SH2 domains, interaction modules and cellular wiring

SH2 domains serve as the prototype for a growing family of protein-interaction modules, characteristic of polypeptides involved in transmitting signals from external and internal cues. The specific interactions of proteins with one another, and with other cellular components such as phospholipids and nucleic acids, provide a very general device to organize cellular behavior. We discuss the idea that rewiring of the cell's interaction network by pathogenic microorganisms and mutant cellular proteins contributes to dysregulation of cell signaling and thus to disease.     

Synthesis of linear and cyclic phosphopeptides as ligands for the N-terminal SH2-domain of protein tyrosine phosphatase SHP-1.

Linear and cyclic phosphopeptides related to the pY2267 binding site of the epithelial receptor tyrosine kinase Ros have been synthesized as ligands for the amino-terminal SH2 (src homology) domain of protein tyrosine phosphatase SHP-1. The synthesis was accomplished by Fmoc-based solid-phase methodology using side-chain unprotected phosphotyrosine for the linear and mono-benzyl protected phosphotyrosine for the cyclic peptides. According to molecular modelling, the incorporation of a glycine residue between Lys (position pY-1 relative to phosphotyrosine) and Asp or Glu (position pY+2) was recommended for the cyclic candidates. The preparation of these peptides was successfully performed by the incorporation of a Fmoc-Xxx(Gly-OAll)-OH (Xxx = Asp, Glu) dipeptide building block that was prepared in solution prior to SPPS. The cyclization was achieved with PyBOP following Alloc/OAll-deprotection. This study demonstrates the usefulness of allyl-type protecting groups for the generation of side-chain cyclized phosphopeptides. Alloc/OAll-deprotection and cyclization are compatible with phosphorylated tyrosine.     

A Phos-tag-based fluorescence resonance energy transfer system for the analysis of the dephosphorylation of phosphopeptides

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules. Here we introduce a novel FRET system for the detection of phosphopeptides using a phosphate-binding tag molecule, Zn²⁺-Phos-tag (1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex) attached with a 7-amino-4-methylcoumarin-3-acetic acid (AMCA). Carboxyfluorescein (FAM)-labeled phospho- and nonphosphopeptides were prepared as the target molecules for the FRET system. A set of FAM (a fluorescent acceptor, λ_em 520 nm) and AMCA (a fluorescent donor, λ_ex 345 nm) is frequently used for a FRET system. The AMCA-labeled Zn²⁺-Phos-tag specifically captured the FAM-labeled phosphopeptide to form a stable 1:1 complex, resulting in efficient FRET. After the FAM-labeled phosphopeptide was dephosphorylated with alkaline phosphatase, the FRET disappeared. Using this FRET system, we demonstrated the detection of the time-dependent dephosphorylation of the FAM-labeled protein-tyrosine phosphatase 1B substrate.     

Synthesis and evaluation of phosphopeptides containing iminodiacetate groups as binding ligands of the Src SH2 domain

Phosphopeptide pTyr-Glu-Glu-Ile (pYEEI) has been introduced as an optimal Src SH2 domain ligand. Peptides, Ac-K(IDA)pYEEIEK(IDA) (1), Ac-KpYEEIEK (2), Ac-K(IDA)pYEEIEK (3), and Ac-KpYEEIEK(IDA) (4), containing 0–2 iminodiacetate (IDA) groups at the N- and C-terminal lysine residues were synthesized and evaluated as the Src SH2 domain binding ligands. Fluorescence polarization assays showed that peptide 1 had a higher binding affinity (K_d = 0.6 μM) to the Src SH2 domain when compared with Ac-pYEEI (K_d = 1.7 μM), an optimal Src SH2 domain ligand, and peptides 2–4 (K_d = 2.9–52.7 μM). The binding affinity of peptide 1 to the SH2 domain was reduced by more than 2-fold (K_d = 1.6 μM) upon addition of Ni²⁺ (300 μM), possibly due to modest structural effect of Ni²⁺ on the protein as shown by circular dichroism experimental results. The binding affinity of 1 was restored in the presence of EDTA (300 μM) (K_d = 0.79 μM). These studies suggest that peptides containing IDA groups may be used for designing novel SH2 domain binding ligands.     

