachment of PKC to mem- the targeting subunit (GM) targets PP-1to choring sites and attenuates ion flow branes clearly requires protein-phospholipid the sarcoplasmic reticulum and to glycogen. on February 23, 2009 through skeletal muscle L-type Ca2ϩ chan- interactions, protein-protein interactions Association with GM or GL has allosteric nels, AMPA-kainate seem to facilitate the differential localiza- effects that modify the substrate specificity ion-channels, and Ca2ϩ-activated Kϩ tion of PKC isoforms inside cells (49). Sev- of the enzyme (3, 52). The PP-1 binding channels (44). Hence, PKA anchoring eral classes of PKC targeting proteins have site on GL has been crystallized in a com- seems to augment rapid cAMP responses been identified. Substrate-binding proteins plex with the PP-1 subunit, and a consensus such as ion-channel modulation. AKAPs (SBPs) bind PKC in the presence of phos- peptide motif, RRVXF, has been identified also orchestrate the role of PKA in more phatidylserine by forming a ternary com- on the various PP-1 targeting subunits (Fig. complicated physiological processes such as plex with the kinase (Fig. 5B). Phosphoryl- 5C) (53). Accordingly, peptide analogs

glucagon-related peptide (GLP-1)–induced ation of SBPs by PKC abolishes the target- have been generated that disturb the loca- www.sciencemag.org insulin secretion from pancreatic ␤ islet ing interaction, suggesting that SBPs may tion of the phosphatase inside cells (54). cells (45). associate with the kinase transiently and The coordination of signaling events at the represent a subclass of PKC substrates that glycogen particle is also achieved by a PP-1 release the enzyme slowly upon completion scaffold protein called PTG, which main- of the phosphotransfer reaction. Receptors tains the phosphatase with some of its ma- A Amphipathic B helix for activated C kinase (RACKs) are not jor substrates, phosphorylase kinase, glyco- Phospholipid necessarily substrates for PKC and bind at gen synthase, and phosphorylase a (55).

one or more sites distinct from the sub- Other PP-1 targeting subunits direct the Downloaded from PKC- AKAP binding strate-binding pocket of the kinase. Thus, it phosphatase to smooth muscle (PP-1M) or protein PKA PKC has been proposed that PKC remains active the nucleus (SDS-22 or NIPP-1), or for Subcellular structure Subcellular structure when bound to a RACK. A third class of association with the p53 binding protein 2 PKC-binding protein, termed PICKs (for [reviewed in (3, 4)]. CDproteins that interact with C kinase), have PP-2A is a heterotrimer consisting of a been cloned in two-hybrid screens in which 36-kD catalytic subunit (C), a 65-kD struc- Targeting the catalytic core of the kinase was used as tural subunit (A), and a regulatory subunit PP-1 subunit PP-2A B subunit bait. One of these proteins, called PICK-1, (B), and is compartmentalized through as- Subcellular structure Subcellular structure is a perinuclear protein that appears to only sociation with its own set of targeting sub- Fig. 5. Targeting proteins for Ser-Thr kinases and recognize determinants in the active en- units. Several families of B subunits have phosphatases. Targeting proteins are depicted zyme, and also contains a PDZ domain. been identified that participate in directing for (A) the cAMP-dependent protein kinase, which This is in keeping with accumulating evi- the subcellular location of the holoenzyme is bound to its AKAPs by an amphipathic helix on dence that subcellular targeting of PKC is to centrosomes, the endoplasmic reticulum, the AKAP; (B) PKC, which is attached to its bind- also mediated through association with oth- golgi, and the nucleus (Fig. 5D). Targeting ing protein through protein-phospholipid interac- tions; (C) PP-1, which binds its targeting subunit er signaling proteins (50). For example, of PP-2A to the microtubule fraction is also through a consensus-binding motif (indicated in AKAP79 colocalizes PKC with PKA and mediated by a specialized B subunit that the single-letter code); and (D) the B subunit of ; gravin binds PKC and PKA; associates with microtubule-associated pro- PP-2A, which binds and targets the A and C sub- and the InaD clusters PKC with phospho- teins such as Tau (56). Targeting proteins unit complex. lipase-C ␤, G proteins, and calmodulin also localize the protein phosphatase 2B

