Acommon focus among molecular and cellular biologists is the identification of proteins that interact with each other. Yeast two-hybrid, cDNA expression library screening, and coimmunoprecipitation experiments are powerful methods for identifying novel proteins that bind to one's favorite protein for the purpose of learning more regarding its cellular function. These same techniques, coupled with truncation and mutagenesis experiments, have been used to define the region of interaction between pairs of proteins. One conclusion from this work is that many interactions occur over short regions, often less than 10 amino acids in length within one protein. For example, mapping studies and 3-dimensional analyses of antigen-antibody interactions have revealed that epitopes are typically 4-7 residues long (1). Other examples include protein-interaction modules, such as Src homology (SH) 2 and 3 domains, phosphotyrosine binding domains (PTB), postsynaptic density/disc-large/ZO1 (PDZ) domains, WW domains, Eps15 homology (EH) domains, and 14-3-3 proteins that typically recognize linear regions of 3-9 amino acids. Each of these domains has been the subject of recent reviews published elsewhere (2 3 4 5 6 7). Among the primary structures of many ligands for protein-protein interactions, the amino acid proline is critical. In particular, SH3, WW, and several new protein-interaction domains prefer ligand sequences that are proline-rich. In addition, even though ligands for EH domains and 14-3-3 domains are not proline-rich, they do include a single proline residue. This review highlights the analysis of those protein-protein interactions that involve proline residues, the biochemistry of proline, and current drug discovery efforts based on proline peptidomimetics.-Kay, B. K., Williamson, M. P., Sudol, M. The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains.

Abstract. GPI-linked protein molecules become Triton-insoluble during polarized sorting to the apical cell surface of epithelial cells. These insoluble complexes, enriched in cholesterol, glycolipids, and GPIlinked proteins, have been isolated by flotation on sucrose density gradients and are thought to contain the putative GPI-sorting machinery. As the cellular origin and molecular protein components of this complex remain unknown, we have begun to characterize these low-density insoluble complexes isolated from MDCK cells. We find that these complexes, which represent 0.4-0.8 % of the plasma membrane, ultrastructurally resemble caveolae and are over 150-fold enriched in a model GPI-anchored protein and caveolin, a caveolar marker protein. However, they exclude many other plasma membrane associated molecules and organellespecific marker enzymes, suggesting that they represent microdomains of the plasma membrane. In addition to caveolin, these insoluble complexes contain a subset of hydrophobic plasma membrane proteins and cytoplasmicaUy-oriented signaling molecules, including: (a) GTP-binding proteins-both small and heterotrimeric; (b) annexin N-an apical calcium-regulated phospholipid binding protein with a demonstrated role in exocytic fusion events; (c) c-Yes-an apically localized member of the Src family of non-receptor type protein-tyrosine kinases; and (d) an unidentified serine-kinase activity. As we demonstrate that caveolin is both a transmembrane molecule and a major phospho-acceptor component of these complexes, we propose that caveolin could function as a transmembrahe adaptor molecule that couples luminal GPIlinked proteins with cytoplasmically oriented signaling molecules during GPI-membrane trafficking or GPImediated signal transduction events. In addition, our results have implications for understanding v-Src transformation and the actions of cholera and pertussis toxins on hetero-trimeric G proteins.

We have used an affinity purification method to identify substrates of protein kinase B/Akt. One protein that associates with 14-3-3 in an Akt-dependent manner is shown here to be the Yes-associated protein (YAP), which is phosphorylated by Akt at serine 127, leading to binding to 14-3-3. Akt promotes YAP localization to the cytoplasm, resulting in loss from the nucleus where it functions as a coactivator of transcription factors including p73. p73-mediated induction of Bax expression following DNA damage requires YAP function and is attenuated by Akt phosphorylation of YAP. YAP overexpression increases, while YAP depletion decreases, p73-mediated apoptosis following DNA damage, in an Akt inhibitable manner. Akt phosphorylation of YAP may thus suppress the induction of the proapoptotic gene expression response following cellular damage.

