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PubMed2002

Application of the yeast two-hybrid system in molecular gerontology.

Most -- if not all -- proteins are bound to interact with other proteins to exert their function, and thus the identification of the interaction partners of a protein is vital in proteomics. The yeast two-hybrid system is a popular and effective tool for studying protein-protein interactions. Although the advantages of the system are manifold, it also has certain drawbacks and limitations. The two-hybrid system has been shown to be extremely useful for placing a protein of unknown function within a functional context, thereby providing information about a putative role of the uncharacterised protein. This concept has also been successfully applied in molecular gerontology.

Aging↗

PubMed2001

The polycomb protein MPc3 interacts with AF9, an MLL fusion partner in t(9;11)(p22;q23) acute leukemias.

Polycomb group (PcG) proteins assemble to form large multiprotein complexes involved in gene silencing. Evidence suggests that PcG complexes are heterogeneous with respect to both protein composition and specific function. MPc3 is a recently described mouse Polycomb (Pc) protein that shares structural homology with at least two other Pc proteins, M33 and MPc2. All three Pc proteins bind another PcG protein, RING1, through a conserved carboxy-terminal C-box motif. Here, data are presented demonstrating that MPc3 also interacts with AF9, a transcriptional activator implicated in the development of acute leukemias. The carboxy-terminus of AF9 is fused to the MLL protein in leukemias characterized by t(9;11)(p22;q23) chromosomal translocations. Importantly, it is the carboxy-terminus of AF9 to which MPc3 binds. The AF9 binding site of MPc3 maps to a central, non-conserved, region of the polypeptide sequence. In contrast to MPc3, data indicate that the Pc protein M33 does not interact with AF9. This finding suggests a potentially unique role for MPc3 in linking a PcG silencing complex to a transcriptional activator protein.

3T3 Cells↗

PubMed2004

Protein interaction networks by proteome peptide scanning.

A substantial proportion of protein interactions relies on small domains binding to short peptides in the partner proteins. Many of these interactions are relatively low affinity and transient, and they impact on signal transduction. However, neither the number of potential interactions mediated by each domain nor the degree of promiscuity at a whole proteome level has been investigated. We have used a combination of phage display and SPOT synthesis to discover all the peptides in the yeast proteome that have the potential to bind to eight SH3 domains. We first identified the peptides that match a relaxed consensus, as deduced from peptides selected by phage display experiments. Next, we synthesized all the matching peptides at high density on a cellulose membrane, and we probed them directly with the SH3 domains. The domains that we have studied were grouped by this approach into five classes with partially overlapping specificity. Within the classes, however, the domains display a high promiscuity and bind to a large number of common targets with comparable affinity. We estimate that the yeast proteome contains as few as six peptides that bind to the Abp1 SH3 domain with a dissociation constant lower than 100 microM, while it contains as many as 50-80 peptides with corresponding affinity for the SH3 domain of Yfr024c. All the targets of the Abp1 SH3 domain, identified by this approach, bind to the native protein in vivo, as shown by coimmunoprecipitation experiments. Finally, we demonstrate that this strategy can be extended to the analysis of the entire human proteome. We have developed an approach, named WISE (whole interactome scanning experiment), that permits rapid and reliable identification of the partners of any peptide recognition module by peptide scanning of a proteome. Since the SPOT synthesis approach is semiquantitative and provides an approximation of the dissociation constants of the several thousands of interactions that are simultaneously analyzed in an array format, the likelihood of each interaction occurring in any given physiological settings can be evaluated. WISE can be easily extended to a variety of protein interaction domains, including those binding to modified peptides, thereby offering a powerful proteomic tool to help completing a full description of the cell interactome.

Animals↗

PubMed2004

Identification of a novel family of G protein-coupled receptor associated sorting proteins.

