Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Protein interaction partner”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 487 records · Page 27Linked to original sources
PubMed1997

A conditionally expressed third partner stabilizes or prevents the formation of a transcriptional activator in a three-hybrid system.

We describe a three-hybrid system that involves three polypeptides that allow or prevent the formation of the transcriptional activator. Beside the two-hybrid fusion proteins, the third partner is under the control of the Met25 promoter, which is positively regulated in medium lacking methionine. We document a situation where such a third partner promotes interaction between two proteins, one fused to a DNA-binding domain and the other fused to an activator domain. This is demonstrated for cdk7-MAT1 interaction stabilized by the presence of cyclin H; these three polypeptides are found either free or associated with the transcription/DNA repair factor TFIIH. We also document the capacity of our system to conditionally inhibit the interaction between two polypeptides that otherwise elicit a positive two-hybrid response. This is demonstrated for Ras-Raf interaction precluded by an excess of Raf. The presence of a methionine-regulated promoter provides an "on" or "off" switch for the formation of the transcriptional activator, thus also providing an excellent control to evaluate the activation or inhibition properties of the third partner.

Artifacts↗
PubMed2000

The protein-tyrosine phosphatase SHP-1 binds to and dephosphorylates p120 catenin.

A prominent tyrosine-phosphorylated protein of approximately 100 kDa (designated pp100) in epidermal growth factor (EGF)-stimulated A431 cells was found to be a main interaction partner of the protein-tyrosine phosphatase SHP-1 in pull-down experiments with a glutathione S-transferase-SHP-1 fusion protein. Binding was largely mediated by the N-terminal SH2 domain of SHP-1 and apparently direct and independent from the previously described association of SHP-1 with the activated EGF receptor. pp100 was partially purified and identified by mass spectrometric analysis of tryptic fragments, partial amino acid sequencing, and use of authentic antibodies as the 3A isoform of the Armadillo repeat protein superfamily member p120 catenin (p120(ctn)). Different p120(ctn) isoforms expressed in human embryonal kidney 293 cells, exhibited differential binding to SHP-1 that correlated partly with the extent of EGF-dependent p120(ctn) tyrosine phosphorylation. Despite strong phosphorylation, p120(ctn) isoforms 3B and 3AB bound, however, less readily to SHP-1. SHP-1 associated transiently with p120(ctn) in EGF-stimulated A431 cells stably transfected with a tetracycline-responsive SHP-1 expression construct, and p120(ctn) exhibited elevated phosphorylation upon a tetracycline-mediated decrease in the SHP-1 level. Functions of p120(ctn), which are regulated by tyrosine phosphorylation, may be modulated by the described SHP-1-p120(ctn) interaction.

Catenins↗
PubMed2004

Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry.

The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions.

Animals↗
PubMed2004

Synergistic transcription activation by Maf and Sox and their subnuclear localization are disrupted by a mutation in Maf that causes cataract.

Crystallin genes are selectively expressed during lens development. Maf and Sox family proteins synergistically enhanced gammaF-crystallin promoter activity in a lens cell line. Mutational analysis of the gammaF-crystallin promoter identified a composite regulatory element containing nonconsensus Maf and Sox recognition sequences. Mutations in these recognition sequences or changes in their spacing eliminated synergistic transcription activation. The transcriptional synergy was also affected by changes in the orientation of the Maf recognition sequence that had no detectable effect on binding affinity. The interaction between Maf and Sox proteins was visualized in living cells by bimolecular fluorescence complementation analysis. The N-terminal region of Maf mediated the interaction with Sox proteins in cells. Synergistic transcription activation required the N-terminal region of Maf as well as the ancillary DNA binding domain and the unique portion of the basic region that mediate specific recognition of the gammaF-crystallin promoter element. A mutation in the ancillary DNA binding domain of Maf (R288P) that has been shown to cause cataract eliminated the transcriptional activity of Maf but had no detectable effect on DNA binding in vitro. Whereas wild-type Maf was uniformly distributed in the nucleoplasm, R288P Maf was enriched in nuclear foci. Cajal bodies and gemini of coiled bodies were closely associated with the foci occupied by R288P Maf. Wild-type Maf formed complexes with Sox proteins in the nucleoplasm, whereas R288P Maf recruited Sox proteins as well as other interaction partners to the nuclear foci. The mislocalization of normal cellular proteins to these foci provides a potential explanation for the dominant disease phenotype of the R288P mutation in Maf.

