Signaling Through Scaffold, Anchoring, and adaptor proteins

Signaling Through Scaffold, Anchoring, and adaptor proteins

(Parte 2 de 3)

IRS-1

SH2 proteins

PI 3'-kinase Grb-2

Shp-2 Nck

Fig. 4. The IRS-1 docking protein. The IRS-1 docking protein contains an NH2-terminal PH domain 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.

on May 28, 2009 w.sciencemag.org Downloaded from strates. The AKAPs have two types of protein sequence that direct PKA function: a conserved amphipathic helix binds the R subunit dimer of the PKA holoenzyme and a specialized targeting region tethers individual 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 subunit. Thus, a pleiotropic protein kinase can rapidly phosphorylate specific targets in response 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. Heterologous expression of AKAP75, or its human homolog AKAP79, redirects PKA to the periphery of HEK 293 cells. This submembrane targeting of PKA enhances cAMP- stimulated phosphorylation of the a1 subunit of the cardiac L-type Ca21 channel and increases ion flow (43). Conversely, microinjection of “anchoring inhibitor peptides,” which compete for the RII-AKAP interaction, displaces the kinase from anchoring sites and attenuates ion flow through skeletal muscle L-type Ca21 channels, AMPA-kainate glutamate receptor ion-channels, and Ca21-activated K1 channels (4). 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 target the type I PKA. However, a family of dual function AKAPs has now been discovered 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-constant interaction is within the physiological concentration range of RI and AKAPs inside cells. Thus, type I PKA anchoring may be relevant under certain conditions where RII concentrations are limiting (47). Certain AKAPs bind multiple signaling enzymes. AKAP79 functions as a signaling scaffold for PKA, PKC, and protein phosphatase 2B at the postsynaptic densities of neurons, whereas AKAP250 (gravin) targets both PKA and PKC to the membrane cytoskeleton and filopodia of cells (48). Anchored signaling scaffolds may permit the integration of signals from distinct second messengers to preferentially control selected phosphorylation events.

The Ca21 phospholipid–dependent protein kinase, PKC, exerts a wide range of biological effects. Various isoforms of PKC are differentially localized inside cells. Whereas the attachment of PKC to membranes clearly requires protein-phospholipid interactions, protein-protein interactions seem to facilitate the differential localization of PKC isoforms inside cells (49). Several classes of PKC targeting proteins have been identified. Substrate-binding proteins (SBPs) bind PKC in the presence of phosphatidylserine by forming a ternary complex with the kinase (Fig. 5B). Phosphorylation of SBPs by PKC abolishes the targeting 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 substrate-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 enzyme, and also contains a PDZ domain. This is in keeping with accumulating evidence that subcellular targeting of PKC is also mediated through association with other signaling proteins (50). For example, AKAP79 colocalizes PKC with PKA and calcineurin; gravin binds PKC and PKA; and the InaD clusters PKC with phospholipase-C b, G proteins, and calmodulin

(Fig. 3). Hence, these proteins may represent an emerging class of mammalian targeting proteins that organize signal transduction events by bringing kinases together.

Phosphatase targeting. The dephosphorylation 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 described for protein kinases. Indeed, a common feature of many intracellular tyrosine phosphatases is the presence of noncatalytic domains that direct the phosphatase to specific compartments. For example, two mammalian 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 recruited into complexes of specific pTyr-containing proteins (51).

Among the Ser-Thr phosphatases, P-1,

P-2A, and P-2B interact with distinct targeting proteins or subunits (3, 4). P-1 associates with glycogen particles in liver through a “glycogen-targeting subunit” (GL), whereas the skeletal muscle form of the targeting subunit (GM) targets P-1t o 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 P-1 binding site on GL has been crystallized in a complex with the P-1 subunit, and a consensus peptide motif, RRVXF, has been identified on the various P-1 targeting subunits (Fig. 5C) (53). Accordingly, peptide analogs have been generated that disturb the location of the phosphatase inside cells (54). The coordination of signaling events at the glycogen particle is also achieved by a P-1 scaffold protein called PTG, which maintains the phosphatase with some of its major substrates, phosphorylase kinase, glycogen synthase, and phosphorylase a (5). Other P-1 targeting subunits direct the phosphatase to smooth muscle (P-1M) or the nucleus (SDS-2 or NIPP-1), or for association with the p53 binding protein 2 [reviewed in (3, 4)].

