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The interest in biosensor-based ligand isolation strategies is to a large extent, a result of fundamental changes in the way novel proteins are isolated and characterized. Advances in genetic screening techniques have led to the identification of novel protein families with little knowledge of function or physiological context (1,2) and a rapidly increasing number of these so-called "orphan proteins" calls for complementary strategies to elucidate their structure and function. Over the past few years, different approaches have been developed to isolate ligands solely on the basis of their affinity for a particular orphan receptor. These include expression cloning strategies to detect cell-membrane bound ligands with tagged receptor extracellular domain (ECD) fusion proteins (3-6) and cell rescue assays to detect soluble ligands (7-9). BIAcore technology has been applied to search for suitable ligand sources of orphan receptors (10-12), but is also well suited to integrated use within a "classical" purification scheme (13,14) and has been exploited as an affinity detector to facilitate the isolation of orphan proteins (15,16). The advantages of this technology include a fast, robot-driven assay that is unaffected by sample toxicity, and the ability to assess specificity and kinetics of the observed interaction even in relatively crude samples, thus minimizing the risk of false positives. [ABSTRACT FROM AUTHOR]

Receptor tyrosine kinases (RTKs) bind to their ligands with high affinity and specificity. Soluble receptor approaches exploit these biological properties to make affinity probes that can be used to detect or to purify the cognate ligands (1,2). In many respects, these soluble receptor reagents resemble antibodies, and they can be used in almost all the same types of procedure. They can also have important advantages over antibodies. They can be used to identify and clone previously unknown ligands of orphan receptors (1-9). They can be produced much more quickly than antibodies. Also, because they exploit natural receptor-ligand interactions, they can give information not available with antibodies, for example permitting quantitative characterization of ligandreceptor binding interactions (1,2,10), or allowing the simultaneous detection of multiple cross-reacting ligands in an embryo (5,11,12). [ABSTRACT FROM AUTHOR]

The interaction of activated growth factor receptors with signaling molecules has attracted enormous attention in the last few years because of the central role these processes play in growth differentiation and proliferation control in cells. The importance of src homology (SH)2 domains in mediating these interactions through binding to specific tyrosine phosphorylation sites on activated receptors has been well established. However, the complexity of intracel-lular signaling, and the apparent redundancy in some systems, has necessitated the development of assays for quantifying these interactions accurately. These in vitro studies have benefited from the linear nature of SH2 domain ligands, i.e., the ability of small synthetic phosphopeptides corresponding to receptor autophosphorylation sites to mimic, in most cases, the specificity and binding properties of the intact receptor. Several assays have been developed employing radioactive peptides or indirect detection of SH2 domains by immunological methods. These assays can determine, with varying degrees of accuracy, the equilibrium dissociation constant, KD (or binding constant, KB = 1/KD) for an interaction, thus providing quantification of its strength. However, additional information about the kinetic rates and thermodynamics can provide a better characterization. Correlated with high-resolution structural detail, these data can provide insight into the balance of bonds that have been made or broken, whether the molecules have undergone conformational change on interacting, or whether water has been released from the binding surface on formation of the complex. Information of this nature can add a further level of detail, helping to address issues such as the specificity of an interaction and the structural-thermodynamic relationship associated with forming the complex, all of which are factors that might be important when attempting pharmaceutical intervention. The use of isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) biosensors has provided a great deal of information on these issues. [ABSTRACT FROM AUTHOR]

The yeast two-hybrid system has been utilized by many laboratories in the characterization of protein-protein interactions that occur in eukaryotic and prokaryotic cell systems. In addition, the two-hybrid system has been used to isolate and characterize novel interacting proteins to aid in the understanding of biochemical signaling pathways for various receptors and enzymes. The interacting components are fused to inert components of the transcriptional apparatus. One of the components is fused to a DNA-binding domain and is generally referred to as the "bait." The second protein is fused to a transcriptional activation domain and is referred to as the "fish" or the "prey." When there is an association between the "bait" and the "prey," the DNA-binding domain and the transcriptional-activation domain are brought into close proximity to activate the transcription of the reporter gene. [ABSTRACT FROM AUTHOR]