During the past few years several new interacting partners for G protein-coupled receptors (GPCRs) have been discovered, suggesting that the activity of these receptors is more complex than previously anticipated. Recently, candidate G protein-coupled receptor associated sorting protein (GASP-1) has been identified as a novel interacting partner for the delta opioid receptor and has been proposed to determine the degradative fate of this receptor. We show here that GASP-1 associates in vitro with other opioid receptors and that the interaction domain in these receptors is restricted to a small portion of the carboxyl-terminal tail, corresponding to helix 8 in the three-dimensional structure of rhodopsin. In addition, we show that GASP-1 interacts with COOH-terminus of several other GPCRs from subfamilies A and B and that two conserved residues within the putative helix 8 of these receptors are critical for the interaction with GASP-1. In situ hybridization and northern blot analysis indicate that GASP-1 mRNA is mainly distributed throughout the central nervous system, consistent with a potential interaction with numerous GPCRs in vivo. Finally, we show that GASP-1 is a member of a novel family comprising at least 10 members, whose genes are clustered on chromosome X. Another member of the family, GASP-2, also interacts with the carboxyl-terminal tail of several GPCRs. Therefore, GASP proteins may represent an important protein family regulating GPCR physiology.

Amino Acid Sequence↗

PubMed2006

Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK).

Present screening methods for protein-protein interactions (PPIs) rely on the overexpression of artificial fusion proteins, making it difficult to assess in vivo relevance. Here we combine stable isotope labeling with amino acids in cell culture (SILAC), RNA interference (RNAi), coimmunoprecipitation and quantitative mass-spectrometry analysis to detect cellular interaction partners of endogenous proteins in mammalian cells with very high confidence. We used this screen to identify interaction partners of beta-catenin and Cbl.

Gene Silencing↗

PubMed2001

The LIS1-related protein NUDF of Aspergillus nidulans and its interaction partner NUDE bind directly to specific subunits of dynein and dynactin and to alpha- and gamma-tubulin.

The NUDF protein of Aspergillus nidulans, which is required for nuclear migration through the fungal mycelium, closely resembles the LIS1 protein required for migration of neurons to the cerebral cortex in humans. Genetic experiments suggested that NUDF influences nuclear migration by affecting cytoplasmic dynein. NUDF interacts with another protein, NUDE, which also affects nuclear migration in A. nidulans. Interactions among LIS1, NUDE, dynein, and gamma-tubulin have been demonstrated in animal cells. In this paper we examine the interactions of the A. nidulans NUDF and NUDE proteins with components of dynein, dynactin, and with alpha- and gamma-tubulin. We show that NUDF binds directly to alpha- and gamma-tubulin and to the first P-loop of the cytoplasmic dynein heavy chain, whereas NUDE binds directly to alpha- and gamma-tubulin, to NUDK (actin-related protein 1), and to the NUDG dynein LC8 light chain. The data suggest a direct role for NUDF in regulation of the dynein heavy chain and an effect on other dynein/dynactin subunits via NUDE. The interactions between NUDE, NUDF, and gamma-tubulin suggest that this protein may also be involved in the regulation of dynein function. Additive interactions between NUDE and dynein and dynactin subunits suggest that NUDE acts as a scaffolding factor between components.

Active Transport, Cell Nucleus↗

PubMed2004

Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies.

OBJECTIVE: There has been considerable interest in understanding determinants of the diverse cardiac phenotypes associated with heterozygous NKX2.5 mutations. We hypothesized that analysis of functional properties of NKX2.5 mutant proteins would result in the ability to classify mutations according to function in a scheme that would help to clarify genotype-phenotype correlations. We analyzed missense mutations in the conserved homeodomain. METHODS: We studied in vitro biochemical characteristics, including nuclear localization, DNA binding, transcriptional activation and protein-protein interaction with transcriptional partners (GATA4, TBX5 and NKX2.5 itself), of eight homeodomain missense mutations. Associated phenotypes include atrioventricular (AV) block (98% penetrance), atrial septal defect (83% penetrance), and additional varied heart malformations. RESULTS: Mutations were present at varied homeodomain locations in the putative nuclear localizing signal (1), helix 2 (1), a turn between helix 2 and 3 (1) and helix 3 (5); a spectrum of biochemical phenotypes was observed. All mutants localized to the nuclei but some exhibited anomalous nuclear distribution. While all mutants exhibited markedly decreased DNA binding and reduced transcriptional activation, interaction with transcriptional partners was varied. CONCLUSION: Each mutant protein had a unique spectrum of observed properties, but our data show that while dominant negative properties could be demonstrated in vitro, the best correlation with clinical phenotypes resulted from the markedly reduced DNA binding shared by all eight homeodomain mutations. This suggests that the principle determinant of the two most common phenotypes associated with homeodomain missense mutations is the total dose of NKX2.5 capable of binding to DNA.

Cell Nucleus↗

PubMed2002

Involvement of TRAF4 in oxidative activation of c-Jun N-terminal kinase.