P-2A is a heterotrimer consisting of a 36-kD catalytic subunit (C), a 65-kD structural subunit (A), and a regulatory subunit (B), and is compartmentalized through association with its own set of targeting subunits. 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 P-2A to the microtubule fraction is also mediated by a specialized B subunit that associates with microtubule-associated proteins such as Tau (56). Targeting proteins also localize the protein phosphatase 2B

PKC- binding protein

Targeting subunitAKAP B subunit

Subcellular structure Subcellular structure

Subcellular structure Subcellular structure P-2A

PKCPKA P-1

Amphipathic helix

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 binding protein through protein-phospholipid interactions; (C) P-1, which binds its targeting subunit through a consensus-binding motif (indicated in the single-letter code); and (D) the B subunit of P-2A, which binds and targets the A and C subunit complex.

on May 28, 2009 w.sciencemag.org Downloaded from

(P-2B, also known as calcineurin). In the brain, the particulate form of the P-2B is inactive, possibly because it is targeted to submembrane sites and inhibited through association with AKAP79 (48). Other studies suggest that cytosolic P-2B associates with one of its physiological substrates, the transcription factor NFATp. T cell activation and increased transcription of genes encoding cytokines require the dephosphorylation-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 activation of receptor tyrosine kinases to changes in gene expression (58). This pathway proceeds from the membranebound guanine nucleotide– binding protein Ras, through the sequential activation 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 successive kinases, are activated in mammalian cells by mitogenic or stress signals.

In Saccharomyces cerevisiae, the pheromone mating response is initiated through G protein–linked receptors that activate a kinase, Ste 20. This leads to the stimulation of the MAPKKK Ste 1, which phosphorylates 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 controlled, 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 upstream 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 individual Ste 5–associated kinases (62). The clustering of successive members in the MAPK cascade favors tight regulation of the pathway by ensuring that signals pass quickly from one kinase to the next, thus preventing “cross-talk” between functionally unrelated 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 Ste7( 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 osmoregulatory 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 1, the MAPKK Pbs2, and the MAPK Hog1. Pbs2, in addition to acting 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 prolinerich 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 emphasized by the observation that although the mating and osmosensory MAPK pathways share a common component, Ste 1, 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 stressactivated (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 enzyme 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, mediated by the 14-3-3 family of adaptor proteins (6). These proteins associate to form homo- and heterodimers with a saddleshaped structure, with each monomer possessing 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, including 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 suggested for c-Raf (6) and Bcr (68). Furthermore, assocation with targets such as c-Raf and Ksr requires recognition of a phosphoserine residue contained within the consensus motif RSX-pSer-XP, in a fashion reminiscent of SH2 domain interactions with pTyr-containing motifs (69).

An important function for 14-3-3 proteins 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 during 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 mammalian 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 phosphorylated nitrate reductase to control nitrogen metabolism in spinach (72). These results suggest that 14-3-3 proteins exert biological effects through regulating oligomeric protein-protein interactions and protein 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 protein-phospholipid interactions, commonly mediated by conserved protein domains. Such protein modules have apparently developed 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 recognition modules—as found in adaptors, anchoring 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 increase 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 association 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 activation, these domains are liberated for association with substrates and cytoskeletal elements. Consequently, modular domains that participate in tyrosine kinase signaling act to localize proteins to specific subcellular sites, to control enzyme activity, to direct the formation of multiprotein complexes, and to directly transduce signals.

The Ser-Thr kinases and phosphatases use a variation on this theme whereby the enzymes appear to be constitutively targeted and colocalized with their substrate at on May 28, 2009 w.sciencemag.org Downloaded from 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 phosphorylation, may directly regulate modular protein-protein interactions. Now that the intricacy of these interactions is understood, the challenge ahead is to understand both the physiological functions and regulation of such signaling networks.

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