The WW domain is a new protein module with two highly conserved tryptophans that binds proline-rich peptide motifs in vitro. It is present in a number of signalling and regulatory proteins, often in several copies. Here we investigate the solution structure of the WW domain of human YAP65 (for Yes kinase-associated protein) in complex with proline-rich peptides containing the core motif PPxY. The structure of the domain with the bound peptide GTPPPPYTVG is a slightly curved, three-stranded, antiparallel beta-sheet. Two prolines pack against the first tryptophan, forming a hydrophobic buckle on the convex side of the sheet. The concave side has three exposed hydrophobic residues (tyrosine, tryptophan and leucine) which form the binding site for the ligand. A non-conserved isoleucine in the amino-terminal flanking region covers a hydrophobic patch and stabilizes the WW domain of human YAP65 in vitro. The structure of the WW domain differs from that of the SH3 domain and reveals a new design for a protein module that uses stacked aromatic surface residues to arrange a binding site for proline-rich peptides.

The WW domain has previously been described as a motif of 38 semiconserved residues found in seemingly unrelated proteins, such as dystrophin, Yesassociated protein (YAP), and two transcriptional regulators, Rsp-5 and FE65. The molecular function of the WW domain has been unknown until this time. Using a functional screen of a cDNA expression library, we have identified two putative ligands of the WW domain of YAP, which we named WBP-1 and WBP-2. Peptide sequence comparison between the two partial clones revealed a homologous region consisting of a proline-rich domain followed by a tyrosine residue (with the shared sequence PPPPY), which we shall call the PY motif. Binding assays and site-specific mutagenesis have shown that the PY motif binds with relatively high affinity and specificity to the WW domain of YAP, with the preliminary consensus XPPXY being critical for binding. Herein, we have implicated the WWV domain with a role in mediating protein-protein interactions, as a variant of the paradigm set by Src homology 3 domains and their proline-rich ligands.The progressive elucidation of the function of Src homology (SH) 2 and SH3 domains, in addition to the identification of their ligands, has in the recent years spawned an era of remarkable developments in the fields of signal transduction and molecular oncology (1-3). As a result of this trend, two new domains, the pleckstrin homology domain and the phosphotyrosine interaction domain, have been identified and are extensively studied in the context of signaling pathways (4-8).The recognition of the existence of modular protein-binding domains has proven to be critical in understanding the intricacies of signal transduction where a variety of events occur. For example, proteins transiently interact with their upstream and downstream partners, substrates are brought into proximity of their catalytic centers, or proteins are localized to their subcellular compartments through the interaction of these domains with their ligands (9).The SH2 domain was first described as a conserved sequence of 100 amino acids in the noncatalytic domain of cytoplasmic tyrosine kinases such as Src and Fps, but it rapidly became recognized in other molecules involved in signaling pathways and cellular transformation including Crk, Ras-GAP, and phospholipase C-,y. When the SH2 domain was later shown to bind with high affinity to phosphotyrosine residues, it was quickly realized that the SH2 domain represented a means by which many molecules sharing a common signaling pathway may associate to regulate signal propagation from the extracellular environment into the cell and its nucleus (10). In contrast, the SH3 domain was originally identified as a 50-amino-acid-long region of homology shared between the Crk and Src oncogene products and phospholipase C-,y (11, 12).Various studies have shown that SH3 domains may mediate protein localization to the plasma membrane or the cytoskeleton, as exemplified by the interaction between Grb2 and Sos that transduces signals from cell surfa...

The ErbB-4 receptor protein-tyrosine kinase is proteolytically processed by membrane proteases in response to the ligand or 12-O-tetradecanoylphorbol-13-acetate stimulation resulting in the cytoplasmic fragment translocating to the cell nucleus. The WW domain-containing co-transcriptional activator Yes-associated protein (YAP) associates physically with the full-length ErbB-4 receptor and functionally with the ErbB-4 cytoplasmic fragment in the nucleus. The YAP⅐ErbB4 complex is mediated by the first WW domain of YAP and the most carboxyl-terminal PPXY motif of ErbB-4. In human tissues, we documented the expression of YAP1 with a single WW domain and YAP2 with two WW domains. It is known that the COOH-terminal fragment of ErbB4 does not have transcriptional activity by itself; however, we show here that in the presence of YAP its transcriptional activity is revealed. There is a difference in the extent of transactivation activity among YAP isoforms: YAP2 is the stronger activator compared with YAP1. This transactivation is abolished by mutations that abrogate the YAP⅐ErbB4 complex formation. The unphosphorylatable mutation that increases the nuclear localization of YAP increases transcription activity. The COOH-terminal fragment of ErbB-4 and full-length YAP2 overexpressed in cells partially co-localize to the nucleus. Our data indicate that YAP is a potential signaling partner of the full-length ErbB4 receptor at the membrane and of the COOH-terminal fragment of ErbB-4 that translocates to the nucleus to regulate transcription.Cells are continuously exposed to diverse stimuli ranging from soluble paracrine and endocrine factors to signaling molecules on neighboring cells. These extracellular signals are transduced to cell nuclei to achieve an appropriate developmental or proliferative response. Receptor protein-tyrosine kinases play pivotal roles in this process. Upon binding of their cognate ligands, the intrinsic protein-tyrosine kinase activity of the receptor is significantly elevated and initiates a network of signaling pathways including the well characterized Ras/mitogen-activated protein kinase and the signal transducers and activators of transcription pathways (1-3).

