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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 mecha- nisms exist to influence where and when protein kinases and phosphatases are activated 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 transduc- tion are often taken for granted. Yet, un- derstanding the mechanisms that allow in- tracellular 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 cor- rect 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 multipro- tein signaling networks (Fig. 1A) or activa- tion 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 typi- fied by the association of autophosphoryl- ated receptor tyrosine kinases with cytoplas- mic proteins that contain specialized protein modules that mediate formation of signaling complexes (Fig. 2) (1). For example, src homology 2 (SH2) domains bind specific phosphotyrosyl residues on activated recep- tors (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 com- plexes of signaling proteins around an acti- vated cell-surface receptor (Fig. 1A). Simi- larly, subcellular organization of serine- threonine kinases and phosphatases occurs

through interactions with the targeting sub- units or anchoring proteins that localize these enzymes (3). In addition, kinase bind- ing 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 signaling scaffolds 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 con- trast the protein modules, adaptor mole- cules, targeting subunits, and anchoring pro- teins that coordinate signaling networks.

Mechanisms for Recognition of Phosphotyrosine and Peptide

Motifs

SH2 domains are protein modules that rec- ognize short, phosphopeptide motifs com- posed of phosphotyrosine (pTyr) followed by three to five COOH-terminal residues, such as those generated by autophosphoryl- ation 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 cou- pled directly or indirectly to downstream signaling molecules, including enzymes that control phospholipid metabolism, Ras-like guanosine triphosphatases (GTPases), pro- tein kinases, transcription factors, and polypeptides that regulate cytoskeletal ar- chitecture 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. Con- version 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 phosphoty- rosine sites (the 12 position) selectively uncouples the receptor from its ability to bind the Grb2 SH2/SH3 adaptor protein and yields a hypomorphic phenotype affect- ing myoblast proliferation and muscle for- mation (6). Thus, the creation of docking sites through autophosphorylation is abso- lutely 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 con- sensus NPXpY (8)] (9). Specificity is con- ferred 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 (11). 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, Cana- da. J. D. Scott, Howard Hughes Medical Institute, Vollum Institute, Portland, OR 97201–3098, USA.

A B Fig. 1. Mechanisms for recruiting or localizing signal transduction com- ponents. (A) Assembly of modular signaling molecules on an activated receptor tyrosine kinase. An extra- cellularsignal inputdrivesautophos- phorylation of the receptor and leads to the recruitment of cyto- plasmic proteins that contain vari- ous protein modules. pY, phospho- tyrosine. (B) A localized signaling complex of three anchored signal- ing enzymes. Each enzyme is inactive when bound to the anchoring protein but is released and activated by different signals. Enz., enzyme.

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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 prin- cipally 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 do- mains, in which the peptide binds as an additional strand to an antiparallel b sheet (13). A distinguishing feature of PDZ do- mains is their recognition of short peptides with a COOH-terminal hydrophobic resi- due and a free carboxylate group, as exem- plified 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 ligand- gated ion channels at synapses, because shaker-type K1 channels and all three class- es 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 hydroxy- amino acid. In particular, a COOH-termi- nal motif (RRESAI) on the inwardly recti- fying K1 channel Kir 2.3 binds the second PDZ domain of a 95-kD postsynaptic den- sity protein called PSD-95. PKA phospho- rylation at the –2 Ser of the PDZ recogni- tion site uncouples the channel from PSD- 95 and results in inhibition of K1 conduc- tance (Fig. 3A) (17).

Many proteins have multiple PDZ do- mains (up to seven in known proteins), which may have at least two important

consequences. An individual PDZ-contain- ing protein could bind several subunits of a particular channel, and thereby induce channel aggregation. This could be en- hanced by the ability of a protein such as PSD-95 to form oligomers through NH2- terminal intermolecular disulfide bonds (18). Furthermore, the individual PDZ do- mains of a protein such as PSD-95 can have distinct binding specificities, leading to the formation of clusters that contain heteroge- neous 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 pep- tide sequences and, indeed, have a propen- sity to undergo homotypic or heterotypic interactions with other PDZ domains (20). Second, proteins with PDZ domains fre- quently contain other interaction modules, including SH3 and LIM domains, and cat- alytic elements such as tyrosine phospha- tase 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 illustrat- ed by InaD, a polypeptide with five PDZ domains that regulates phototransduction in Drosophila melanogaster photoreceptors (Fig. 3B) (21). InaD associates through dis- tinct 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 chan- nel by PLC-b in response to stimulation of rhodopsin and deactivation through phos- phorylation of TRP by PKC (22). 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 left- handed polyproline type II helix (Fig. 2D) (23). A principal role of SH3 domains is in forming functional oligomeric complexes at defined subcellular sites, frequently in con- junction with other modular domains (2). There are certain parallels between SH3 and PDZ domains. Proteins can have multiple SH3 domains, potentially allowing cluster- ing of several distinct ligands, and Ser or Thr phosphorylation adjacent to the proline-rich ligand may influence SH3 domain interac- tions (24). Serine phosphorylation of a PDZ or SH3 recognition site results in uncoupling of signaling proteins, in contrast to the au- tophosphorylation of receptor tyrosine ki- nases, which promotes the assembly of sig- naling complexes at the SH2 acceptor site.

