Target-assisted iterative screening applied to random peptide libraries unveiled a novel and atypical recognition consensus shared by CIN85/SETA/Ruk SH3 domains, PX(P/A)XXR. Confirmed by mutagenesis and in vitro binding experiments, the novel consensus allowed for the accurate mapping of CIN85 SH3 binding sites within known CIN85 interactors, c-Cbl, BLNK, Cbl-b, AIP1/Alix, SB1, and CD2 proteins, as well as the prediction of CIN85 novel-interacting partners in protein databases. Synaptojanin 1, PAK2, ZO-2, and TAF II 70, which contain CIN85 SH3 recognition consensus sites, were selectively precipitated from mouse brain lysates by CIN85 SH3 domains in glutathione S-transferase pulldown experiments. A direct interaction of synaptojanin 1 and PAK2 with CIN85 SH3 domains was confirmed by Far Western blotting.One of the initial approaches to study a novel gene product is the identification of its interacting partners. As increasingly more proteins become functionally characterized it is more and more probable that the identification of interacting partners of a novel protein will suggest a possible function for this protein by associative reasoning and, thus, provide a framework for its characterization. Using this approach, a novel CIN85/CMS adaptor protein family was functionally implicated in a remarkably wide spectrum of different cellular processes such as the down-regulation of receptor tyrosine kinases and endocytosis (through the association of CIN85 with Cbl/Cbl-b and endophilins) (1, 2), apoptosis (through the association of CIN85 with p85-phosphatidylinositol 3-kinase and AIP1/Alix) (3, 4), and adhesion phenomena (through the association of CMS with p130Cas and the interactions of CD2AP/CMS with CD2 and cortactin) (5-7). However, the mapping of CIN85/CMS SH3 domains binding sites within the characterized CIN85/CMS interactors has remained an elusive goal, giving rise to discordant conclusions in the literature. The absence of information on the recognition properties of CIN85 SH3 domains hampers the further characterization of its interactions and the understanding of the biological role of this protein, which has emerged as a highly connected node linking functionally diverse protein interaction sub-networks. Intriguingly, it has been suggested that CIN85/CMS may have unique function(s) in higher eukaryotes, as CIN85 orthologues of the human and rat proteins were not found in Caenorhabditis elegans, Drosophila melanogaster, or yeast (8).At least five discrete protein interaction modules can be inferred from the analysis of the CIN85 amino acid sequence. These are three consecutive SH3 domains at the N terminus followed by a central proline-rich region and a coiled-coil domain situated at the C terminus (Fig. 1). CIN85 was shown to homodimerize through its coiled-coil domains, and a number of proteins interacting with the CIN85 proline-rich region were identified including Grb2, p130Cas , endophilins, p85-phosphatidylinositol 3-kinase, and cortactin (1,3,7,9,10). The SH3 domains of CIN85 bound c-Cbl, Cbl-b,...

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The present work is intended to demonstrate that most of the paradoxes, controversies, and contradictions accumulated in molecular and cell biology over many years of research can be readily resolved if the cell and living systems in general are re-interpreted within an alternative paradigm of biological organization that is based on the concepts and empirical laws of nonequilibrium thermodynamics. In addition to resolving paradoxes and controversies, the proposed re-conceptualization of the cell and biological organization reveals hitherto unappreciated connections among many seemingly disparate phenomena and observations, and provides new and powerful insights into the universal principles governing the emergence and organizational dynamics of living systems on each and every scale of biological organizational hierarchy, from proteins and cells to economies and ecologies.

Syntrophins, a family of intracellular peripheral membrane proteins of the dystrophin-associated protein complex (DAPC), each contain a single PDZ domain. Syntrophin PDZ domains bind C-terminal peptide sequences with the consensus R/K-E-S/T-X-V-COOH, an interaction that mediates association of skeletal muscle sodium channels with the DAPC. Here, we have isolated cyclic peptide ligands for syntrophin PDZ domains from a library of combinatorial peptides displayed at the N terminus of protein III of bacteriophage M13. Affinity selection from a library of X 10 C peptides yielded ligands with the consensus X-(R/K)-E-T-C-L/M-A-G-X-⌿-C, where ⌿ represents any hydrophobic amino acid. These peptides contain residues (underlined) similar to the C-terminal consensus sequence for binding to syntrophin PDZ domains and bind to the same site on syntrophin PDZ domains as C-terminal peptides, but do not bind to other closely related PDZ domains. PDZ binding is dependent on the formation of an intramolecular disulfide bond in the peptides, since treatment with dithiothreitol, or substitution of either of the two cysteines with alanines, eliminated this activity. Furthermore, amino acid replacements revealed that most residues in the phage-selected peptides are required for binding. Our results define a new mode of binding to PDZ domains and suggest that proteins containing similar conformationally constrained sequences may be ligands for PDZ domains. PDZ1 domains are 80 -90-amino acid modules present in numerous eukaryotic proteins. They were first described as a series of three internal, repeated elements within the postsynaptic density (PSD)-95 protein (1). In fact, the name PDZ is derived from three proteins first recognized to contain repeats of this domain: PSD-95; the Drosophila discs-large tumor suppressor protein, Dlg; and the mammalian tight-junction protein zona occludens-1, ZO-1 (2-5). PDZ domains have since been identified in a large number of multifunctional proteins, many of which are associated with specialized regions of cell to cell contact such as tight junctions, septate junctions, and synaptic junctions (6). The PDZ domain may be an evolutionarily old domain, as it has been detected in mammalian, nematode, yeast, plant, and bacterial genomic sequences by computer analysis (7).PDZ domains mediate protein-protein interactions by at least two distinct mechanisms. Certain PDZ domains bind directly to specific recognition sequences at the C terminus of transmembrane proteins. For example, the second PDZ domain of PSD-95 interacts with an S/T-X-V-COOH motif in N-methyl-D-aspartate receptor 2B subunits (8, 9) and in Shaker-type potassium channels (10). PDZ domains can also form heterotypic dimers with other PDZ domains. For instance, the Nterminal region of nNOS, which itself contains a PDZ domain, binds directly to PDZ domains in both PSD-95 and ␣ 1 -syntrophin, a component of the dystrophin-associated protein complex (DAPC) (11). A third possible mode of interaction, in which the consensus binding sequence is locat...