Animals↗
PubMed2004

SDPpred: a tool for prediction of amino acid residues that determine differences in functional specificity of homologous proteins.

SDPpred (Specificity Determining Position prediction) is a tool for prediction of residues in protein sequences that determine the proteins' functional specificity. It is designed for analysis of protein families whose members have biochemically similar but not identical interaction partners (e.g. different substrates for a family of transporters). SDPpred predicts residues that could be responsible for the proteins' choice of their correct interaction partners. The input of SDPpred is a multiple alignment of a protein family divided into a number of specificity groups, within which the interaction partner is believed to be the same. SDPpred does not require information about the secondary or three-dimensional structure of proteins. It produces a set of the alignment positions (specificity determining positions) that determine differences in functional specificity. SDPpred is available at http://math.genebee.msu.ru/~psn/.

Algorithms↗
PubMed1995

The K protein domain that recruits the interleukin 1-responsive K protein kinase lies adjacent to a cluster of c-Src and Vav SH3-binding sites. Implications that K protein acts as a docking platform.

The heterogeneous ribonucleoprotein particle (hnRNP) K protein interacts with multiple molecular partners including DNA, RNA, serine/threonine, and tyrosine kinases and the product of the proto-oncogene, Vav. The K protein is phosphorylated in vivo and in vitro on serine/threonine residues by an interleukin 1 (IL-1)-responsive kinase with which it forms a complex. In this study we set out to map the K protein domains that bind kinases. We demonstrate that the K protein contains a cluster of at least three SH3-binding sites (P1, PPGRGGRPMPPSRR, amino acids 265-278; P2, PRRGPPPPPPGRG, 285-297; and P3, RARNLPLPPPPPPRGG, 303-318) and that each one of these sites is capable of selectively engaging c-Src and Vav SH3 domains but not SH3 domains of Abl, p85 phosphatidylinositol 3-kinase, Grb-2, and Csk. We demonstrate that the K protein domain that recruits and is phosphorylated in an RNA-dependent manner by the IL-1-responsive kinase, designated KPK for K protein kinase, is contained within the 338-425-amino acid stretch and thus is contiguous but does not include the cluster of the SH3-binding sites. K protein and KPK co-immunoprecipitate from cell extracts with either c-Src or Vav, suggesting that K protein-KPK-c-Src and K protein-KPK-Vav complexes exist in vivo. Furthermore, in the context of K protein, c-Src can reactivate KPK in vitro. The succession of kinase-binding sites contained within the K protein that allow it to form multienzyme complexes and facilitate kinase cross-talk suggest that K protein may serve as a docking platform that promotes molecular interactions occurring during signal transduction.

Amino Acid Sequence↗
PubMed1998

Pontin52, an interaction partner of beta-catenin, binds to the TATA box binding protein.

beta-catenin, the vertebrate homolog of the Drosophila Armadillo protein, has been shown to have dual cellular functions, as a component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. At Wnt signaling, beta-catenin becomes stabilized in the cytoplasm and subsequently available for interaction with transcription factors of the lymphocyte enhancer factor-1/T-cell factor family, resulting in a nuclear localization of beta-catenin. Although beta-catenin does not bind DNA directly, its carboxyl- and amino-terminal regions exhibit a transactivating activity still not well understood molecularly. Here we report the identification of an interaction partner of beta-catenin, a nuclear protein designated Pontin52. Pontin52 binds beta-catenin in the region of Armadillo repeats 2-5 and, more importantly, also binds the TATA box binding protein. We provide evidence for an in vivo multiprotein complex composed of Pontin52, beta-catenin, and lymphocyte enhancer factor-1/T-cell factor. Our results suggest involvement of Pontin52 in the nuclear function of beta-catenin.

Amino Acid Sequence↗
PubMed2003

Looking for an insulin pill? Use the BRET methodology!