WW 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). The WW domains of the E3 ubiquitin protein ligase Nedd4 bind such proline-rich motifs in an amiloride- sensitive epithelial Na1 channel, likely lead- ing to channel degradation (Fig. 3A) (26). Channel mutations that disrupt this interac- tion cause a human hypertensive disorder, Liddle’s syndrome (27). WW domains may also regulate catalytic function, as suggested by a structural analysis of the peptidyl-prolyl cis-trans isomerase Pin1. Pin1, which inter- acts with cell cycle components such as the protein kinase NIMA, possesses an NH2- terminal WW domain that forms one part of a ligand-binding surface, which includes an a helix from the rotamase domain. The WW domain may thereby contribute to substrate

A B C

D E F

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

Drosophila eye

TRP Ca2+ channel

InaC InaD

GuK

CaN PKA

PKC

CaM

PDZ

PDZ

PDZ

PDZ PDZ

B

A Amiloride-sensitive

Na channel Kir 2.3 K+

channels AMPA

receptor L-type Ca2+ channel

β

Rhodopsin

Nedd4

PSD 95 AKAP 79

Eye PKC

Fig. 3. Localization of signaling molecules with ion channels. (A) Modulation of certain ion chan- nels may be coordinated by modular docking proteins or scaffold proteins that tether sig- naling enzymes in proximity to ion channels. Each ion channel is identified above, and the docking or anchoring proteins are indicated below. CaN, cal- cineurin. (B) In Drosophila eye, InaD coordinates the location of PKC and phospholipase b in re- lation to the channel. Calmodu- lin (CaM) and InaC also associ- ate with the channel.

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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 reg- ulators creates a recognition site for Pin1, which in turn could modify their confor- mation and functional properties (29). A conserved domain, EVH1, has recently been identified in profilin-binding pro- teins 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 tar- geting. Not all localization of signaling mod- ules is directed by interaction with other proteins. Pleckstrin homology (PH) do- mains bind the charged headgroups of spe- cific polyphosphoinositides and may there- by regulate the subcellular targeting of sig- naling proteins to specific regions of the plasma membrane (Fig. 4) (31). This posi- tions such proteins for interactions with regulators or targets. In this way, PH do- mains can couple the actions of phosphati- dyl inositol (PI) kinases, inositol phospha- tases, and phospholipases to the regulation of intracellular signaling (32). This is illus- trated 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 (33). Be- cause the p85 regulatory subunit of PI 39- kinase possesses SH2 domains that control enzyme activation by tyrosine phosphoryl- ation, it seems that the SH2 and PH do- mains of distinct proteins can act sequen- tially 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 activa- tion of specific signaling proteins. For ex- ample, 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 adja- cent 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 signal- ing is to use adaptor proteins that provide additional docking sites for modular signal- ing 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 autophosphoryl- ation site on a specific receptor (Fig. 4). Once it is associated with the appropriate activated receptor, the docking protein be- comes phosphorylated at multiple sites that interact with specific SH2 domains of sig- naling 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 phos- phorylation of IRS-1 and mitogenic signal- ing, suggesting that membrane targeting of IRS-1 and physical binding to its receptor tyrosine kinase facilitate insulin-mediated signaling (Fig. 4) (37). Another membrane- associated 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 con- sequently is phosphorylated by a broad range of receptor tyrosine kinases (11). 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-binding motifs. Commonly, a spe- cific 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 do- mains, including Drk (the Drosophila or- tholog of Grb-2) and corkscrew (an SH2- containing tyrosine phosphatase), was origi- nally identified in a genetic screen for com-

ponents of RTK signaling pathways in Dro- sophila (41). This genetic analysis supports the notion that SH2-docking proteins aug- ment and regulate tyrosine kinase signaling.