2078 SCIENCE ⅐ VOL. 278 ⅐ 19 DECEMBER 1997 ⅐ www.sciencemag.org ARTICLE (PP-2B, also known as calcineurin). In the cytoplasmic anchor for the MAPK. lates the activity of the Cdc2 protein kinase brain, the particulate form of the PP-2B is Recently, a second yeast scaffold protein and thereby controls entry into mitosis. inactive, possibly because it is targeted to called Pbs2p was identified that coordinates Phosphorylation of Cdc25C at Ser216 dur- submembrane sites and inhibited through components of the S. cerevisiae osmoregula- ing interphase creates a 14-3-3 binding site association with AKAP79 (48). Other stud- tory pathway. One activator of this pathway and inhibits Cdc25C biological activity ies suggest that cytosolic PP-2B associates is the transmembrane osmosensor Sho1, (70). A distinct function of 14-3-3 proteins with one of its physiological substrates, the which has a cytoplasmic SH3 domain and may be to potentiate the survival of mam- NFATp. activa- activates a MAPK cascade containing the malian cells through inducible recognition tion and increased transcription of genes MAPKKK Ste 11, the MAPKK Pbs2, and of phosphorylated Bad, a death inducer, encoding require the dephospho- the MAPK Hog1. Pbs2, in addition to act- thereby disrupting a heteromeric complex rylation-dependent translocation of the ing as a MAPKK, also appears to serve a between Bad and Bcl-XL, an antagonist of NFAT complex into the nucleus, possibly scaffolding function by interacting with cell death (71). By contrast, in plants, 14- with the phosphatase still attached (57). Sho1, Ste11, and Hog1 (64). Indeed, the 3-3 binds and inhibits the activity of phos-

Coordination of MAP kinase cascades. NH2-terminal region of Pbs2 has a proline- phorylated nitrate reductase to control ni- Ras signal transduction pathways link ac- rich motif that binds the Sho1 SH3 do- trogen metabolism in spinach (72). These tivation of receptor tyrosine kinases to main; genetic evidence indicates that this results suggest that 14-3-3 proteins exert changes in gene expression (58). This interaction is necessary for activation of the biological effects through regulating oligo- pathway proceeds from the membrane- pathway in response to osmotic stress. The meric protein-protein interactions and pro- bound guanine nucleotide–binding pro- importance of the Ste 5 and Pbs2 scaffolds tein localization, as well as through control tein Ras, through the sequential activa- in maintaining signaling specificity is em- of enzymatic activity. tion of the cytoplasmic Ser-Thr kinases phasized by the observation that although Raf [a mitogen-activated protein kinase the mating and osmosensory MAPK path- Conclusions and Perspectives (MAPK) kinase kinase or MAPKKK], ways share a common component, Ste 11, Mek (a MAPK kinase or MAPKK), and they show no cross-talk. Cellular responses to external and intrinsic Erk (a MAPK), and leads to specific gene So far, there is little evidence to suggest signals are organized and coordinated expression in the nucleus. Distinct MAPK that Ste 5 orthologs exist in mammalian through specific protein-protein and pro- cassettes, each composed of three succes- cells, but it seems likely that mammalian tein-phospholipid interactions, commonly sive kinases, are activated in mammalian scaffolding proteins for MAPK pathways mediated by conserved protein domains. cells by mitogenic or stress signals. exist. Subcellular targeting of the stress- Such protein modules have apparently de- on February 23, 2009 In , the phero- activated (Jun) protein kinase (termed JNK veloped to recognize determinants that are mone mating response is initiated through or SAPK) is achieved, in part, through likely to be exposed within their molecular –linked receptors that activate a association with JIP-1, an SH3-containing partners. By the repeated use of these rather kinase, Ste 20. This leads to the stimulation protein that prevents nuclear translocation simple lock-and-key recognition events, a of the MAPKKK Ste 11, which phospho- of JNK and inhibits the bound kinase (65). complex and diverse regulatory network of rylates and activates Ste 7 (a MAPKK), This latter property is reminiscent of the molecular interactions can be assembled. which in turn phosphorylates and activates AKAP signaling scaffolds, where each en- The covalent association of these recogni- the MAPK homologs Fus3 or Kss1 (59). zyme is maintained in the inactive state by tion modules—as found in adaptors, an-