Protein-protein interactions form the basis of most signal transduction pathways. In the case of pathways initiated by receptor tyrosine kinases (RTKs), phosphotyrosine residues present on the cytoplasmic domain of the activated receptor form docking sites for adaptor molecules and enzymes containing src homology (SH)2 and phosphotyrosine binding (PTB) domains (1). Association of these signaling components initiates cascades of interactions and reactions that determine the cell's response to the stimulus. Because these initial interactions are responsible for the biological properties of different receptors, there has been considerable interest in identifying potential binding proteins with a view to understanding the molecular basis of signaling diversity. [ABSTRACT FROM AUTHOR]

Activation of receptor tyrosine kinases (RTXs) leads to autophosphorylation of the intracellular region of the receptor on specific tyrosine residues, thus creating binding sites for a variety of signaling proteins (1). These interactions are mediated by protein modules such as src homology (SH)2 and phosphotyrosine binding (PTB) domains, which target specific tyrosine phosphorylated peptide sequences (2). This chapter describes how the physical association of an RTK with particular substrates can be exploited in an expression cloning procedure and novel receptor targets identified. [ABSTRACT FROM AUTHOR]

Growth factor receptors endowed with intrinsic tyrosine-kinase activity (receptor tyrosine kinases, RTKs) are capable of intracellular signal transduction through their ability to autophosphorylate and to phosphorylate cellular proteins, globally referred to as substrates (refs. 1-3 and references therein). [ABSTRACT FROM AUTHOR]

The far Western gel overlay assay is a highly sensitive tool for the detection of direct protein-protein interactions. The ability to distinguish direct associations between two proteins makes this technique ideal not only for identifying potential binding partners of a protein, but also for characterizing interaction domains and binding sites. The assay relies on the ability of the protein of interest to interact with target proteins that have been immobilized on nitrocelulose filters. Potential interactions can be monitored under various types of conditions by manipulation of the protein that is used as the probe. Such manipulations may include mutagenesis, or protein modifications such as phosphorylation. Detection of phosphorylation-dependent interactions is particularly relevant in light of the fact that many signaling proteins are modified by phosphorylation, which in turn affects activity, localization, and most importantly, the ability to form protein complexes (seerefs. 1-4). We describe here the standard protocol for the far-Western gel overlay assay, with modifications that enable the detection of phosphorylation-dependent protein interactions. This technique has been used successfully by our laboratory, as well as others to identify potential phosphorylation-dependent interactions between a number of signaling proteins (5-7). [ABSTRACT FROM AUTHOR]

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is fast becoming the marker of choice for protein localization and gene expression studies. The rapid rise in popularity stems from several advantages over traditional markers such as β-galactosidase and alkaline phosphatase, which typically require the addition of exogenous substrates for detection. The unique intrinsic spectral properties of GFP allow it to be directly visualized in fixed or live cells by fluorescence microscopy without the addition of cofactors or exogenous substrates (1-8). [ABSTRACT FROM AUTHOR]

The activation of T cells results in both tyrosine and serine/threonine phos-phorylations of a number of substrates within minutes of T-cell receptor (TCR) ligation (1,2). The phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) on the (ξ-chain within the TCR complex act as binding sites for the N-terminal SH2 domains of ZAP-70 (3). This relocation brings ZAP-70 proximal to the CD4-associated p56lck kinase, which activates the kinase activity of ZAP-70 by phosphorylation at Y493 (4). The activated ZAP-70 is then able to transduce a signal via an as yet uncharacterized cascade, which may involve SLP-76 (5), vav (6) PLC-Γ( (7) p120/130 (8) and LAT (9). The major role of ZAP-70 in T-cell signal transduction has been highlighted in ZAP-70 deficient patients who suffer severe combined immunodeficiency disease (SCID) like symptoms (10). [ABSTRACT FROM AUTHOR]