Infected cell protein 0 (ICP0) is a 775-residue multifunctional herpes simplex virus protein associated with numerous functions related to transactivation of gene expression and repression of host defenses to infection. We report that an uncharted domain of ICP0 located between residues 245 and 510 contains multiple SH3 domain binding motifs similar to those required for binding to CIN85, the M r 85,000 protein that interacts with Cbl. CIN85 and Cbl are involved in endocytosis and negative regulation of numerous receptor tyrosine kinases. We report that ICP0 binds CIN85 in a reciprocal manner and that the complexes pulled down by ICP0 also contain Cbl. We tested the role of ICP0 in the down-regulation of receptor tyrosine kinases by using epidermal growth factor receptor (EGFR) as a prototypic receptor. In transfection assays, ICP0, in the absence of other viral genes, down-regulated EGF-dependent expression of a reporter gene (luciferase). ICP0 also down-regulated both total and cell surface levels of EGFR in EGF-independent manner. In wild-type virus-infected cells, the surface levels of EGFR were also decreased in the absence of EGF stimulation. Stimulation by EGF enhanced the decrease in surface EGFR. We conclude that ICP0 encodes SH3 domain binding sites that function to downregulate signaling pathways associated with receptor tyrosine kinases. The results suggest that ICP0 precludes signaling to the infected cells through the receptor tyrosine kinases.endocytosis ͉ receptor tyrosine kinase ͉ ubiquitin ligase T he infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is a multifunctional 775-aa protein expressed immediately after infection. As reviewed in detail elsewhere, in transduced cells, ICP0 enhances the expression of genes introduced by infection or transfection (reviewed in ref. 1). In infected cultured cells, ICP0 plays an important role at low multiplicities of infection but no obvious role at high multiplicities.ICP0 does play a key role in enabling viral replication and spread in experimental animal systems. ICP0 has been shown to interact with a large number of cellular proteins. Among these proteins are cyclin D3, cdc34, the transcriptional factor BMAL1, the translation elongation factor 1␦, and the ubiquitin specific protease USP7 (1). ICP0 is encoded in three exons of 19, 221, and 533 codons, respectively. The functions identified to date map in sequences encoded by exon 2 and the carboxyl terminal 160 amino acids of exon 3.In this article, we report a function of ICP0 mapping in the uncharted domain of sequences encoded in exon 3. Specifically, we report that ICP0 contains several putative SH3 domain binding sites. The recurrent motif of these binding sites conforms to the PX(P͞A)XXR recognition consensus of SH3 domains of CIN85 (2), the M r 85,000 adapter protein that binds Cbl, a multifunctional oncoprotein that also functions as an E3 ubiquitin ligase (ref. 3 and references therein). We thus report that ICP0 binds CIN85 in a reciprocal manner and that the complex containing I...

The cell, as a molecular system, is often interpreted in terms of complex clockworks, and the design charts of mechanical and electrical engineering are assumed to provide adequate approximations for the description of cellular organization. However, a growing body of experimental evidence obtained through the observation and analysis of real-time dynamics of fluorescently labeled molecules inside living cells is increasingly inconsistent with the classico-mechanistic perception of the cell. An overview of recent studies favors an emerging alternative image of the cell as a dynamic integrated system of interconnected and interdependent metastable molecular organizations realized through stochasticity and self-organization.

One of the major goals of researchers in the field of apoptosis is to understand the molecular mechanisms of the various components of the apoptotic pathways, with the hope to identify targets for novel cancer therapies. The discovery of a TNF‐related, apoptosis‐inducing ligand, TRAIL, that kills transformed cells with great specificity in vitro, has provided the hope that TRAIL may be used to induce cell death in tumor cells without affecting normal tissues. However, TRAIL signaling is very complex and a clear understanding of its function is necessary before it can be used in cancer therapy. Complexity of TRAIL‐induced signaling is apparent from its ubiquitous expression, its ability to interact with five receptors, and its tumor‐selective induction of apoptosis. The signaling events that mediate the tumor selectivity of TRAIL‐induced apoptosis and the biological functions of each of the TRAIL receptors are not well characterized. This review will focus on the complexity of TRAIL and the role of c‐FLIP in mediating TRAIL function.

Once the sequence of a genome is in hand, understanding the function of its encoded proteins becomes a task of paramount importance. Much like the biochemists who first outlined different biochemical pathways, many genomic scientists are engaged in determining which proteins interact with which proteins, thereby establishing a protein interaction network. While these interactions have evolved in regard to their specificity, affinity and cellular function over billions of years, it is possible in the laboratory to isolate peptides from combinatorial libraries that bind to the same proteins with similar specificity, affinity and primary structures, which resemble those of the natural interacting proteins. We have termed this phenomenon`convergent evolution'. In this review, we highlight various examples of convergent evolution that have been uncovered in experiments dissecting protein^protein interactions with combinatorial peptides. Thus, a fruitful approach for mapping protein^protein interactions is to isolate peptide ligands to a target protein and identify candidate interacting proteins in a sequenced genome by computer analysis. ß