This signaling pathway can be tightly con- the anchoring protein. choring proteins, and docking proteins— www.sciencemag.org trolled, because each enzyme associates The 14-3-3 proteins. Growing evidence allows a single polypeptide to bind multiple with a docking site on a scaffold protein suggests that Ser phosphorylation induces protein ligands. This can be used to couple called Ste 5 (60). There may be additional specific protein-protein interactions, medi- an activated receptor to several downstream components of the complex, because up- ated by the 14-3-3 family of adaptor pro- targets and biochemical pathways or to in- stream activators of the pathways such as teins (66). These proteins associate to form crease the affinity and specificity with the G protein ␤ subunit, Ste 4, and possibly homo- and heterodimers with a saddle- which a single partner is engaged. As an Ste 20 interact with Ste 5 (61). Ste 5 may shaped structure, with each monomer pos- added complexity, a single module can bind serve two functions. Dimerization of Ste 5, sessing an extended groove that provides a either to a motif within the same molecule Downloaded from which requires an NH2-terminal RING-H2 likely site for peptide binding (Fig. 2F) (67). or in an intermolecular fashion to other domain, can facilitate intersubunit auto- Mammalian 14-3-3 proteins can associate proteins. Thus, the intramolecular associa- phosphorylation and activation of individ- with a number of signaling molecules, in- tion of the SH2 and SH3 domains of Src ual Ste 5–associated kinases (62). The clus- cluding the c-Raf and Ksr Ser-Thr protein family kinases with internal binding sites tering of successive members in the MAPK kinases, Bcr, PI 3Ј-kinase, and polyomavirus both represses Src kinase activity and cascade favors tight regulation of the path- middle T antigen. Through dimerization, blocks SH2/SH3 domain association with way by ensuring that signals pass quickly the proteins also may function to bridge the heterologous polypeptides. During Src acti- from one kinase to the next, thus prevent- interaction of two binding partners, as sug- vation, these domains are liberated for as- ing “cross-talk” between functionally unre- gested for c-Raf (66) and Bcr (68). Further- sociation with substrates and cytoskeletal lated MAPK units in the same cell. In more, assocation with targets such as c-Raf elements. Consequently, modular domains addition to the indirect association of Ste and Ksr requires recognition of a phospho- that participate in tyrosine kinase signaling 7 and Fus3 or Kss1 through the Ste 5 serine residue contained within the consen- act to localize proteins to specific subcellu- scaffolding protein, these two kinases may sus motif RSX-pSer-XP, in a fashion remi- lar sites, to control enzyme activity, to di- interact directly in the absence of Ste 5 niscent of SH2 domain interactions with rect the formation of multiprotein complex- through an NH2-terminal peptide motif in pTyr-containing motifs (69). es, and to directly transduce signals. Ste7(63). Such interactions involving a An important function for 14-3-3 pro- The Ser-Thr kinases and phosphatases MAPKK and its cognate MAPK could teins in cell cycle control is suggested by use a variation on this theme whereby the increase the fidelity of the pathway and their ability to recognize a pSer motif in enzymes appear to be constitutively target- might allow the MAPKK to serve as a human Cdc25C, a phosphatase that regu- ed and colocalized with their substrate at