We previously found that the angiogenic factors TNFalpha and HIV-1 Tat activate an NAD(P)H oxidase in endothelial cells, which operates upstream of c-Jun N-terminal kinase (JNK), a MAPK involved in the determination of cell fate. To further understand oxidant-related signaling pathways, we screened lung and endothelial cell libraries for interaction partners of p47(phox) and recovered the orphan adapter TNF receptor-associated factor 4 (TRAF4). Domain analysis suggested a tail-to-tail interaction between the C terminus of p47(phox) and the conserved TRAF domain of TRAF4. In addition, TRAF4, like p47(phox), was recovered largely in the cytoskeleton/membrane fraction. Coexpression of p47(phox) and TRAF4 increased oxidant production and JNK activation, whereas each alone had minimal effect. In addition, a fusion between p47(phox) and the TRAF4 C terminus constitutively activated JNK, and this activation was decreased by the antioxidant N-acetyl cysteine. In contrast, overexpression of the p47(phox) binding domain of TRAF4 blocked endothelial cell JNK activation by TNFalpha and HIV-1 Tat, suggesting an uncoupling of p47(phox) from upstream signaling events. A secondary screen of endothelial cell proteins for TRAF4-interacting partners yielded a number of proteins known to control cell fate. We conclude that endothelial cell agonists such as TNFalpha and HIV-1 Tat initiate signals that enter basic signaling cassettes at the level of TRAF4 and an NAD(P)H oxidase. We speculate that endothelial cells may target endogenous oxidant production to specific sites critical to cytokine signaling as a mechanism for increasing signal specificity and decreasing toxicity of these reactive species.

Acetylcysteine↗

PubMed1999

Expression of the transcriptional repressor protein Kid-1 leads to the disintegration of the nucleolus.

The rat Kid-1 gene codes for a 66-kDa protein with KRAB domains at the NH2 terminus and two Cys2His2-zinc finger clusters of four and nine zinc fingers at the COOH terminus. It was the first KRAB-zinc finger protein for which a transcriptional repressor activity was demonstrated. Subsequently, the KRAB-A domain was identified as a widespread transcriptional repressor motif. We now present a biochemical and functional analysis of the Kid-1 protein in transfected cells. The full-length Kid-1 protein is targeted to the nucleolus and adheres tightly to as yet undefined nucleolar structures, leading eventually to the disintegration of the nucleolus. The tight adherence and nucleolar distribution can be attributed to the larger zinc finger cluster, whereas the KRAB-A domain is responsible for the nucleolar fragmentation. Upon disintegration of the nucleolus, the nucleolar transcription factor upstream binding factor disappears from the nucleolar fragments. In the absence of Kid-1, the KRIP-1 protein, which represents the natural interacting partner of zinc finger proteins with a KRAB-A domain, is homogeneously distributed in the nucleus, whereas coexpression of Kid-1 leads to a shift of KRIP-1 into the nucleolus. Nucleolar run-ons demonstrate that rDNA transcription is shut off in the nucleolar fragments. Our data demonstrate the functional diversity of the KRAB and zinc finger domains of Kid-1 and provide new functional insights into the regulation of the nucleolar structure.

Animals↗

PubMed2003

Surface plasmon resonance spectroscopy (SPR) interaction studies of the circadian-controlled tomato LHCa4*1 (CAB 11) protein with its promoter.

Feedback regulation is an important biochemical mechanism which is also able to direct the circadian timing at the transcriptional level. Independent investigations highlighted a conserved ca. 10 nucleotide motif present in many circadian regulated Lhc genes. Two of such nucleotide motifs exist within 119 nucleotides of the Lhca4*1 promoter from tomato. This promoter fragment was used as a bait in a yeast one hybrid screen and interestingly a clone encoding with sequence identity to the LHCa4*1 protein was isolated as an interaction partner. The LHCa4*1 protein was heterologous expressed and binding to the 119bp promoter fragment was demonstrated by surface plasmon resonance spectroscopy (SPR, Biacore). This result allows to postulate an autoregulatory feedback loop involved in expression of the Lhca4*1 gene.

Amino Acid Sequence↗

PubMed2006

Activator of G-protein signaling in asymmetric cell divisions of the sea urchin embryo.