Signaling Networks for Serine-Threonine Phosphorylation

The importance of protein-protein interac- tions is not confined to signaling steps con- trolled by tyrosine phosphorylation. Al- though some Ser-Thr kinases and phospha- tases were originally thought to be consti- tutively attached to intracellular loci through association with targeting subunits or anchoring proteins, it is now clear that some are also recruited into signaling net- works (3). Apparently, protein modules control the location and assembly of these Ser-Thr kinase signaling networks, provid- ing an intriguing parallel with tyrosine ki- nase signaling events.

Protein kinase A and protein kinase C an- choring. 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 reg- ulatory (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, where- as 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

N P E Y–P

IRS-1

SH2 proteins

PI 3'-kinase

Grb-2

Shp-2

Nck

Phospholipid

Fig. 4. The IRS-1 docking protein. The IRS-1 docking protein contains an NH2-terminal PH do- main that potentially mediates interactions with the membrane and a PTB domain that binds a specific juxtamembrane Tyr autophosphorylation site in the insulin receptor. The kinase domain of the activated insulin receptor phosphorylates Tyr residues in IRS-1 that act as docking sites for multiple SH2-domain signaling proteins.

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strates. The AKAPs have two types of pro- tein sequence that direct PKA function: a conserved amphipathic helix binds the R subunit dimer of the PKA holoenzyme and a specialized targeting region tethers indi- vidual PKA-AKAP complexes to specific subcellular structures (Fig. 5A) (42). As a consequence, PKA is held by the AKAP in an inactive state at a defined intracellular location, where it is poised to respond to cAMP by the local release of active C sub- unit. Thus, a pleiotropic protein kinase can rapidly phosphorylate specific targets in re- sponse to a defined signal.

Information on the AKAP motifs that direct R subunit–binding and subcellular localization has been exploited to alter the distribution of PKA inside cells. Heterolo- gous expression of AKAP75, or its human homolog AKAP79, redirects PKA to the periphery of HEK 293 cells. This submem- brane targeting of PKA enhances cAMP- stimulated phosphorylation of the a1 sub- unit of the cardiac L-type Ca21 channel and increases ion flow (43). Conversely, microinjection of “anchoring inhibitor pep- tides,” which compete for the RII-AKAP interaction, displaces the kinase from an- choring sites and attenuates ion flow through skeletal muscle L-type Ca21 chan- nels, AMPA-kainate glutamate receptor ion-channels, and Ca21-activated K1 channels (44). Hence, PKA anchoring seems to augment rapid cAMP responses such as ion-channel modulation. AKAPs also orchestrate the role of PKA in more complicated physiological processes such as glucagon-related peptide (GLP-1)–induced insulin secretion from pancreatic b islet cells (45).

AKAPs were thought to exclusively tar- get the type II PKA. However, a family of dual function AKAPs has now been discov- ered that bind RI or RII (46). Although in vitro studies indicate that RI binds several AKAPs, with affinity one one-hundredth that of RII, the micromolar binding-con- stant interaction is within the physiological concentration range of RI and AKAPs in- side cells. Thus, type I PKA anchoring may be relevant under certain conditions where RII concentrations are limiting (47). Cer- tain AKAPs bind multiple signaling en- zymes. AKAP79 functions as a signaling scaffold for PKA, PKC, and protein phos- phatase 2B at the postsynaptic densities of neurons, whereas AKAP250 (gravin) tar- gets both PKA and PKC to the membrane cytoskeleton and filopodia of cells (48). Anchored signaling scaffolds may permit the integration of signals from distinct sec- ond messengers to preferentially control se- lected phosphorylation events.

The Ca21 phospholipid–dependent pro- tein kinase, PKC, exerts a wide range of biological effects. Various isoforms of PKC are differentially localized inside cells. Whereas the attachment of PKC to mem- branes clearly requires protein-phospholipid interactions, protein-protein interactions seem to facilitate the differential localiza- tion of PKC isoforms inside cells (49). Sev- eral classes of PKC targeting proteins have been identified. Substrate-binding proteins (SBPs) bind PKC in the presence of phos- phatidylserine by forming a ternary com- plex with the kinase (Fig. 5B). Phosphoryl- ation of SBPs by PKC abolishes the target- ing interaction, suggesting that SBPs may associate with the kinase transiently and represent a subclass of PKC substrates that release the enzyme slowly upon completion of the phosphotransfer reaction. Receptors for activated C kinase (RACKs) are not necessarily substrates for PKC and bind at one or more sites distinct from the sub- strate-binding pocket of the kinase. Thus, it has been proposed that PKC remains active when bound to a RACK. A third class of PKC-binding protein, termed PICKs (for proteins that interact with C kinase), have been cloned in two-hybrid screens in which the catalytic core of the kinase was used as bait. One of these proteins, called PICK-1, is a perinuclear protein that appears to only recognize determinants in the active en- zyme, and also contains a PDZ domain. This is in keeping with accumulating evi- dence that subcellular targeting of PKC is also mediated through association with oth- er signaling proteins (50). For example, AKAP79 colocalizes PKC with PKA and calcineurin; gravin binds PKC and PKA; and the InaD clusters PKC with phospho- lipase-C b, G proteins, and calmodulin