Protein phosphorylation on histidine occurs in a large number of processes in prokaryotes and in an unknown number of processes in eukaryotes. The functions of protein phosphohistidine in prokaryotes are much more clearly understood (1,2. The methods presented here have been developed for the study of protein histidine phosphorylation in eukaryotes where phosphohistidine is found as an intermediate in some enzyme reactions and may also be involved in signaling mechanisms (3). In prokaryotes, two types of protein phosphorylation on histidine have been characterized in detail. The first, historically, is the phosphoenolpyruvate-sugar phosphotransferase system (4,5). In this system, phosphohistidine acts as a phosphate donor and the initial phosphate donor is phosphoenolpyruvate, so this is not a protein kinase system. The second prokaryotic system is the twocomponent regulatory system (1,2). The core of this system comprises three protein domains, which may or may not be on different polypeptides. The first domain is the protein kinase domain, the second is the histidine substrate for this kinase and the third contains an aspartate residue that removes the phosphate from phosphohistidine. Genes homologous to those of the two-component system have been identified in lower eukaryotes (1,6,7). [ABSTRACT FROM AUTHOR]

Normal cellular proliferation is under the tight control of both positive and negative regulators that determine whether a particular cell can progress through the cell cycle (seerefs. 1-3 for reviews). This carefully ordered progression of the mammalian cell cycle is controlled by the sequential formation, activation, and deactivation of specific cell cycle-regulatory molecules (Fig. 1) that exist as a series of complexes consisting of a catalytic kinase subunit (known as the cyclin-dependent kinase or CDK) and a regulatory cyclin subunit. These cyclin:CDK complexes form part of the positive regulatory machinery that drives the cell through the cycle. Most cyclin mRNAs and proteins show a dramatic fluctuation in their expression during the cell cycle. For example,G1/s cyclin, cyclin, cyclin E is unstable, peaks during lateG1 and disappears rapidly thereafter, whereas cyclins A and B accumulate transiently at the onset of S phase and in late G2 respectively, followed by their rapid degradation (1,2). In contrast, expression of the various CDK molecules remains relatively constant throughout the cell cycle. [ABSTRACT FROM AUTHOR]

Two isoforms of the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) occur in mammalian cells, GSK-3α (53 kDa) and GSK-3β (47 kDa), which are 85% identical over the entire protein and 95% identical within the catalytic domain (1). GSK-3 is ubiquitously expressed in mammalian tissues and GSK-3 homologs have been identified in every eukaryotic species tested to date (2), strongly suggesting that it plays a central role in cellular regulation. [ABSTRACT FROM AUTHOR]

The JAK/STAT pathway is activated by a wide range of ligands including cytokines and growth factors (1,2; see Table 1). This pathway was discovered by two independent approaches that initially identified the role of JAKs and STATs in interferon signaling. The first, a biochemical approach, used promoter elements to isolate and purify STATs (3) and the second, a genetic approach, led to the isolation of mutants defective in their response to interferon (4). The latter resulted in eight mutant cell lines (summarized in Table 2) which were complemented by components of the interferon pathway (5-9). These mutants have been fundamental in demonstrating a role for JAKs and STATs in cytokine signaling and in understanding the mechanisms of activating the JAK/STAT pathway. [ABSTRACT FROM AUTHOR]

