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PubMed1999

Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light.

Chemical cross-linking is a potentially useful technique for probing the architecture of multiprotein complexes. However, analyses using typical bifunctional cross-linkers often suffer from poor yields, and large-scale modification of nucleophilic side chains can result in artifactual results attributable to structural destabilization. We report here the de novo design and development of a type of protein cross-linking reaction that uses a photogenerated oxidant to mediate rapid and efficient cross-linking of associated proteins. The process involves brief photolysis of tris-bipyridylruthenium(II) dication with visible light in the presence of the electron acceptor ammonium persulfate and the proteins of interest. Very high yields of cross-linked products can be obtained with irradiation times of <1 second. This chemistry obviates many of the problems associated with standard cross-linking reagents.

2,2'-Dipyridyl↗

PubMed1999

Yeast HOS3 forms a novel trichostatin A-insensitive homodimer with intrinsic histone deacetylase activity.

Histone deacetylases such as human HDAC1 and yeast RPD3 are trichostatin A (TSA)-sensitive enzymes that are members of large, multiprotein complexes. These contain specialized subunits that help target the catalytic protein to histones at the appropriate DNA regulatory element, where the enzyme represses transcription. To date, no deacetylase catalytic subunits have been shown to have intrinsic activity, suggesting that noncatalytic subunits of the deacetylase complex are required for their enzymatic function. In this paper we describe a novel yeast histone deacetylase HOS3 that is relatively insensitive to the histone deacetylase inhibitor TSA, forms a homodimer when expressed ectopically both in yeast and Escherichia coli, and has intrinsic activity when produced in the bacterium. Most HOS3 protein can be found associated with a larger complex in partially purified yeast nuclear extracts, arguing that the HOS3 homodimer may be dissociated from a very large nuclear structure during purification. We also demonstrate, using a combination of mass spectrometry, tandem mass spectrometry, and proteolytic digestion, that recombinant HOS3 has a distinct specificity in vitro for histone H4 sites K5 and K8, H3 sites K14 and K23, H2A site K7, and H2B site K11. We propose that while factors that interact with HOS3 may sequester the catalytic subunit at specific cellular sites, they are not required for HOS3 histone deacetylase activity.

Amino Acid Sequence↗

PubMed1999

SEM1, a homologue of the split hand/split foot malformation candidate gene Dss1, regulates exocytosis and pseudohyphal differentiation in yeast.

The exocyst is an essential multiprotein complex mediating polarized secretion in yeast. Here we describe a gene, SEM1, that can multicopy-suppress exocyst mutants sec3-2, sec8-9, sec10-2, and sec15-1. SEM1 is highly conserved among eukaryotic species. Its human homologue, DSS1, has been suggested as a candidate gene for the split hand/split foot malformation disorder. SEM1 is not an essential gene. However, its deletion rescued growth of the temperature-sensitive exocyst mutants sec3-2, sec8-9, sec10-1, and sec15-1 at the restrictive temperature. Cell fractionation showed that Sem1p is mainly cytosolic but also associates with the microsomal fraction. In linear sucrose gradients, Sem1p cosedimented with the exocyst component Sec8p. In diploid cells that normally do not form pseudohyphae (S288C background), deletion of SEM1 triggered pseudohyphal growth. This phenotype was abolished after reintroduction of either SEM1 or the mouse homologue Dss1 into the cells. In diploids that have normal capacity for pseudohyphal growth (Sigma1278b background), deletion of SEM1 enhanced filamentous growth. The functionality of both SEM1 and Dss1 in a differentiation process in yeast suggests that Dss1 indeed could be the gene affected in the split hand/split foot malformation disorder. These results characterize SEM1 as a regulator of both exocyst function and pseudohyphal differentiation and suggest a unique link between these two cellular functions in yeast.

Amino Acid Sequence↗

PubMed1999

Factor-specific modulation of CREB-binding protein acetyltransferase activity.