An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein phosphopeptides

Sequestration of calcium phosphate by caseins occurs in the Golgi region of mammary secretory cells during lactation, where it helps to prevent calcification of the gland and to deliver high concentrations of calcium and phosphate to the neonate in the form of milk. Calcium phosphate nanoclusters are formed when a core of amorphous calcium phosphate is sequestered within a shell of casein or casein phosphopeptides. The nanoclusters can form spontaneously from a supersaturated solution or by dispersion of a precipitate of calcium phosphate, demonstrating that they are thermodynamically stable complexes. The average size and chemical composition of the complexes are largely independent of the solution conditions (pH, temperature, peptide concentration, salt composition and rate of reaction) under which they form. Larger, metastable, colloidal particles can form if there is not enough of the phosphopeptide to sequester all the calcium phosphate, or, transiently, if the salt and peptide solutions are mixed together without sufficient care. A thermodynamic model of the sequestration process is presented which makes use of an invariant ion activity product observed in nanocluster-containing solutions. In any given solution that has thermodynamic stability, the extent of the sequestration reaction can be calculated from the empirical formula of the nanoclusters using the criterion that the solution should have the equilibrium value of the invariant ion activity product. Other members of the paralogous group of secretory calcium-binding phosphoproteins to which caseins belong may also be able to sequester calcium phosphate in biological fluids such as saliva and in the extracellular matrix of mineralizing tissues.Abbreviations -PP _s1-casein 5P (f59–79) - -PP -casein 4P (f1–25) - ACP amorphous calcium phosphate - Cit citrate - CPN calcium phosphate nanocluster - CPP commercial phosphopeptide - IAP ion activity product - MWCO molecular weight cut-off - PP phosphopeptide - SAXS small-angle X-ray scattering - SCPP secretory calcium-binding phosphoprotein - UF ultrafiltrate     

Src homology domains of v-Src stabilize an active conformation of the tyrosine kinase catalytic domain

To examine the interactions between Src homology,domains and the tyrosine kinase catalytic domain of v-Src, various combinations of domains have been expressed in bacteria as fusion proteins. Constructs containing the isolated catalytic domain, SH2 + catalytic domain, and SH3 + SH2 + catalytic domains were active in autophosphorylation assays. For the catalytic domain of v-Src, but not for v-Abl, addition of exogenous Src SH3-SH2 domains stimulated the autophosphorylation activity. In contrast to results for autophosphorylation, constructs containing Src homology domains were more active towards a synthetic peptide substrate than the isolated catalytic domain. The ability of the SH2 and SH3 domains of v-Src to stabilize an active enzyme conformation was also confirmed by refolding after denaturation in guanidinium hydrochloride. Collectively the data suggest that, in addition to their roles in intermolecular protein-protein interactions, the Src homology regions of v-Src exert a positive influence on tyrosine kinase function, potentially by maintaining an active conformation of the catalytic domain.     

快速构建蛋白质结构域克隆库的方法

对蛋白质组学的研究有许多不同的切入方法 .从研究的生物学意义和可行性考虑 ,提出从蛋白结构域入手进行蛋白质组学研究 .SH2 (Srchomology 2 )结构域是细胞信号转导中重要的元件之一 ,人SH2结构域共有约 12 0种 ,对其进行研究将深刻揭示细胞信号转导的规律 .为了得到人所有的SH2结构域序列及克隆 ,首先在公共数据库里检索出了人所有的SH2结构域序列 ,利用国际上现有的共享资源IMAGE(IntegratedMolecularAnalysisofGenomesandTheirExpression)克隆为PCR模板 ,解决了从cDNA文库中难以克隆低丰度结构域的问题 .利用有方向性的TOPO克隆技术提高克隆效率 ,从而快速高效地构建了包括 6 0个SH2结构域的克隆库 .克隆库可以方便地转换到GATEWAY系统具有各种用途的载体上 ,为SH2结构域的蛋白质组学研究奠定了坚实的基础     

Identification of Novel Crk-associated Substrate (p130Cas) Variants with Functionally Distinct Focal Adhesion Kinase Binding Activities