The mitogen-activated protein kinases (MAPKs) correspond to a family of serine/threonine kinases that can be divided into three subtypes, ERK, JNK/ SAPK, and p38 MAPK. They are activated by a complex set of upstream kinases organized into parallel kinase cascades. This introduction will not discuss these upstream kinases, but Fig. 1 illustrates the complexity of these cascades (1-4). Note that there appears to be cell-line specific variation within this circuitry, especially in relation to upstream activators of these cascades, although this remains a contentious issue. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGF-β) family members, which include TGF-βs, activins, and bone morphogenetic proteins (BMPs), elicit their multifunctional effects by binding to and complex formation of type I and type II serine/threonine kinase receptors (see Fig. 1). Each family member signals via distinct combinations of type I and type II receptors, both of which are required for signaling. Upon formation of the heteromeric receptor complex, the type I receptor is phosphorylated by the type II receptor kinase. Phosphorylation occurs predominantly in a region rich in glycine and serine residues (GS domain) in the juxtamembrane domain of the type I receptor, which possibly leads to a conformational change and thereby activates the type I receptor kinase (see Fig. 1) (1-3). The activated type I receptor propagates the signal downstream through transient interaction with, and phosphorylation of, particular Smoeand mad related protein (Smad) molecules (1-3). Certain Smads are phosphorylated directly by activated type I receptors in a differential manner; they are therefore termed pathway-restricted Smads. Whereas Smad2 and Smad3 act in TGF-β and activin pathways, Smad1, Smad5, and Smad8 are thought to act in BMP pathways. Phosphorylation occurs at the two most C-terminal serine residues in a conserved C-terminal Ser-Ser-X-Ser motif (see Fig. 2). Pathway-restricted Smads oligomerize with Smad4, which acts as a common mediator in TGF-β, activin, and BMP signaling. After translocation to the nucleus, the oligomers interact with DNA directly, or in complex with other DNA-binding proteins, and control transcription of target genes (see Figs. 1 and 2). Recently, inhibitory Smads, Smad6, and Smad7, have been identified that antagonize TGF-β family signaling (3). [ABSTRACT FROM AUTHOR]

After incubation of cells with 32P-labeled inorganic phosphate, it is possible to identify in vivo radiolabeled phosphoproteins. Generally, after the cells are lysed the phosphoprotein can be separated by sodium dodecyl sulfate (SDS) gel electrophoresis and a rough estimate of the size of the phosphoprotein can be gainnted. In order to determine the phosphorylated residues in the protein, the radiolabeled band can be transferred to a membrane, hydrolyzed with trypsin (or another suitable enzyme), and the two-dimensional (2D) map establishnted. The phosphopeptides observed on the 2D map can be tentatively correlated with expected tryptic fragments, based on their hydrophobicity and charge. A number of protocols have been developed to refine the correlation of the expected fragments to phosphopeptides present on the 2D map. For example, phospho-amino acid analysis of individual species present on the 2D map can be used to identify the type of phosphorylated residues present. In addition, manual Edman degradation can be performed on phosphopeptides after they are removed from the 2D map in order to identify the position of the 32P-containing residue. This information can be used to help determine the position of the phosphorylated residue when more than one such residue is present in the tentatively assigned fragment sequence. Armed with this information, mutational analysis can confirm the site of phosphorylation, again using 2D map analysis. Although the above protocols have been successfully utilized, difficulties can arise. For example, the 2D map may not have sufficient resolution to separate two different phosphopeptides. As a result, the manual Edman analysis may not take into account the heterogeneous nature of the sample extractnted. Alternatively, ambiguous results can be obtained from manual Edmanwhen more than five cycles are required to discriminate between two possible "tentatively assigned" fragments. Partial oxidation of cysteine, methionine and tryptophan residue-containing peptides may also complicate the interpretation of the 2D map. Finally, the correlation between the pI and the hydrophobicity of the fragments may not be sufficient information to direct the mutational analysis. Many of these problems can be resolved by high-sensitivity mass spectrometry (MS) analysis. Analysis of peptide mixtures with MS will generally clarify whether a sample is heterogeneous and delineate modifications such as phosphorylation and oxidation. [ABSTRACT FROM AUTHOR]