(Fig. 3). Hence, these proteins may repre- sent an emerging class of mammalian tar- geting proteins that organize signal trans- duction events by bringing kinases together.

Phosphatase targeting. The dephosphoryl- ation of proteins is of equal importance to protein phosphorylation for the regulation of cellular behavior, and the functions of protein phosphatases seem to be controlled by targeting interactions like those de- scribed for protein kinases. Indeed, a com- mon feature of many intracellular tyrosine phosphatases is the presence of noncatalytic domains that direct the phosphatase to spe- cific compartments. For example, two mam- malian tyrosine phosphatases, Shp-1 and Shp-2, contain tandem NH2-terminal SH2 domains that both regulate phosphatase ac- tivity and allow these enzymes to be recruit- ed into complexes of specific pTyr-contain- ing proteins (51).

Among the Ser-Thr phosphatases, PP-1, PP-2A, and PP-2B interact with distinct targeting proteins or subunits (3, 4). PP-1 associates with glycogen particles in liver through a “glycogen-targeting subunit” (GL), whereas the skeletal muscle form of the targeting subunit (GM) targets PP-1 to the sarcoplasmic reticulum and to glycogen. Association with GM or GL has allosteric effects that modify the substrate specificity of the enzyme (3, 52). The PP-1 binding site on GL has been crystallized in a com- plex with the PP-1 subunit, and a consensus peptide motif, RRVXF, has been identified on the various PP-1 targeting subunits (Fig. 5C) (53). Accordingly, peptide analogs have been generated that disturb the loca- tion of the phosphatase inside cells (54). The coordination of signaling events at the glycogen particle is also achieved by a PP-1 scaffold protein called PTG, which main- tains the phosphatase with some of its ma- jor substrates, phosphorylase kinase, glyco- gen synthase, and phosphorylase a (55). Other PP-1 targeting subunits direct the phosphatase to smooth muscle (PP-1M) or the nucleus (SDS-22 or NIPP-1), or for association with the p53 binding protein 2 [reviewed in (3, 4)].

PP-2A is a heterotrimer consisting of a 36-kD catalytic subunit (C), a 65-kD struc- tural subunit (A), and a regulatory subunit (B), and is compartmentalized through as- sociation with its own set of targeting sub- units. Several families of B subunits have been identified that participate in directing the subcellular location of the holoenzyme to centrosomes, the endoplasmic reticulum, golgi, and the nucleus (Fig. 5D). Targeting of PP-2A to the microtubule fraction is also mediated by a specialized B subunit that associates with microtubule-associated pro- teins such as Tau (56). Targeting proteins also localize the protein phosphatase 2B

A

C D

B

PKC- binding protein

Targeting subunit

AKAP

B subunit

Subcellular structure Subcellular structure

Subcellular structure Subcellular structure

PP-2A

PKCPKA

PP-1

Amphipathic helix

Phospholipid

Fig. 5. Targeting proteins for Ser-Thr kinases and phosphatases. Targeting proteins are depicted for (A) the cAMP-dependent protein kinase, which is bound to its AKAPs by an amphipathic helix on the AKAP; (B) PKC, which is attached to its bind- ing protein through protein-phospholipid interac- tions; (C) PP-1, which binds its targeting subunit through a consensus-binding motif (indicated in the single-letter code); and (D) the B subunit of PP-2A, which binds and targets the A and C sub- unit complex.

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(PP-2B, also known as calcineurin). In the brain, the particulate form of the PP-2B is inactive, possibly because it is targeted to submembrane sites and inhibited through association with AKAP79 (48). Other stud- ies suggest that cytosolic PP-2B associates with one of its physiological substrates, the transcription factor NFATp. T cell activa- tion and increased transcription of genes encoding cytokines require the dephospho- rylation-dependent translocation of the NFAT complex into the nucleus, possibly with the phosphatase still attached (57).