Elevated levels of p130^Cas (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1 gene) are associated with aggressiveness of breast tumors. Following phosphorylation of its substrate domain, p130^Cas promotes the integration of protein complexes involved in multiple signaling pathways and mediates cell proliferation, adhesion, and migration. In addition to the known BCAR1-1A (wild-type) and 1C variants, we identified four novel BCAR1 mRNA variants, generated by alternative first exon usage (1B, 1B1, 1D, and 1E). Exons 1A and 1C encode for four amino acids (aa), whereas 1D and 1E encode for 22 aa and 1B1 encodes for 50 aa. Exon 1B is non-coding, resulting in a truncated p130^Cas protein (Cas1B). BCAR1-1A, 1B1, and variant 1C mRNAs were ubiquitously expressed in cell lines and a survey of human tissues, whereas 1B, 1D, and 1E expression was more restricted. Reconstitution of all isoforms except for 1B in p130^Cas-deficient murine fibroblasts induced lamellipodia formation and membrane ruffling, which was unrelated to the substrate domain phosphorylation status. The longer isoforms exhibited increased binding to focal adhesion kinase (FAK), a molecule important for migration and adhesion. The shorter 1B isoform exhibited diminished FAK binding activity and significantly reduced migration and invasion. In contrast, the longest variant 1B1 established the most efficient FAK binding and greatly enhanced migration. Our results indicate that the p130^Cas exon 1 variants display altered functional properties. The truncated variant 1B and the longer isoform 1B1 may contribute to the diverse effects of p130^Cas on cell biology and therefore will be the target of future studies.     

Regulation of ADL6 activity by its associated molecular network

Plant dynamin-like proteins consist of a group of high molecular weight GTPase with diverse structural arrangements and cellular localizations. In addition, unlike animal dynamins, there was no evidence for the involvement of any plant dynamin-like protein in clathrin-mediated vesicle trafficking. In this study we demonstrate that ADL6 (Arabidopsis dynamin-like protein 6), due to its domain arrangement, behaves similarly to the animal dynamins. The association of ADL6 with clathrin-coated vesicles was demonstrated by co-fractionation and immunocytochemical studies. ADL6 also interacted via its C-terminus with gamma-adaptin, an adaptor protein of clathrin-coated vesicles. Our results suggest that ADL6 participates in clathrin-mediated vesicle trafficking originating from the Golgi. In addition, our studies demonstrate that ADL6 intrinsic GTPase activity is regulated by its association with acidic phospholipids and an SH3 (Src homology 3)-containing protein.     

Structural and biophysical investigation of the interaction of a mutant Grb2 SH2 domain (W121G) with its cognate phosphopeptide

The adaptor protein Grb2 is a key element of mitogenetically important signaling pathways. With its SH2 domain it binds to upstream targets while its SH3 domains bind to downstream proteins thereby relaying signals from the cell membranes to the nucleus. The Grb2 SH2 domain binds to its targets by recognizing a phosphotyrosine (pY) in a pYxNx peptide motif, requiring an Asn at the +2 position C‐terminal to the pY with the residue either side of this Asn being hydrophobic. Structural analysis of the Grb2 SH2 domain in complex with its cognate peptide has shown that the peptide adopts a unique β‐turn conformation, unlike the extended conformation that phosphopeptides adopt when bound to other SH2 domains. TrpEF1 (W121) is believed to force the peptide into this unusual conformation conferring this unique specificity to the Grb2 SH2 domain. Using X‐ray crystallography, electron paramagnetic resonance (EPR) spectroscopy, and isothermal titration calorimetry (ITC), we describe here a series of experiments that explore the role of TrpEF1 in determining the specificity of the Grb2 SH2 domain. Our results demonstrate that the ligand does not adopt a pre‐organized structure before binding to the SH2 domain, rather it is the interaction between the two that imposes the hairpin loop to the peptide. Furthermore, we find that the peptide adopts a similar structure when bound to both the wild‐type Grb2 SH2 domain and a TrpEF1Gly mutant. This suggests that TrpEF1 is not the determining factor for the conformation of the phosphopeptide.     