CREB-binding proteins (CBP) and p300 are essential transcriptional coactivators for a large number of regulated DNA-binding transcription factors, including CREB, nuclear receptors, and STATs. CBP and p300 function in part by mediating the assembly of multiprotein complexes that contain additional cofactors such as p300/CBP interacting protein (p/CIP), a member of the p160/SRC family of coactivators, and the p300/CBP associated factor p/CAF. In addition to serving as molecular scaffolds, CBP and p300 each possess intrinsic acetyltransferase activities that are required for their function as coactivators. Here we report that the adenovirus E1A protein inhibits the acetyltransferase activity of CBP on binding to the C/H3 domain, whereas binding of CREB, or a CREB/E1A fusion protein to the KIX domain, fails to inhibit CBP acetyltransferase activity. Surprisingly, p/CIP can either inhibit or stimulate CBP acetyltransferase activity depending on the specific substrate evaluated and the functional domains present in the p/CIP protein. While the CBP interaction domain of p/CIP inhibits acetylation of histones H3, H4, or high mobility group by CBP, it enhances acetylation of other substrates, such as Pit-1. These observations suggest that the acetyltransferase activities of CBP/p300 and p/CAF can be differentially modulated by factors binding to distinct regions of CBP/p300. Because these interactions are likely to result in differential effects on the coactivator functions of CBP/p300 for different classes of transcription factors, regulation of CBP/p300 acetyltransferase activity may represent a mechanism for integration of diverse signaling pathways.

Acetylation↗

PubMed2000

IFN consensus sequence binding protein potentiates STAT1-dependent activation of IFNgamma-responsive promoters in macrophages.

IFNgamma, once called the macrophage-activating factor, stimulates many genes in macrophages, ultimately leading to the elicitation of innate immunity. IFNgamma's functions depend on the activation of STAT1, which stimulates transcription of IFNgamma-inducible genes through the GAS element. The IFN consensus sequence binding protein (icsbgamma or IFN regulatory factor 8), encoding a transcription factor of the IFN regulatory factor family, is one of such IFNgamma-inducible genes in macrophages. We found that macrophages from ICSBP-/- mice were defective in inducing some IFNgamma-responsive genes, even though they were capable of activating STAT1 in response to IFNgamma. Accordingly, IFNgamma activation of luciferase reporters fused to the GAS element was severely impaired in ICSBP-/- macrophages, but transfection of ICSBP resulted in marked stimulation of these reporters. Consistent with its role in activating IFNgamma-responsive promoters, ICSBP stimulated reporter activity in a GAS-specific manner, even in the absence of IFNgamma treatment, and in STAT1 negative cells. Indicative of a mechanism for this stimulation, DNA affinity binding assays revealed that endogenous ICSBP was recruited to a multiprotein complex that bound to GAS. These results suggest that ICSBP, when induced by IFNgamma through STAT1, in turn generates a second wave of transcription from GAS-containing promoters, thereby contributing to the elicitation of IFNgamma's unique activities in immune cells.

Animals↗

PubMed2000

The serine and threonine residues in the Ig-alpha cytoplasmic tail negatively regulate immunoreceptor tyrosine-based activation motif-mediated signal transduction.

The B cell antigen receptor (BCR) is a multiprotein complex consisting of the membrane-bound Ig molecule and the Ig-alpha/Ig-beta heterodimer. On BCR engagement, Ig-alpha and Ig-beta become phosphorylated not only on tyrosine residues of the immunoreceptor tyrosine-based activation motif but also on serine and threonine residues. We have mutated all serine and threonine residues in the Ig-alpha tail to alanine and valine, respectively. The mutated Ig-alpha sequence was expressed either as a single-chain Fv/Ig-alpha molecule or in the context of the complete BCR. In both cases, the mutated Ig-alpha showed a stronger tyrosine phosphorylation than the wild-type Ig-alpha and initiated increased signaling on stimulation. These findings suggest that serine/threonine kinases can negatively regulate signal transduction from the BCR.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2.

PTEN is a tumor suppressor gene mutated in human cancers. Although many mutations target the phosphatase domain, others create a truncated protein lacking the C-terminal PDZ-binding motif or a protein that extends beyond the PDZ-binding motif. Using the yeast two-hybrid system, we isolated a membrane-associated guanylate kinase family protein with multiple PDZ domains [AIP-1 (atrophin interacting protein 1), renamed MAGI-2 (membrane associated guanylate kinase inverted-2)]. MAGI-2 contains eight potential protein-protein interaction domains and is localized to tight junctions in the membrane of epithelial cells. PTEN binds to MAGI-2 through an interaction between the PDZ-binding motif of PTEN and the second PDZ domain of MAGI-2. MAGI-2 enhances the ability of PTEN to suppress Akt activation. Furthermore, certain PTEN mutants have reduced stability, which is restored by adding the minimal PDZ-binding motif back to the truncated protein. We propose that MAGI-2 improves the efficiency of PTEN signaling through assembly of a multiprotein complex at the cell membrane.

Gene Expression Regulation↗

PubMed1995

Identification of the protein 4.1 binding interface on glycophorin C and p55, a homologue of the Drosophila discs-large tumor suppressor protein.

Protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. In this investigation, synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The results show that the 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila discs-large tumor suppressor protein. Our data provide new insights into how protein 4.1, glycophorin C, p55, and their non-erythroid homologues, interact with the cytoskeleton to exert their physiological effects.

Amino Acid Sequence↗

PubMed1995

The 23-kDa acidic protein in reticulocyte lysate is the weakly bound component of the hsp foldosome that is required for assembly of the glucocorticoid receptor into a functional heterocomplex with hsp90.

The heat shock proteins hsp90 and hsp70 have been immunopurified from rabbit reticulocyte lysate in a multiprotein complex that acts as a self-sufficient protein folding machine. This immunopurified "foldosome" directs the assembly of the glucocorticoid receptor-hsp90 complex and refolds the receptor to the steroid binding state (Hutchison, K.A., Dittmar, K.D., and Pratt, W.B. (1994) J. Biol. Chem. 269, 27894-27899). Extensive washing of the immunoadsorbed foldosome eliminates a weakly bound component required for receptor heterocomplex assembly and folding. This protein factor is contained in a Centricon C-100 filtrate of lysate which reconstitutes the receptor activating activity of the washed foldosome. This hsp90-associated protein folding system is present in both animal and plant cells, and the Centricon C-100 fraction of rabbit reticulocyte lysate potentiates receptor folding directed by wheat germ lysate. We have used this ability to stimulate wheat germ lysate-directing folding of the glucocorticoid receptor as a rapid assay for the factor. We demonstrate that the activity segregates with the 23-kDa acidic protein component of the hsp90 foldosome when rabbit reticulocyte lysate is fractionated by ammonium sulfate precipitation and ion exchange chromatography. Immunoadsorption of the Centricon C-100 filtrate with a monoclonal antibody against p23 eliminates its ability to stimulate the wheat germ heterocomplex assembly/receptor folding system, and the activity is replaced by purified, bacterially expressed p23. Immunodepletion of p23 also eliminates the ability of the Centricon C-100 filtrate to reconstitute receptor activating activity of the washed foldosome and addition of purified, bacterially expressed p23 restores its activity, confirming that p23 is the weakly bound component of the foldosome complex required for refolding of the receptor to the steroid binding conformation.

Animals↗

PubMed1996

Specific interactions between the human RAD51 and RAD52 proteins.

Processing of DNA damage by the DNA double-strand break repair pathway in mammalian cells is accomplished by multiprotein complexes. However, the nature of these complexes and details of the molecular interactions are not fully understood. Interaction of the yeast RAD51 and RAD52 proteins plays a crucial role in yeast DNA homologous recombination and DNA double-strand break repair. Here, specific interactions between human RAD51 and RAD52 proteins are demonstrated both in vivo, using the yeast two-hybrid system and immunoprecipitation of insect cells co-infected with RAD51 and RAD52 recombinant viruses, and in vitro, using affinity chromatography with purified recombinant proteins. These results suggest that RAD52 may modulate the catalytic activities of RAD51 protein such as homologous pairing and strand exchange through a direct physical interaction. In addition, the domain in RAD52 that mediates this interaction was determined in vitro and in vivo. The RAD51-interacting region (amino acids 291-330) of the human RAD52 protein shows no homology with the yeast RAD52 protein, indicating that the interaction between RAD51 and RAD52 is species-specific.

Base Sequence↗

PubMed1996

Csk is constitutively associated with a 60-kDa tyrosine-phosphorylated protein in human T cells.

The protein-tyrosine kinase Csk is one of the main down-regulators of the Src family of kinases. Csk may be involved in the down-regulation of T cell receptor (TCR) signaling by C-terminal tyrosine phosphorylation of Lck and Fyn; however, it is not known how Csk activity is regulated or how it targets these Src family members. We used Jurkat T cells and normal human T cells to examine proteins that bind to the SH2 domain of Csk. In both Jurkat and normal T cells, the Src homology 2 (SH2) domain of Csk bound constitutively to a tyrosine-phosphorylated protein of 60 kDa (p60). The 60-kDa protein was detected in Csk immunoprecipitates from both unstimulated and CD3-stimulated cells. In addition to p60, a protein of 190 kDa coprecipitated with Csk, and both proteins were phosphorylated on tyrosine residues by the immunocomplex. Small amounts of GTPase-activating protein (GAP) were detected in anti-Csk immunoprecipitates, suggesting that p60 may be a GAP-associated protein. Our data demonstrate that the SH2 domain of Csk specifically associates with at least two tyrosine-phosphorylated proteins in normal human T cells, that this association is independent of TCR/CD3 activation, and that Csk may be a part of a multiprotein complex containing GAP.