An asymmetric fourth cell division in the sea urchin embryo results in formation of daughter cells, macromeres and micromeres, with distinct sizes and fates. Several lines of functional evidence presented here, including pharmacological interference and dominant negative protein expression, indicate that heterotrimeric G protein Gi and its interaction partner, activator of G-protein signaling (AGS), are necessary for this asymmetric cell division. Inhibition of Gi signaling by pertussis toxin interferes with micromere formation and leads to defects in embryogenesis. AGS was isolated in a yeast two-hybrid screen with G alpha i as bait and was expressed in embryos localized to the cell cortex at the time of asymmetric divisions. Introduction of exogenous dominant-negative AGS protein, containing only G-protein regulatory (GPR) domains, selectively prevented the asymmetric division in normal micromere formation. These results support the growing evidence that AGS is a universal regulator of asymmetric cell divisions in embryos.

Animals↗

PubMed2005

Synthesis and application of fluorescent ras proteins for live-cell imaging.

Semisynthetic Ras proteins are efficient probes for cell-biology experiments. With a Bodipy FL fluorophore introduced at an appropriate site on the Ras peptide by solid-phase synthesis, the resulting Ras chimera is processed by the cellular machinery and the intracellular localization of the protein can then be visualized by means of confocal laser fluorescence microscopy at relatively low concentrations. The absence of a large N-terminal protein tag overcomes possible interferences in the interaction with cellular partner proteins. The fluorescence emission from Bodipy FL is continuous and disappears only after irreversible bleaching. These characteristics make Ras proteins with nonprotein fluorophores suitable for biophysical analysis. The easy accessibility of the lipopeptide moiety by chemical synthesis opens up numerous options for further biological investigations.

Amino Acid Sequence↗

PubMed2004

MAPKAP kinase 3pK phosphorylates and regulates chromatin association of the polycomb group protein Bmi1.

Polycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation. Although the mechanisms involved in PcG-mediated repression are beginning to unravel, little is known about the regulation of PcG function. We showed previously that PcG complexes are phosphorylated in vivo, which regulates their association with chromatin. The nature of the responsible PcG kinases remained unknown. Here we present the novel finding that the PcG protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways and implicated in differentiation and developmental processes. We identified 3pK as an interaction partner of PcG proteins, in vitro and in vivo, by yeast two-hybrid interaction and co-immunoprecipitation, respectively. Activation or overexpression of 3pK resulted in phosphorylation of Bmi1 and other PcG members and their dissociation from chromatin. Phosphorylation and subsequent chromatin dissociation of PcG complexes were expected to result in de-repression of targets. One such reported Bmi1 target is the Cdkn2a/INK4A locus. Cells overexpressing 3pK showed PcG complex/chromatin dissociation and concomitant de-repression of p14(ARF), which was encoded by the Cdkn2a/INK4A locus. Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction. We speculate that phosphorylation may not only affect chromatin association but, in addition, the function of individual complex members. Our findings linked for the first time MAPK signaling pathways to the Polycomb transcriptional memory system. This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcG-mediated repression as a dynamically controlled process.

Cell Line↗

PubMed2001

The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia.

We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23). The rearrangement resulted in a 5'-MLL/FBP17-3' fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.

Artificial Gene Fusion↗

PubMed2002

Interaction of the Arabidopsis E2F and DP proteins confers their concomitant nuclear translocation and transactivation.

E2F transcription factors are required for the progression and arrest of the cell cycle in animals. Like animals, plants have evolved to conserve the E2F family. The Arabidopsis genome encodes E2F and DP proteins that share a high similarity with the animal E2F and DP families. Here, we show that Arabidopsis E2F and DP proteins are not predominantly localized to the nucleus in analyses with green fluorescent protein, and that the complete nuclear localization of some members is driven by the co-expression of their specific partner proteins. Both AtE2F1 and AtE2F3 were translocated to the nucleus and transactivate an E2F reporter gene when co-expressed with DPa but not DPb. In contrast, AtE2F2 was inactive for both nuclear translocation and transactivation even when Dpa or DPb was co-expressed. Because the DNA binding activities of the three E2Fs are equally stimulated by the interaction with DPa or DPb in vitro, the observed transactivation of AtE2F1 and AtE2F3 is DPa specific and nuclear import dependent. A green fluorescent protein fusion with an AtE2F3 mutant, in which a conserved nuclear export signal-like sequence in the dimerization domain was deleted, was localized to the nucleus. Thus, the concomitant nuclear translocation seems to be conferred by the DPa interaction to release an activity that inhibits an intrinsic nuclear import activity of AtE2Fs. Furthermore, the nuclear translocation of AtE2F3 stimulated by DPa was abolished by the deletion of the N-terminal region of AtE2F3, which is conserved among all the E2F proteins identified in plants to date. Replacement of the N-terminal region of AtE2F3 with a canonical nuclear localization signal only partially mimicked the effect of the DPa co-expression, demonstrating the function of plant E2F distinct from that observed for animal E2Fs. These observations suggest that the function of plant E2F and DP proteins is primarily controlled by their nuclear localization mediated by the interaction with specific partner proteins.