Structural and thermodynamic basis for the interaction of the Src SH2 domain with the activated form of the PDGF beta-receptor

Recruitment of the Src kinase to the activated form of the platelet-derived growth factor (PDGF) receptor involves recognition of a unique sequence motif in the juxtamembrane region of the receptor by the Src homology 2 (SH2) domain of the enzyme. This motif contains two phosphotyrosine residues separated by one residue (sequence pYIpYV where pY indicates a phosphotyrosine). Here, we provide the thermodynamic and structural basis for the binding of this motif by the Src SH2 domain. We show that the second phosphorylation event increases the free energy window for specific interaction and that the physiological target is exquisitely designed for the task of recruiting specifically an SH2 domain which otherwise demonstrates very little intrinsic ability to discriminate sequences C-terminal to the first phosphorylation event. Surprisingly, we show that water plays a role in the recognition process.     

Structural basis for differential recognition of tyrosine-phosphorylated sites in the linker for activation of T cells (LAT) by the adaptor Gads

The transmembrane protein, linker for activation of T cells (LAT), is essential for T-cell activation and development. Phosphorylation of LAT at multiple tyrosines creates binding sites for the adaptors Gads and Grb2, leading to nucleation of multiprotein signaling complexes. Human LAT contains five potential binding sites for Gads, of which only those at Tyr171 and Tyr191 appear necessary for T-cell function. We asked whether Gads binds preferentially to these sites, as differential recognition could assist in assembling defined LAT-based complexes. Measured calorimetrically, Gads-SH2 binds LAT tyrosine phosphorylation sites 171 and 191 with higher affinities than the other sites, with the differences ranging from only several fold weaker binding to no detectable interaction. Crystal structures of Gads-SH2 complexed with phosphopeptides representing sites 171, 191 and 226 were determined to 1.8-1.9 A resolutions. The structures reveal the basis for preferential recognition of specific LAT sites by Gads, as well as for the relatively greater promiscuity of the related adaptor Grb2, whose binding also requires asparagine at position +2 C-terminal to the phosphorylated tyrosine.     

The Src module: an ancient scaffold in the evolution of cytoplasmic tyrosine kinases

Tyrosine kinases were first discovered as the protein products of viral oncogenes. We now know that this large family of metazoan enzymes includes nearly one hundred structurally diverse members. Tyrosine kinases are broadly classified into two groups: the transmembrane receptor tyrosine kinases, which sense extracellular stimuli, and the cytoplasmic tyrosine kinases, which contain modular ligand-binding domains and propagate intracellular signals. Several families of cytoplasmic tyrosine kinases have in common a core architecture, the “Src module,” composed of a Src-homology 3 (SH3) domain, a Src-homology 2 (SH2) domain, and a kinase domain. Each of these families is defined by additional elaborations on this core architecture. Structural, functional, and evolutionary studies have revealed a unifying set of principles underlying the activity and regulation of tyrosine kinases built on the Src module. The discovery of these conserved properties has shaped our knowledge of the workings of protein kinases in general, and it has had important implications for our understanding of kinase dysregulation in disease and the development of effective kinase-targeted therapies.     

Alternative splicing of ADAM15 regulates its interactions with cellular SH3 proteins

A Disintegrin And Metalloprotease (ADAM15) is a member of the adamalysin protein family and has been associated with cancer, possibly via its role in ectodomain shedding of cadherins. Alternative mRNA splicing generates several ADAM15 isoforms containing different combinations of putative Src homology‐3 (SH3) domain binding sites in their cytosolic tails. Here we present a comprehensive characterization of SH3 binding potential of different ADAM15 isoforms. Alternative use of ADAM15 exons was found to profoundly influence selection of SH3‐containing cellular partner proteins, including the avid interactions with nephrocystin and sorting nexin‐33 (SNX33 a.k.a. SNX30). Specifically, strong co‐precipitation of nephrocystin from cell lysates was specific to ADAM15 isoforms i4, i5, and i6. These isoforms contain one or both of the two almost identical proline‐rich regions encoded by exons 20 and 21, wherein the residues RxLPxxP were found to be indispensable for nephrocystin SH3 binding. Similarly, robust cellular association with SNX33 was observed only for ADAM15 isoforms containing the most carboxyterminal proline cluster lacking in isoforms i1 and i3. Thus, alternative mRNA splicing provides a versatile mechanism for regulation of intracellular protein interactions and thereby likely the cellular functions of ADAM15, which could explain the association with cancer of some but not all ADAM15 isoforms. J. Cell. Biochem. 108: 877–885, 2009. © 2009 Wiley‐Liss, Inc.     

