Principles of cell signaling - UT Southwestern

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39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 600 activated protein kinase (MAPK) cascade, including a MAP3K (Ste11p), a MAP2K (Ste7p) and a MAPK (Fus3p). (The MAPK cascade will be discussed in 14.32 MAPKs are central to many signaling pathways). The function of Ste5p is partially retained even if the positions of its binding sites for the kinases are shuffled in the linear sequence of the protein, indicating that a major role is to bring the enzymes into proximity, rather than to precisely orient them. Ste5p also binds to the subunits of the heterotrimeric G protein that mediates the actions of mating pheromones, linking the membrane signal to the intracellular transducers. Yeast that lack Ste5p cannot mate, demonstrating that Ste5p is required for this biological function (but not all functions) carried out by the pathway. In addition to facilitating signaling in their own pathways, scaffolds can enhance signaling specificity by limiting interactions with other signaling proteins. Scaffolds thus insulate components of a signaling pathway both from activation by inappropriate signals and from producing incorrect outputs. For example, the mating and osmosensing pathways in yeast share several components, including the MAP3K Ste11p, but each pathway maintains specificity because it employs different scaffolds that restrict signal transmission. In contrast, the presence of excess scaffold can inhibit signaling because the individual signaling components will more frequently bind to distinct scaffold proteins rather than forming a functional complex. Such dilution among scaffolds causes separation rather than concentration of the components, preventing their productive interaction. 14.9 Independent, modular domains specify proteinprotein interactions Key concepts • Protein interactions may be mediated by small, conserved domains. • Modular interaction domains are essential for signal transmission. • Adaptors consist exclusively of binding domains or motifs. Modular protein interaction domains or motifs occur in many signaling proteins and confer the ability to bind structural motifs in other molecules, including proteins, lipids, and nucleic acids. Some of these domains are listed in FIGURE 14.9. In contrast to scaffolds, which bind specific proteins with considerable selectivity, modular interaction domains generally recognize not a single molecule but a group of targets that share related structural features. Modular interaction domains important for signal transduction were first discovered in the protein tyrosine kinase proto-oncogene Src, which contains a protein tyrosine kinase domain and two domains named Src homology (SH) 2 and 3 domains. The modular SH2 and SH3 domains were originally identified by comparison of Src to two other tyrosine kinases, Fps and Abl. One or both of these domains appear in numerous proteins and both are critically involved in protein-protein interactions. SH3 domains, which consist of approximately 50 residues, bind to specific short proline-rich sequences. Many cytoskeletal proteins and proteins found in focal adhesion complexes contain SH3 domains and proline rich sequences, suggesting that this targeting motif may send proteins with these domains to these sites of action within cells. In contrast to phosphotyrosine-SH2 binding, the proline-rich binding sites for SH3 domains are present in resting and activated cells. However, SH3-proline interactions may be negatively regulated by phosphorylation within the proline-rich motif. SH2 domains, which consist of approximately 100 residues, bind to Tyr phosphorylated proteins, such as cytoplasmic tyrosine kinases and receptor tyrosine kinases. Thus, Tyr phosphorylation regulates the appearance of SH2 binding sites and, thereby, regulates a set of protein-protein interactions in a stimulusdependent manner. A clever strategy was used to identify the binding specificity of SH2 domains. An isolated recombinant SH2 domain was incubated with cell lysates and then recovered from the lysates using a purification tag. The proteins associated with the SH2 domain were some of the same proteins that were recognized by antiphosphotyrosine antibodies. By this and other methods, it was discovered that SH2 domains recognize sequences surrounding Tyr phosphorylation sites and require phosphorylation of the included Tyr for high affinity binding. Information on specific amino acid sequences that recognize and bind to modular binding domains is being accumulated as these individual interactions are identified. In addition, screening programs using cDNA and/or peptide libraries to assess binding capabilities 600 CHAPTER 14 Principles of cell signaling
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 601 Characteristics of some common modular protein domains Domain Characteristics Cellular involvement 14-3-3 Binds protein phosphoserine or phosphothreonine Bromo CARD C1 C2 EF hand F-Box FHA HECT LIM PDZ PH RING SAM SH2 Binds acetylated lysine residues Dimerization Binds phorbol esters or diacylglycerol Binds phospholipids Binds calcium Binds Skp1 in a ubiquitin-ligase complex Binds protein phosphothreonine or phosphoserine Binds E2 ubiquitin-conjugating enzymes to transfer ubiquitin to the substrate or to ubiquitin chains Zinc-binding cysteine-rich motif that forms two tandemly repeated zinc fingers Binds to the C-terminal 4-5 residues of proteins that have a hydrophobic residue at the terminus; may bind to PIP2 Binds to specific phosphoinositides, esp. PI-4,5-P 2 , PI-3,4-P 2 or PI-3,4,5-P 3 . Binds zinc and may be found in E3 ubiquitin ligases Homo- and heterooligomerization Binds to protein phosphotyrosine (pY) Protein sequestration Chromatin-associated proteins Caspase activation Recruitment to membranes Signal transduction, vesicular trafficking Calcium-dependent processes Ubiquitination Various; DNA damage FYVE Binds to PI(3)P Membrane trafficking, TGF- signaling Ubiquitination Wide variety of processes Scaffolding diverse protein complexes often at the membrane Recruitment to membranes and motility Ubiquitination, transcription Wide variety of processes Tyrosine protein kinase signaling SH3 Binds to PXXP motifs Various processes TPR WW Degenerate sequence of ~34 amino acids with residues WL/GYAFAP; forms a scaffold Binds proline-rich sequences Wide variety of processes Alternative to SH3; vesicular trafficking FIGURE 14.9 The table describes a subset of known modular protein interaction domains found in many proteins. Interactions mediated by these domains are essential to controlling cell function. Few if any of these domains exist in prokaryotes. Adapted from the Pawson Lab, Protein Interaction Domains, Mount Sinai Hospital (http://pawsonlab.mshri.on.ca/). yield such motifs. Consensus target sequences for individual domains have been identified based on the sequence specificity of their binding to arrayed sequences. These consensus sequences can then be used to predict whether the domain will bind a site in a candidate protein. Adaptor proteins, which lack enzymatic activity, link signaling molecules and target them in a manner that is responsive to extracellular signals. Adaptor proteins are generally made up of two or more modular interaction domains or the complementary recognition motifs. Unlike scaffolds, adaptors are usually 14.9 Independent, modular domains specify protein-protein interactions 601
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Principles of cell signaling - UT Southwestern

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