Active Transport, Cell Nucleus↗

PubMed1998

Identification of the LMO4 gene encoding an interaction partner of the LIM-binding protein LDB1/NLI1: a candidate for displacement by LMO proteins in T cell acute leukaemia.

The T cell oncogenes LMO1 and LMO2 are activated by distinct chromosomal translocations in childhood T cell acute leukaemias. Transgenic mouse models of this disease demonstrate that enforced expression of Lmo1 and Lmo2 cause T cell leukaemias with long latency and that Lmo2 expression leads to an inhibition of the T cell differentiation programme, prior to overt disease. These functions appear to be partly mediated by interaction of LMO1 or LMO2 with the LIM-binding protein LDB1/ NLI1. We have now identified a new member of the Lmo family, designated Lmo4, via its interaction with Ldb1. Lmo4 is widely expressed in mouse tissues, including adult thymus (mainly CD4, CD8-double positive T cells) and embryonic thymus (mainly CD4, CD8-double negative T cells). These characteristics imply that Ldb1-Lmo4 interaction may function in the T cell developmental programme and that enforced expression of LMO1 or LMO2 by chromosomal translocations or transgenesis may displace Lmo4 from this complex and thereby influence T cell differentiation prior to T cell tumour occurrence.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Exploiting the co-evolution of interacting proteins to discover interaction specificity.

Protein interactions are fundamental to the functioning of cells, and high throughput experimental and computational strategies are sought to map interactions. Predicting interaction specificity, such as matching members of a ligand family to specific members of a receptor family, is largely an unsolved problem. Here we show that by using evolutionary relationships within such families, it is possible to predict their physical interaction specificities. We introduce the computational method of matrix alignment for finding the optimal alignment between protein family similarity matrices. A second method, 3D embedding, allows visualization of interacting partners via spatial representation of the protein families. These methods essentially align phylogenetic trees of interacting protein families to define specific interaction partners. Prediction accuracy depends strongly on phylogenetic tree complexity, as measured with information theoretic methods. These results, along with simulations of protein evolution, suggest a model for the evolution of interacting protein families in which interaction partners are duplicated in coupled processes. Using these methods, it is possible to successfully find protein interaction specificities, as demonstrated for >18 protein families.

Amino Acid Sequence↗

PubMed2004

RNA-protein interactions in the yeast three-hybrid system: affinity, sensitivity, and enhanced library screening.

The yeast three-hybrid system has become a useful tool in analyzing RNA-protein interactions. An RNA sequence is tested in combination with an RNA-binding protein linked to a transcription activation domain (AD). A productive RNA-protein interaction activates a reporter gene in vivo. The system has been used to test candidate RNA-protein pairs, to isolate mutations in each interacting partner, and to identify proteins that bind a given RNA sequence. However, the relationship between reporter gene activation and in vitro affinity of an RNA-protein interaction has not been examined systematically. This limits interpretation of the data and complicates the development of new strategies. Here, we analyze several key parameters of the three-hybrid system, using as a model the interaction of a PUF protein, FBF-1, with a range of RNA targets. We compare activation of two reporter genes as a function of the in vitro affinity of the interaction. HIS3 and LacZ expression levels are directly related to affinity over a 10-fold range of Kd. Expression of the reporter genes also is directly related to the abundance of the activation domain fusion protein. We describe a new yeast strain, YBZ1, that simplifies screening of cDNA/AD libraries. This strain possesses a tandem, head-to-tail dimer of a high-affinity variant of MS2 coat protein, fused to a monomer of the LexA DNA-binding protein. We show that the use of this strain in cDNA library screens increases the number of genuine, sequence-specific positives detected, and at the same time reduces the background of false, RNA-independent positives.

Adaptor Proteins, Signal Transducing↗