Insulin exerts its biological effects through a plasma membrane receptor that possesses a tyrosine-kinase activity. This tyrosine-kinase activity depends on the autophosphorylation of the receptor on tyrosine residues and on its dephosphorylation by protein tyrosine-phosphatases. The discovery of pharmacological agents that specifically stimulate the autophosphorylation of the insulin receptor or inhibit its dephosphorylation will be of great importance for the treatment of insulin resistant or insulin deficient patients. Bioluminescence Resonance Energy Transfer (BRET) has developed in recent years as a new technique to study protein-protein interactions. In the BRET technique, one partner is fused to Renilla luciferase, whereas the other partner is fused to a fluorescent protein (e.g. YFP, Yellow Fluorescent Protein). The luciferase is excited by addition of its substrate, coelenterazine. If the two partners interact, resonance energy transfer occurs between the luciferase and the YFP, and a fluorescent signal, emitted by the YFP, can be detected. Our work indicates that this methodology could be an important tool for the search of molecules that activate insulin receptor autophosphorylation or that inhibit its dephosphorylation. Indeed, we first showed that the activation of the insulin receptor by different ligands can be monitored using a chimeric receptor with one B-subunit fused to Renilla luciferase and the other B-subunit fused to YFP. The conformational changes induced by different ligands could be detected as an energy transfer (BRET signal) between the luciferase and the YFP, that reflects the activation state of the receptor. This methodology allows for rapid analysis of the effects of agonists on insulin receptor activity and may therefore be used in high-throughput screening for the discovery of molecules with insulin-like properties. More recently, we demonstrated that the BRET methodology could also be used to monitor the interaction of the insulin receptor with protein tyrosine-phosphatase 1B, one of the main tyrosine-phosphatase that controls its activity. HEK cells were co-transfected with the insulin receptor fused to Renilla luciferase and a substrate-trapping mutant of PTP1B (PTP1B-D181A) fused to YFP. Insulin-induced BRET signal could be followed in real time for more than 30 min. Therefore, this methodology can also be used in high-throughput screening for the search of molecules that will specifically disrupt the interaction between the insulin receptor and PTP1B.

Administration, Oral↗
PubMed2002

DAP-like kinase interacts with the rat homolog of Schizosaccharomyces pombe CDC5 protein, a factor involved in pre-mRNA splicing and required for G2/M phase transition.

DAP-like kinase (Dlk, also termed ZIP kinase) is a leucine zipper-containing serine/threonine-specific protein kinase with as yet unknown biological function(s). Interaction partners so far identified are either transcription factors or proteins that can support or counteract apoptosis. Thus, Dlk might be involved in regulating transcription or, more generally, survival or apoptosis. Here we report on a new interaction partner, the rat homolog of Schizosaccharomyces pombe CDC5 protein, a presumptive transcription and splicing factor involved in the G(2)/M transition. In vitro, rat CDC5 forms complexes with, but is not phosphorylated by, Dlk. Rather, it was phosphorylated by an associated kinase which was identified as CK2. The interaction domain of Dlk was mapped to the leucine zipper, while that of CDC5 was mapped to the C-terminal region between residues 500 and 802. In vivo, both proteins co-localize perfectly in distinct speckle-like structures in the nucleus, some of which overlap with promyelocytic leukemia protein. Interestingly, splicing factor SC35, which also resides in speckles, was partially displaced upon overexpression of either CDC5 or Dlk, perhaps due to phosphorylation by Dlk. Together with previous data, these results suggest that Dlk might play a role in coordinating specific transcription and splicing events.

Animals↗
PubMed2002

The t(3;21) fusion product, AML1/Evi-1 blocks AML1-induced transactivation by recruiting CtBP.

AML1/Evi-1 is a chimeric protein that is derived from t(3;21), found in blastic transformation of chronic myelogenous leukemia. It is composed of the N-terminal AML1 portion with the DNA-binding Runt domain and the C-terminal Evi-1 portion. It has been shown to dominantly repress AML1-induced transactivation. The mechanism for it has been mainly attributed to competition with AML1 for the DNA-binding and for the interaction with PEBP2beta (CBFbeta), a partner protein which heterodimerizes with AML1. It was recently found that Evi-1 interacts with C-terminal binding protein (CtBP) to repress TGFbeta-induced transactivation. Here, we demonstrate that AML1/Evi-1 interacts with CtBP in SKH1 cells, a leukemic cell line which endogenously overexpresses AML1/Evi-1 and that AML1/Evi-1 requires the interaction with CtBP to repress AML1-induced transactivation. The association with CtBP is also required when AML1/Evi-1 blocks myeloid differentiation of 32Dcl3 cells induced by granulocyte colony-stimulating factor. Taken together, it is suggested that one of the mechanisms for AML1/Evi-1-associated leukemogenesis should be an aberrant recruitment of a corepressor complex by the chimeric protein.

Alcohol Oxidoreductases↗
PubMed2005

Purification and properties of rab6 interacting proteins.