www.sciencemag.org ⅐ SCIENCE ⅐ VOL. 278 ⅐ 19 DECEMBER 1997 2079 their sites of action. Often, these anchored 37. L. Yenush et al., J. Biol. Chem. 271, 24300 (1996). 1475 (1997). enzymes only become activated when their 38. K. M. Weidner et al., Nature 384, 173 (1996); Y. 56. E. Sontag et al., Neuron 17, 1201 (1996). Yamanashi and D. Baltimore, Cell 88, 205 (1997); N. 57. F. Shibasaki, E. R. Price, D. Milan, F. McKeon, Na- stimulating second messengers and signals Carpino et al., ibid., p. 197; S. J. Holland et al., ture 382, 370 (1996); C. Loh et al., J. Biol. Chem. become available. Recent data suggest that EMBO J. 16, 3877 (1997). 271, 10884 (1996). serine phosphorylation, like tyrosine phos- 39. H. Kouhara et al., Cell 89, 693 (1997). 58. C. J. Marshall, Cell 80, 179 (1995). 40. P. van der Geer et al., Curr. Biol. 6, 1435 (1996). phorylation, may directly regulate modular 59. I. Herskowitz, ibid., p. 187. 41. T. Raabe et al., Cell 85, 911 (1996). 60. K.-Y. Choi et al., Cell 78, 499 (1994); J. A. Printen protein-protein interactions. Now that the 42. M. L. Dell’Acqua and J. D. Scott, J. Biol. Chem. 272, and G. F. Sprague Jr., Genetics 138, 609 (1994); S. intricacy of these interactions is under- 12881 (1997). Marcus et al., Proc. Natl. Acad. Sci. U.S.A. 91, 7762 stood, the challenge ahead is to understand 43. T. Gao et al., Neuron 19, 185 (1997). (1994). 44. C. Rosenmund et al., Nature 368, 853 (1994); B. D. 61. M. S. Whiteway et al., Science 269, 1572 (1995). both the physiological functions and regu- Johnson, T. Scheuer, W. A. Catterall, Proc. Natl. 62. D. Yablonski, I. Marbach, A. Levitzki, Proc. Natl. lation of such signaling networks. Acad. Sci. U.S.A. 91, 11492 (1994). Acad. Sci. U.S.A. 93, 13864 (1996). 45. L. B. Lester, L. K. Langeberg, J. D. Scott, Proc. Natl. 63. L. Bardwell and J. Thorner, Trends Biochem. Sci. 21, Acad. Sci. U.S.A., in press. 373 (1996). REFERENCES AND NOTES 46. L. J. Huang et al., J. Biol. Chem. 272, 8057 (1997). ______64. F. Posas and H. Saito, Science 276, 1702 (1997). 47. K. A. Burton et al., Proc. Natl. Acad. Sci. U.S.A. 94, 65. M. Dickens et al., ibid. 277, 693 (1997). 1. D. Anderson et al., Science 250, 979 (1990). 11067 (1997). 2. T. Pawson, Nature 373, 573 (1995); G. B. Cohen, R. 48. V. M. Coghlan et al., Science 267, 108 (1995); T. M. 66. D. Morrison, ibid. 266, 56 (1994). Ren, D. Baltimore, Cell 80, 237 (1995). Klauck et al., ibid. 271, 1589 (1996); J. B. Nauert et 67. B. Xiao et al., Nature 376, 188 (1995); D. Liu et al., 3. M. Hubbard and P. Cohen, Trends Biochem. Sci. 18, al., Curr. Biol. 7, 52 (1997). ibid., p. 191. 172 (1993); D. Mochly-Rosen, Science 268, 247 49. D. Mochly-Rosen, H. Khaner, J. Lopez, Proc. Natl. 68. S. Braselmann and F. McCormick, EMBO J. 14, (1995); M. C. Faux and J. D. Scott, Trends Biochem. Acad. Sci. U.S.A. 88, 3997 (1991); C. Chapline et al., 4839 (1995). Sci. 21, 312 (1996). J. Biol. Chem. 268, 6858 (1993). 69. A. J. Muslin et al., Cell 84, 889 (1996); H. M. Xing, K. 4. M. C. Faux and J. D. Scott, Cell 85, 8 (1996). 50. J. Staudinger et al., J. Cell Biol. 128, 263 (1995). Kornfeld, A. J. Muslin, Curr. Biol. 7, 294 (1997). 5. Z. Songyang et al., ibid. 72, 767 (1993); G. Waksman 51. N. K. Tonks and B. G. Neel, Cell 87, 365 (1997); L. J. 70. C.-Y. Peng et al., Science 277, 1501 (1997). et al., ibid., p. 779; S. M. Pascal et al., ibid. 77, 461 Mauro and J. E. Dixon, Trends Biochem. Sci. 19, 71. J. Zha et al., Cell 87, 619 (1996). (1994). 151 (1994). 72. G. Moorhead et al., Curr. Biol. 6, 1104 (1996). 6. F. Maina et al., ibid. 87, 531 (1996). 52. M. J. Hubbard et al., Eur. J. Biochem. 189, 243 73. T.P. is a Terry Fox Cancer Scientist of the National 7. P. Blaikie et al., J. Biol. Chem. 269, 32031 (1994); (1990). Cancer Institute of Canada and J.D.S. is supported W. M. Kavanaugh and L. T. Williams, Science 266, 53. M. P. Egloff et al., EMBO J. 16, 1876 (1997). by NIH grant GM48231. We thank R. Frank for tech- 1862 (1994); T. A. Gustafson et al., Mol. Cell. Biol. 54. D. F. Johnson et al., Eur. J. Biochem. 239, 317 nical assistance. Because of space limitations, we 15, 2500 (1995). (1996). were not able to acknowledge the contributions of all 8. Single-letter abbreviations for the amino acid resi- 55. J. A. Printen, M. J. Brady, A. R. Saltiel, Science 275, investigators to this growing field. dues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N,

Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, on February 23, 2009 Trp; and Y, Tyr. X indicates any amino acid. 9. P. van der Geer et al., Curr. Biol. 5, 404 (1995); W. M. RESEARCH ARTICLES Kavanaugh, C. W. Turck, L. T. Williams, Science 268, 1177 (1995). 10. T. Trub et al., J. Biol. Chem. 270, 18205 (1995); P. van der Geer et al., Proc. Natl. Acad. Sci. U.S.A. 93, 963 (1996). Large-Cage Zeolite Structures 11. M. M. Zhou et al., Nature 378, 584 (1995). 12. J. P. Borg, J. Ooi, E. Levy, B. Margolis, Mol. Cell. Biol. 16, 6229 (1996); S. C. Li et al., Proc. Natl. Acad. with Multidimensional Sci. U.S.A. 94, 7204 (1997).