A bewildering array of receptor and nonreceptor protein tyrosine kinases have been uncovered during the past two decades. During this time it has become clear that the autophosphorylation of these protein kinases on tyrosine residues has two major effects on their function. First, the phosphorylation event often enhances the activity of the protein kinase such that it may then phosphorylate other intracellular substrates on tyrosines; this is the case for the insulin receptor and pp60c-src, for example. However, a more widespread role among the tyrosine kinases is the generation of a phosphotyrosine binding site for effector proteins that contain either src homology 2(SH2) or "Phospho Tyrosine Binding" (PTB) domains. It is these latter effector proteins that then further transmit the signal into the cell interior (1,2). [ABSTRACT FROM AUTHOR]

Signals transmitted between cells activate intracellular signaling pathways that are precisely regulated, controlled, and organized. Ultimately, the intracellular biochemical events culminate in a specific cellular response(s). A major mechanism for intracellular signal transduction in both eukaryotic and prokaryotic cells is protein phosphorylation (1). Beside being fast and reversible, protein phosphorylation is tightly regulated and highly specific, and allows signals to be sustained or attenuated via amplification, feedback, and crosstalk (2). The specificity in signaling is, in part, achieved by two means. Catalytic specificity of protein kinases and phosphatases provides the basis for site-specific phosphorylation and dephosphorylation, respectively (3). Specific tyrosine and serine/threonine phosphorylation sites and their surrounding sequences, in turn, provide selective binding sites for conserved protein modules found in most cytoplasmic signaling molecules (2,4,5). Identifying the sites of protein phosphorylation and the nature of the phosphorylated residue is therefore paramount in the study of signal transduction. [ABSTRACT FROM AUTHOR]

Changes in the tyrosine phosphorylation state of a protein in response to external stimuli can have profound effects on cellular signal transduction. The addition of a phosphate group to a tyrosine residue can change a protein' activation state or create a high affinity binding site for other proteins. Conversely, removal of a phosphate group can also change the catalytic activity of an enzyme. Tyrosine phosphorylation of cellular proteins is a rare event that can be increased growth factor addition or cellular attachment to extracellular matrix. Therefore, it is important to be able to observe changes in tyrosine phosphorylation of particular proteins under the influence of different stimuli. Tyrosine phosphorylation of proteins is difficult to detect unless external stimuli are present; even then, many proteins are phosphorylated only in response to one stimulus. Therefore, it is necessary to concentrate the protein of interest in order to observe the phosphorylation state changes between stimulated and unstimulated cells. 32P-labeling of cellular proteins can be used; however, phosphoserine and phosphothreonine are also detected along with phosphotyrosine. Phosphoamino acid analysis can be helpful, but it is not quantitative because acid hydrolysis, which breaks down the proteins into individual amino acids, can remove the phosphate group from the tyrosine. Therefore, other methods of detecting changes in tyrosine phosphorylation states have been developed. [ABSTRACT FROM AUTHOR]

Protein tyrosine kinases (PTKs) play prominent roles in the regulation of fundamental biological processes including normal cell growth and survival, cell differentiation, and development. Across vertebrate and invertebrate species, both nonreceptor (cytoplasmic) and receptor (transmembrane) type PTKs have been identified, making them one of the most extensively examined family of proteins. Currently, genes encoding at least 50 receptor and 33 nonreceptor vertebrate PTKs have been cloned (1,2), several by techniques that exploit the structural and functional conservation of the kinase catalytic domain. [ABSTRACT FROM AUTHOR]

Many aspects of cellular metabolism are regulated by reversible phosphorylation of proteins. Several amino acid residues within proteins are subject to such posttranslational regulatory events, the best characterized of which are tyrosine, serine, and threonine. Transfer of phosphate to such residues is mediated by protein kinases that catalyze the transfer of phosphate from adenosine triphosphate (ATP) to specific relevant amino acid residues. This fundamental property of protein kinases is utilized and elaborated upon in many different contexts to generate protein kinase mediated signaling cascades by which extracellular stimuli are accurately perceived and elicit appropriate cellular responses. [ABSTRACT FROM AUTHOR]