A crucial step in the characterization of novel partners of Rab proteins is the confirmation that they indeed interact together by techniques other than the yeast two-hybrid assay used to discover them. Some methods and clues that would help to discriminate between putative interactors are summarized. Pull-down, co-immunoprecipitation, and gel filtration experiments are described as ways of checking protein-protein interaction in vitro and in vivo.

Adaptor Proteins, Signal Transducing↗
PubMed2003

The cyclin-dependent kinase 11(p46) isoform interacts with RanBPM.

We identified Ran-binding protein (RanBPM) as an interacting partner of the caspase-processed C-terminal domain of cyclin-dependent kinase 11 (CDK11(p46)) by using the yeast two-hybrid system. CDK11(p110) protein kinases are members of the cyclin-dependent kinase superfamily. During staurosporine-, Fas-, and tumor necrosis factor alpha-induced apoptosis caspase-processed activated CDK11(p46) is generated from larger CDK11(p110) isoforms. CDK11(p46) promotes apoptosis when it is ectopically expressed in human cells. However, the mechanism of signal transduction through CDK11(p46) is still unclear. In this study, we demonstrate that CDK11(p46) directly interacts with RanBPM in vitro and in human cells. RanBPM contains a conserved SPRY (repeats in splA and Ryr) domain and is localized both in the nucleus and cytoplasm. The SPRY domain of RanBPM is responsible for the association between CDK11(p46) and RanBPM. Furthermore, we show that CDK11(46) phosphorylates RanBPM.

Adaptor Proteins, Signal Transducing↗
PubMed2004

VAAMANA--a BEL1-like homeodomain protein, interacts with KNOX proteins BP and STM and regulates inflorescence stem growth in Arabidopsis.

Plant shoot growth depends on the activity of the shoot apical meristem (SAM), where organ primordia are initiated. In turn, the function of the SAM depends on the activities of homeodomain (HD) proteins of the knotted1-like homeobox (KNOX) class [Long et al., Nature 379 (1996) 66; Vollbrecht et al., Development 127 (2000) 3161]. In plants, KNOX proteins have been shown to interact specifically with the BEL1-like (BELL) class of homeodomain proteins [Bellaoui et al., Plant Cell 13 (2001) 2455; Muller et al., Plant 27 (2001) 13; Smith et al., Proc. Natl. Acad. Sci. USA 99 (2002) 9579], through a domain conserved between plants and animals. We have isolated a mutation in a BELL homeobox gene VAAMANA (VAN) that causes a dwarf phenotype. In addition, van inflorescence stems have clusters of cauline leaves; typically three are produced at each node. VAN interacts specifically with the class I KNOX proteins SHOOTMERISTEMLESS (STM), BREVIPEDICELLUS (BP), and KNAT6 (K6), and nuclear localisation of a VAN-GFP fusion depends on co-expression of STM or BP in tobacco leaves. This suggests that localisation of VAN, like that of the animal PBC homeodomain protein [Rieckhof et al., Cell 91 (1997) 171; Berthelsen et al., Genes Dev. 13 (1999) 946], is also regulated by interaction with a partner homeodomain protein.

Amino Acid Sequence↗
PubMed2002

Photoaffinity labeling in drug discovery and developments: chemical gateway for entering proteomic frontier.

One of the major events occurring at biological interfaces is the specific recognition of bioactive ligands by their receptor proteins. The elucidation of interacting partners is an immediate entrance into the discovery of medicinal leads. The method of photoaffinity labeling enables the direct probing of target protein through a covalent bond introduced between a ligand and its specific receptor. Thus, the photoaffinity labeling is applied in two stages of drug discovery and development processes. First, the method is useful for the screening of early leads. If the binding site analysis of target protein is important for defining a particular pharmacophore, the photoaffinity labeling will give the structural information at the contact point of drugs with receptors. Second, emerging new technologies, combinatorial chemistry, recombinant DNA techniques, and high-throughput analysis, are extending the potential of photoaffinity labeling to become a rapid and more sensitive means for the identification of drug-receptor pairs as well as the elucidation of molecular recognition mechanism at drug-receptor interfaces. This review focuses on several recent impacts of photoaffinity labeling as a useful tool for drug discovery and developments.

Animals↗
PubMed2005

Interacting proteins and differences in nuclear transport reveal specific functions for the NAP1 family proteins in plants.