Construction of a non-redundant human SH2 domain database

Domain database is essential for domain property research. Eliminating redundant information in database query is very important for database quality. Here we report the manual construction of a non-redundant human SH2 domain database. There are 119 human SH2 domains in 110 SH2-containing proteins. Human SH2s were aligned with ClustalX, and a homologous tree was generated. In this tree, proteins with similar known function were classified into the same group. Some proteins in the same group have been reported to have similar binding motifs experimentally. The tree might provide clues about possible functions of hypothetical proteins for further experimental verification.     

Regulation of EGF-induced phospholipase C-gamma1 translocation and activation by its SH2 and PH domains

Translocation of phospholipase C-γ1 is essential for its function in response to growth factors. However, in spite of recent progress, the phospholipase C-γ1 translocation pattern and the molecular mechanism of the translocation are far from fully understood. Contradictory results were reported as to which domain, PH or SH2, controls the epidermal growth factor-induced translocation of phospholipase C-γ1. In this communication, we studied epidermal growth factor-induced translocation of phospholipase C-γ1 by using comprehensive approaches including biochemistry, indirect fluorescence and live fluorescence imaging. We provided original evidence demonstrating that: (i) endogenous phospholipase C-γ1, similar to YFP-tagged phospholipase C-γ1, translocated to endosomes following its initial translocation from cytosol to the plasma membrane in response to epidermal growth factor; (ii) phospholipase C-γ1 remained phosphorylated in endosomes, but phospholipase C-γ1 activity is not required for its translocation, which suggests a signaling role for phospholipase C-γ1 in endosomes; (iii) the PH domain was not required for the initial translocation of phospholipase C-γ1 from cytosol to the plasma membrane, but it stabilizes phospholipase C-γ1 in the membrane at a later time; (iv) the function of the phospholipase C-γ1 PH domain in stabilizing phospholipase C-γ1 membrane association is very important in maintaining the activity of phospholipase C-γ1; and (v) the role of the PH domain in phospholipase C-γ1 membrane association and activation is dependent on PI3K activity. We conclude that the phospholipase C-γ1 SH2 and PH domains coordinate to determine epidermal growth factor-induced translocation and activation of phospholipase C-γ1.     

Grb2 dominantly associates with dynamin II in human hepatocellular carcinoma HepG2 cells

The two SH3 domains and one SH2 domain containing adaptor protein Grb2 is an essential element of the Ras signaling pathway in multiple systems. The SH2 domain of Grb2 recognizes and interacts with phosphotyrosine residues on activated tyrosine kinases, whereas the SH3 domains bind to several proline‐rich domain‐containing proteins such as Sos1. To define the difference in Grb2‐associated proteins in hepatocarcinoma cells, we performed coprecipitation analysis using recombinant GST‐Grb2 fusion proteins and found that several protein components (p170, p125, p100, and p80) differently associated with GST‐Grb2 proteins in human Chang liver and hepatocarcinoma HepG2 cells. Sos1 and p80 proteins dominantly bind to Grb2 fusion proteins in Chang liver, whereas p100 remarkably associate with Grb2 in HepG2 cells. Also GST‐Grb2 SH2 proteins exclusively bound to the p46^Shc, p52^Shc, and p66^Shc are important adaptors of the Ras pathway in HepG2 cells. The p100 protein has been identified as dynamin II. We observed that the N‐SH3 and C‐SH3 domains of Grb2 fusion proteins coprecipitated with dynamin II besides Sos1. These results suggest that dynamin II may be a functional molecule involved in Grb2‐mediated signaling pathway on Ras activation for tumor progression and differentiation of hepatocarcinoma cells. J. Cell. Biochem. 84: 150–155, 2002. © 2001 Wiley‐Liss, Inc.