Coordination of MAP kinase cascades. Ras signal transduction pathways link ac- tivation of receptor tyrosine kinases to changes in gene expression (58). This pathway proceeds from the membrane- bound guanine nucleotide– binding pro- tein Ras, through the sequential activa- tion of the cytoplasmic Ser-Thr kinases Raf [a mitogen-activated protein kinase (MAPK) kinase kinase or MAPKKK], Mek (a MAPK kinase or MAPKK), and Erk (a MAPK), and leads to specific gene expression in the nucleus. Distinct MAPK cassettes, each composed of three succes- sive kinases, are activated in mammalian cells by mitogenic or stress signals.

In Saccharomyces cerevisiae, the phero- mone mating response is initiated through G protein–linked receptors that activate a kinase, Ste 20. This leads to the stimulation of the MAPKKK Ste 11, which phospho- rylates and activates Ste 7 (a MAPKK), which in turn phosphorylates and activates the MAPK homologs Fus3 or Kss1 (59). This signaling pathway can be tightly con- trolled, because each enzyme associates with a docking site on a scaffold protein called Ste 5 (60). There may be additional components of the complex, because up- stream activators of the pathways such as the G protein b subunit, Ste 4, and possibly Ste 20 interact with Ste 5 (61). Ste 5 may serve two functions. Dimerization of Ste 5, which requires an NH2-terminal RING-H2 domain, can facilitate intersubunit auto- phosphorylation and activation of individ- ual Ste 5–associated kinases (62). The clus- tering of successive members in the MAPK cascade favors tight regulation of the path- way by ensuring that signals pass quickly from one kinase to the next, thus prevent- ing “cross-talk” between functionally unre- lated MAPK units in the same cell. In addition to the indirect association of Ste 7 and Fus3 or Kss1 through the Ste 5 scaffolding protein, these two kinases may interact directly in the absence of Ste 5 through an NH2-terminal peptide motif in Ste 7 (63). Such interactions involving a MAPKK and its cognate MAPK could increase the fidelity of the pathway and might allow the MAPKK to serve as a

cytoplasmic anchor for the MAPK. Recently, a second yeast scaffold protein

called Pbs2p was identified that coordinates components of the S. cerevisiae osmoregula- tory pathway. One activator of this pathway is the transmembrane osmosensor Sho1, which has a cytoplasmic SH3 domain and activates a MAPK cascade containing the MAPKKK Ste 11, the MAPKK Pbs2, and the MAPK Hog1. Pbs2, in addition to act- ing as a MAPKK, also appears to serve a scaffolding function by interacting with Sho1, Ste11, and Hog1 (64). Indeed, the NH2-terminal region of Pbs2 has a proline- rich motif that binds the Sho1 SH3 do- main; genetic evidence indicates that this interaction is necessary for activation of the pathway in response to osmotic stress. The importance of the Ste 5 and Pbs2 scaffolds in maintaining signaling specificity is em- phasized by the observation that although the mating and osmosensory MAPK path- ways share a common component, Ste 11, they show no cross-talk.

So far, there is little evidence to suggest that Ste 5 orthologs exist in mammalian cells, but it seems likely that mammalian scaffolding proteins for MAPK pathways exist. Subcellular targeting of the stress- activated (Jun) protein kinase (termed JNK or SAPK) is achieved, in part, through association with JIP-1, an SH3-containing protein that prevents nuclear translocation of JNK and inhibits the bound kinase (65). This latter property is reminiscent of the AKAP signaling scaffolds, where each en- zyme is maintained in the inactive state by the anchoring protein.

The 14-3-3 proteins. Growing evidence suggests that Ser phosphorylation induces specific protein-protein interactions, medi- ated by the 14-3-3 family of adaptor pro- teins (66). These proteins associate to form homo- and heterodimers with a saddle- shaped structure, with each monomer pos- sessing an extended groove that provides a likely site for peptide binding (Fig. 2F) (67). Mammalian 14-3-3 proteins can associate with a number of signaling molecules, in- cluding the c-Raf and Ksr Ser-Thr protein kinases, Bcr, PI 39-kinase, and polyomavirus middle T antigen. Through dimerization, the proteins also may function to bridge the interaction of two binding partners, as sug- gested for c-Raf (66) and Bcr (68). Further- more, assocation with targets such as c-Raf and Ksr requires recognition of a phospho- serine residue contained within the consen- sus motif RSX-pSer-XP, in a fashion remi- niscent of SH2 domain interactions with pTyr-containing motifs (69).