Nucleosome assembly protein 1 (NAP1) is conserved from yeast to human and facilitates the in vitro assembly of nucleosomes as a histone chaperone. Inconsistent with their proposed function in the nucleus, however, many NAP1 proteins had been reported to localize in the cytoplasm. We investigated the subcellular localization of tobacco (Nicotiana tabacum) and rice (Oryza sativa) NAP1 family proteins first by identification of interacting partners and by direct examination of the localization of green fluorescent protein-tagged proteins. Through treatment of tobacco cells with leptomycin B and mutagenesis of nuclear export signal, we demonstrated that Nicta;NAP1;1 and Orysa;NAP1;1 shuttle between the cytoplasm and the nucleus. Together with the demonstration that tobacco NAP1 proteins bind histone H2A and H2B, our results support the current model and provide additional evidence that function of NAP1 as histone chaperones appears to be conserved in plants. In addition, we show that tobacco NAP1 proteins interact with tubulin and the mitotic cyclin Nicta;CYCB1;1, suggesting a role for NAP1 in microtubule dynamics. Interestingly, in spite of their high homology with the above NAP1 proteins, the other three tobacco proteins and Orysa;NAP1;2 did not show nucleocytoplasmic shuttling and were localized only in the cytoplasm. Moreover, Orysa;NAP1;3 that lacks a typical nuclear localization signal sequence was localized in both the cytoplasm and the nucleus. Finally, we show that only Orysa;NAP1;3 could be phosphorylated by casein kinase 2alpha in vitro. However, this phosphorylation was not responsible for nuclear import of Orysa;NAP1;3 as being demonstrated through mutagenesis studies. Together, our results provide an important step toward elucidating the molecular mechanism of function of the NAP1 family proteins in plants.

Active Transport, Cell Nucleus↗
PubMed2002

Identification and characterization of AGTRAP, a human homolog of murine Angiotensin II Receptor-Associated Protein (Agtrap).

Several classes of cytoplasmic proteins have been found to interact specifically with the carboxyl-terminal cytoplasmic region of the angiotensin II type 1 (AT(1)) receptor to regulate different aspects of AT(1) receptor physiology. The murine Angiotensin II Receptor-Associated Protein (Agtrap) is a new member of them. We have recently cloned a new human gene cDNA that codes for a homolog of the murine Agtrap protein from a human fetal brain cDNA library. The deduced polypeptide product of the cDNA is 22 kDa in size, and its DNA and amino acid sequences are 85 and 77% identical to those of the mouse Agtrap gene, respectively. Hence we have named it the human Angiotensin II Receptor-Associated Protein (AGTRAP) gene. The mRNA of AGTRAP was most abundantly expressed in kidney, heart, pancreas and thyroid. Using the yeast two-hybrid screening of a human fetal brain cDNA library, we have identified a new interaction partner of the human AGTRAP protein, RACK1 (Receptor of Activated Protein C Kinase). The AGTRAP-RACK1 interaction was confirmed by GST fusion protein pull-down assays, co-immunoprecipitation and surface plasmon resonance. We suggest that the AGTRAP-RACK1 interaction may help to recruit signaling complex to the AT(1) receptor to affect AT(1) receptor signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2006

The fellowship of the rings: distinct pools of proliferating cell nuclear antigen trimer at work.

The proliferating cell nuclear antigen (PCNA) is a homotrimeric ring-shaped protein that, by encircling DNA, may function as a sliding platform for proteins participating in various DNA transactions. PCNA plays a fundamental role in DNA replication and repair, but also in postreplicative events, like DNA methylation, chromatin assembly and remodeling, sister chromatid cohesion, and coordinates these activities with cell cycle control. However, relevant aspects of PCNA function are still not well understood, like the role of PCNA in the association with partner proteins, and how multiple protein interactions are orchestrated. Based on emerging evidence, I suggest that 1) PCNA interacting proteins may be reclassified in three major categories, namely, a) cell cycle control; b) DNA replication/repair; c) chromatin regulation/transcription. 2) PCNA is a negative regulator, rather than a processivity/recruitment factor, of chromatin-modifying enzymes. 3) At DNA replication sites, PCNA function may be envisaged with a model of "dynamic hand-off" of interacting partners that rapidly and transiently exchange in a mutually exclusive manner, while cyclin-dependent kinase (Cdk) 2 (CDK2) is stably bound to PCNA. The partner exchange might occur through a conformational change of the PCNA/protein/DNA complex allowing CDK2 to phosphorylate the partner protein, thereby enabling its hand-off from PCNA.

Cyclin-Dependent Kinase 2↗
PubMed2001

A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment.

We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

Adaptor Proteins, Signal Transducing↗