Due to its small size and compact fold, the WW domain became an attractive model for studies of protein stability and design [7^11]. Speci¢c residues have been identi¢ed that play a critical role in the structure and function of the domain and also in modulating its stability. In fact, the WW domain is the ¢rst protein module that has been successfully designed de novo, demonstrating the signi¢cant insight we already have regarding its fold [12]. Besides, the WW domain sequence is well conserved in length, even in its loops, which is a remarkable feature of this domain, compared with others, making protein modeling a useful tool for generating three-dimensional representations of their sequences. Nevertheless, attempts to predict binding targets for a speci¢c WW domain sequence or even for one of its subgroups or classes, with a good probability, have not been made so far.Based on the pattern of semi-conserved residues, WW domain sequences have been classi¢ed into three groups as described previously [12]. Group I contains the C-terminal tryptophan and the N-terminal proline, Group II sequences lack the N-terminal proline and ¢nally Group III with sequences without the second tryptophan. In another classi¢cation, based on the ligand predilection, WW domains were divided into two major and two minor groups [5]. One major group (Group I) binds polypeptides with the minimal core consensus PPxY, whereas the other binds ligands with the PPLP motif usually embedded in a long stretch of prolines (Group II). Group III WW domains select poly-P motifs £anked by R or K, whereas Group IV WW domains bind to short sequences with phospho-S or phospho-T followed by P, in a phosphorylation-dependent manner [5]. A sequence alignment of some selected WW sequences combining binding preferences and sequence conservation is shown in Fig. 1.In this contribution we will review the structural characteristics of WW domain^ligand complexes determined so far. On the basis of four WW domain structures in complex with di¡erent peptides and two structures of free WW domains [3,12^16], a three-dimensional structure has been modeled for the Npw38 WW domain that allows us to compare binding properties of WW and SH3 domains. WW domain as a phosphate-dependent SH3 domain?WW domains have the ability to bind proline-rich cores and/or phospho-SP/phospho-TP-containing motifs [5]. It is interesting that such a small and well-conserved module has a surprisingly large repertoire of potential ligands. The dissociation constants (K d ) for WW^ligand complexes lie in the high nM to low mM range for proline-rich ligands, and in the low mM range for phospho-SP-or phospho-TP-containing ligands [5].

The Hippo pathway in Drosophila controls the size and shape of organs. In the fly, activation of this pathway conveys growthinhibitory signals and promotes apoptosis in epithelial cells. We "reconstituted" the Hippo pathway in a human epithelial cell line and showed that, in contrast to flies, the activation of this pathway results in anti-apoptotic signals. We have shown that in human embryonic kidney (HEK) 293 cells, the complex formation between transcriptional co-activators YAPs (Yes kinase-associated proteins) and Lats kinases requires the intact WW domains of YAPs, as well as intact Pro-Pro-AA-Tyr (where AA is any amino acid) motifs in Lats kinases. These kinases cooperate with the upstream Mst2 kinase to phosphorylate YAPs at Ser-127. Overexpression of YAP2 in HEK293 cells promoted apoptosis, whereas the Mst2/Lats1-induced phosphorylation of YAP partially rescued the cells from apoptotic death. Apoptotic signaling of YAP2 was mediated via stabilization of p73, which formed a complex with YAP2. All components of the Hippo pathway that we studied were localized in the cytoplasm, with the exception of YAP, which also localized in the nucleus. The localization of YAP2 in the nucleus was negatively controlled by the Lats1 kinase. Our apoptotic "readout" of the Hippo pathway in embryonic kidney cells represents a useful experimental system for the identification of the putative upstream receptor, membrane protein, or extracellular factor that initiates an entire signaling cascade and ultimately controls the size of organs.