Amino Acid Sequence↗

PubMed1996

Phosphatidylinositol 3-kinase is an early intermediate in the G beta gamma-mediated mitogen-activated protein kinase signaling pathway.

The beta gamma-subunit of Gi mediates mitogen-activated protein (MAP) kinase activation through a signaling pathway involving Shc tyrosine phosphorylation, subsequent formation of a multiprotein complex including Shc, Grb2, and Sos, and sequential activation of Ras, Raf, and MEK. The mechanism by which G beta gamma mediates tyrosine phosphorylation of Shc, however, is unclear. This study assesses the role of phosphatidylinositol 3-kinase (PI-3K) in G beta gamma-mediated MAP kinase activation. We show that Gi-coupled receptor- and G beta gamma-stimulated MAP kinase activation is attenuated by the PI-3K inhibitors wortmannin and LY294002 or by over expression of a dominant negative mutant of the p85 subunit of PI-3K. Wortmannin and LY294002 also inhibit Gi-coupled receptor-stimulated Ras activation. The PI-3K inhibitors do not affect MAP kinase activation stimulated by over-expression of Sos, a constitutively active mutant of Ras, or a constitutively active mutant of MEK. These results demonstrate that PI-3K activity is required in the G beta gamma-mediated MAP kinase signaling pathway at a point upstream of Sos and Ras activation.

Androstadienes↗

PubMed1996

Drosophila alpha-catenin and E-cadherin bind to distinct regions of Drosophila Armadillo.

Adherens junctions are multiprotein complexes mediating cell-cell adhesion and communication. They are organized around a transmembrane cadherin, which binds a set of cytoplasmic proteins required for adhesion and to link the complex to the actin cytoskeleton. Three components of Drosophila adherens junctions, analogous to those in vertebrates, have been identified: Armadillo (homolog of beta-catenin), Drosophila E-cadherin (DE-cadherin), and alpha-catenin. We carried out the first analysis of the interactions between these proteins using in vitro binding assays, the yeast two-hybrid system, and in vivo assays. We identified a 76-amino acid region of Armadillo that is necessary and sufficient for binding alpha-catenin and found that the N-terminal 258 amino acids of alpha-catenin interact with Armadillo. A large region of Armadillo, spanning six central Armadillo repeats, is required for DE-cadherin binding, whereas only 41 amino acids of the DE-cadherin cytoplasmic tail are sufficient for Armadillo binding. Our data complement and extend results obtained in studies of vertebrate adherens junctions, providing a foundation for understanding how junctional proteins assemble and a basis for interpreting existing mutations and creating new ones.

Amino Acid Sequence↗

PubMed1997

Constitutive binding of the transcription factor interleukin-2 (IL-2) enhancer binding factor to the IL-2 promoter.

A positive regulatory element in the interleukin-2 (IL-2) promoter, designated the antigen receptor response element-2, is essential for the induction of IL-2 gene expression upon the binding of an inducible multiprotein complex of proteins known as nuclear factor of activated T cells. In the current study, we demonstrated that the winged-helix transcription factor IL-2 enhancer binding factor (ILF) is constitutively expressed in both resting and activated Jurkat cells and binds to two adjacent sequence motifs immediately downstream of the binding site for NFAT. One of these elements has a high degree of homology with consensus binding sites for a variety of winged-helix DNA binding proteins, and the second site functions to modulate ILF binding. Mutagenesis of each of the two sequence elements required for ILF binding decreased IL-2 promoter activity when assayed in transfection assays. Although ILF bound constitutively to the IL-2 promoter, it was not detected as a component of the NFAT complex. These results suggest that important regulatory sequences in the IL-2 promoter are bound by ILF and that this binding may be involved in the control of IL-2 gene expression.

Binding Sites↗

PubMed1997

Allosteric mechanism of induction of CytR-regulated gene expression. Cytr repressor-cytidine interaction.

Transcription from cistrons of the Escherichia coli CytR regulon is activated by E. coli cAMP receptor protein (CRP) and repressed by a multiprotein complex composed of CRP and CytR. De-repression results when CytR binds cytidine. CytR is a homodimer and a LacI family member. A central question for all LacI family proteins concerns the allosteric mechanism that couples ligand binding to the protein-DNA and protein-protein interactions that regulate transcription. To explore this mechanism for CytR, we analyzed nucleoside binding in vitro and its coupling to cooperative CytR binding to operator DNA. Analysis of the thermodynamic linkage between sequential cytidine binding to dimeric CytR and cooperative binding of CytR to deoP2 indicates that de-repression results from just one of the two cytidine binding steps. To test this conclusion in vivo, CytR mutants that have wild-type repressor function but are cytidine induction-deficient (CID) were identified. Each has a substitution for Asp281 or neighboring residue. CID CytR281N was found to bind cytidine with three orders of magnitude lower affinity than wild-type CytR. Other CytR mutants that do not exhibit the CID phenotype were found to bind cytidine with affinity similar to wild-type CytR. The rate of transcription regulated by heterodimeric CytR composed of one CytR281N and one wild-type subunit was compared with that regulated by wild-type CytR under inducing conditions. The data support the conclusion that the first cytidine binding step alone is sufficient to induce.