13. D. A. Doyle et al., Cell 85, 1067 (1996). www.sciencemag.org 14. E. Kim et al., Nature 378, 85 (1995); Z. Songyang et 12-Ring Channels al., Science 275, 73 (1997). 15. J. S. Simske et al., Cell 85, 195 (1996). 16. J. Chevesich, A. J. Kreuz, C. Montell, Neuron 18,95 Xianhui Bu, Pingyun Feng, Galen D. Stucky (1997); H.-C. Kornau, L. T. Schenker, M. B. Kennedy, P. H. Seeburg, Science 269, 1737 (1995); Zeolite type structures with large cages interconnected by multidimensional 12-ring H. Dong et al., Nature 386, 279 (1997); P. R. Brake- man et al., ibid., p. 284. (rings of 12 tetrahedrally coordinated atoms) channels have been synthesized; more 17. N. A. Cohen et al., Neuron 17, 759 (1996). than a dozen large-pore materials were created in three different topologies with

18. Y. Hsueh, E. Kim, M. Sheng, ibid. 18, 803 (1997). aluminum (or gallium), cobalt (or manganese, magnesium, or zinc), and phosphorus Downloaded from 19. M. Irie et al., Science 277, 1511 (1997). 20. J. E. Brenman et al., Cell 84, 757 (1996). at the tetrahedral coordination sites. Tetragonal UCSB-8 has an unusually large cage 21. B. Shieh and M. Zhu, Neuron 16, 991 (1996). built from 64 tetrahedral atoms and connected by an orthogonal channel system with 22. S. Tsunoda et al., Nature 388, 243 (1997). 12-ring apertures in two dimensions and 8-ring apertures in the third. Rhombohedral 23. S. Feng, J. K. Chen, H. Yu, J. A. Simon, S. L. Schre- UCSB-10 and hexagonal UCSB-6 are structurally related to faujasite and its hexagonal iber, Science 266, 1241 (1994). 24. D. Chen et al., J. Biol. Chem. 271, 6328 (1996). polymorph, respectively, and have large cages connected by 12-ring channels in all 25. M. Sudol, Prog. Biophys. Mol. Biol. 65, 113 (1996); three dimensions. M. J. Macias et al., Nature 382, 646 (1996). 26. M. T. Bedford et al., EMBO J. 16, 2376 (1997). 27. P. M. Snyder et al., Cell 83, 969 (1995). 28. R. Ranganathan et al., ibid. 89, 875 (1997). 29. M. B. Yaffe et al., Science 278, 1957 (1997). Extensive research has led to the synthesis ly (2). A number of structures with 12-ring 30. K. Niebuhr et al., EMBO J. 16, 5433 (1997). 31. J. E. Harlan et al., Nature 371, 168 (1994); M. A. of zeolitic materials with previously unseen channels (for example, AlPO4-5, MAPSO- Lemmon et al., Proc. Natl. Acad. Sci. U.S.A. 92, compositions and framework topologies (1), 46, and CoAPO-50) have also been report- 10472 (1995). including ultralarge-pore structures VPI-5, ed (1). Other open framework structures 32. A. Toker and L. Cantley, Nature 387, 673 (1997). AlPO 8, and UTD 1, which have pores that have large cages or pore sizes include 33. J. K. Klarlund et al., Science 275, 1927 (1997); P. 4- - Chardin et al., Nature 384, 481 (1996). formed of 18-,14-, and 14-rings, respective- JDF-20, cloverite (3), and unusually low- 34. K. Salim et al., EMBO J. 15, 6241 (1996); M. Fukuda density vanadium phosphate frameworks et al., J. Biol. Chem. 271, 30303 (1996). X. Bu and P. Feng are in the Department of Chemistry (4). 35. M. Vihinen et al., FEBS Lett. 413, 205 (1997); M. and G. D. Stucky is in the Department of Chemistry and Hyvonen and M. Saraste, EMBO J. 16, 3396 (1997). Department of Materials, University of California, Santa Unlike faujasite or its hexagonal poly- 36. X. J. Sun et al., Mol. Cell. Biol. 13, 7418 (1993). Barbara, CA 93106, USA. morph, known materials with a zeolite

2080 SCIENCE ⅐ VOL. 278 ⅐ 19 DECEMBER 1997 ⅐ www.sciencemag.org