An important function for 14-3-3 pro- teins in cell cycle control is suggested by their ability to recognize a pSer motif in human Cdc25C, a phosphatase that regu-

lates the activity of the Cdc2 protein kinase and thereby controls entry into mitosis. Phosphorylation of Cdc25C at Ser216 dur- ing interphase creates a 14-3-3 binding site and inhibits Cdc25C biological activity (70). A distinct function of 14-3-3 proteins may be to potentiate the survival of mam- malian cells through inducible recognition of phosphorylated Bad, a death inducer, thereby disrupting a heteromeric complex between Bad and Bcl-XL, an antagonist of cell death (71). By contrast, in plants, 14- 3-3 binds and inhibits the activity of phos- phorylated nitrate reductase to control ni- trogen metabolism in spinach (72). These results suggest that 14-3-3 proteins exert biological effects through regulating oligo- meric protein-protein interactions and pro- tein localization, as well as through control of enzymatic activity.

Conclusions and Perspectives

Cellular responses to external and intrinsic signals are organized and coordinated through specific protein-protein and pro- tein-phospholipid interactions, commonly mediated by conserved protein domains. Such protein modules have apparently de- veloped to recognize determinants that are likely to be exposed within their molecular partners. By the repeated use of these rather simple lock-and-key recognition events, a complex and diverse regulatory network of molecular interactions can be assembled. The covalent association of these recogni- tion modules—as found in adaptors, an- choring proteins, and docking proteins— allows a single polypeptide to bind multiple protein ligands. This can be used to couple an activated receptor to several downstream targets and biochemical pathways or to in- crease the affinity and specificity with which a single partner is engaged. As an added complexity, a single module can bind either to a motif within the same molecule or in an intermolecular fashion to other proteins. Thus, the intramolecular associa- tion of the SH2 and SH3 domains of Src family kinases with internal binding sites both represses Src kinase activity and blocks SH2/SH3 domain association with heterologous polypeptides. During Src acti- vation, these domains are liberated for as- sociation with substrates and cytoskeletal elements. Consequently, modular domains that participate in tyrosine kinase signaling act to localize proteins to specific subcellu- lar sites, to control enzyme activity, to di- rect the formation of multiprotein complex- es, and to directly transduce signals.

The Ser-Thr kinases and phosphatases use a variation on this theme whereby the enzymes appear to be constitutively target- ed and colocalized with their substrate at

ARTICLE

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their sites of action. Often, these anchored enzymes only become activated when their stimulating second messengers and signals become available. Recent data suggest that serine phosphorylation, like tyrosine phos- phorylation, may directly regulate modular protein-protein interactions. Now that the intricacy of these interactions is under- stood, the challenge ahead is to understand both the physiological functions and regu- lation of such signaling networks.

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RESEARCH ARTICLES

Large-Cage Zeolite Structures with Multidimensional

12-Ring Channels Xianhui Bu, Pingyun Feng, Galen D. Stucky

Zeolite type structures with large cages interconnected by multidimensional 12-ring (rings of 12 tetrahedrally coordinated atoms) channels have been synthesized; more than a dozen large-pore materials were created in three different topologies with aluminum (or gallium), cobalt (or manganese, magnesium, or zinc), and phosphorus at the tetrahedral coordination sites. Tetragonal UCSB-8 has an unusually large cage built from 64 tetrahedral atoms and connected by an orthogonal channel system with 12-ring apertures in two dimensions and 8-ring apertures in the third. Rhombohedral UCSB-10 and hexagonal UCSB-6 are structurally related to faujasite and its hexagonal polymorph, respectively, and have large cages connected by 12-ring channels in all three dimensions.

Extensive research has led to the synthesis of zeolitic materials with previously unseen compositions and framework topologies (1), including ultralarge-pore structures VPI-5, AlPO4-8, and UTD-1, which have pores formed of 18-, 14-, and 14-rings, respective-

ly (2). A number of structures with 12-ring channels (for example, AlPO4-5, MAPSO- 46, and CoAPO-50) have also been report- ed (1). Other open framework structures that have large cages or pore sizes include JDF-20, cloverite (3), and unusually low- density vanadium phosphate frameworks (4).

Unlike faujasite or its hexagonal poly- morph, known materials with a zeolite

X. Bu and P. Feng are in the Department of Chemistry and G. D. Stucky is in the Department of Chemistry and Department of Materials, University of California, Santa Barbara, CA 93106, USA.

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