Allosteric Regulation↗

PubMed1997

Molecular cloning of a new interferon-induced PML nuclear body-associated protein.

Transcriptional induction of genes is an essential part of the cellular response to interferons. We have established a cDNA library from human lymphoblastoid Daudi cells treated for 16 h with human alpha/beta-interferon (IFN) and made use of differential screening to search for as yet unidentified IFN-regulated genes. In the course of this study, we have isolated a human cDNA that codes for a 20-kDa protein sharing striking homology with the product of the Xenopus laevis XPMC2 gene. This new gene is induced by both type I and II IFNs in various cell lines and will be referred to as ISG20 for interferon-stimulated gene product of 20 kDa. Confocal immunofluorescence analysis of the subcellular localization of ISG20 protein reveals that it is closely associated with PML and SP100 gene products within the large nuclear matrix-associated multiprotein complexes termed the PML nuclear bodies.

Amino Acid Sequence↗

PubMed1997

Identification of elongin C sequences required for interaction with the von Hippel-Lindau tumor suppressor protein.

Elongin C is a 112-amino acid protein that is found in mammalian cells as a positive regulatory subunit of heterotrimeric RNA polymerase II elongation factor Elongin (SIII) and as a component of a multiprotein complex containing the von Hippel-Lindau (VHL) tumor suppressor protein. As a subunit of the Elongin complex, Elongin C interacts directly with the transcriptionally active Elongin A subunit and potently induces its elongation activity; in addition, Elongin C interacts with the ubiquitin-like Elongin B subunit, which regulates the interaction of Elongin C with Elongin A. As a component of the VHL complex, Elongin C interacts directly with both Elongin B and the VHL protein. Binding of the VHL protein to Elongin C was found to prevent Elongin C from interacting with and activating Elongin A in vitro, leading to the proposal that one function of the VHL protein may be to regulate RNA polymerase II elongation by negatively regulating the Elongin complex. In this report, we identify Elongin C sequences required for its interaction with the VHL protein. We previously demonstrated that the ability of Elongin C to bind and activate Elongin A is sensitive to mutations in the C-terminal half of Elongin C, as well as to mutations in an N-terminal Elongin C region needed for formation of the Elongin BC complex. Here we show that interaction of Elongin C with the VHL tumor suppressor protein depends strongly on sequences in the C terminus of Elongin C but is independent of the N-terminal Elongin C region required for binding to Elongin B and for binding and activation of Elongin A. Taken together, our results are consistent with the proposal that the VHL protein negatively regulates Elongin C activation of the Elongin complex by sterically blocking the interaction of C-terminal Elongin C sequences with Elongin A. In addition, our finding that only a subset of Elongin C sequences required for its interaction with Elongin A are critical for binding to VHL may offer the opportunity to develop reagents that selectively interfere with Elongin and VHL function.

Amino Acid Sequence↗

PubMed1997

Resistance to diverse drugs and ultraviolet light conferred by overexpression of a novel human 26 S proteasome subunit.

We have investigated the usefulness of the fission yeast Schizosaccharomyces pombe as a model organism for the discovery of novel modes of drug resistance in human cells. In fission yeast, overexpression of the essential pad1(+) gene confers pleiotropic drug resistance through a pathway involving an AP-1 transcription factor encoded by pap1(+). We have identified POH1, a human pad1 homologue that can substitute fully for pad1(+) and induce AP-1-dependent drug resistance in fission yeast. POH1 also confers P-glycoprotein-independent resistance to taxol (paclitaxel), doxorubicin, 7-hydroxystaurosporine, and ultraviolet light when transiently overexpressed in mammalian cells. Poh1 is a previously unidentified component of the human 26 S proteasome, a multiprotein complex that degrades proteins targeted for destruction by the ubiquitin pathway. Hence, Poh1 is part of a conserved mechanism that determines cellular susceptibility to cytotoxic agents, perhaps by influencing the ubiquitin-dependent proteolysis of transcription factors.

Amino Acid Sequence↗