JAB1/CSN5 interacts with the GAL4 DNA binding domain: A note of caution about two-hybrid interactions

doi:10.1016/S0300-9084(01)01329-3

Biochimie

Volume 83, Issue 10, October 2001, Pages 969-971

https://doi.org/10.1016/S0300-9084(01)01329-3 Get rights and content

Abstract

The Jun activation domain binding protein 1 (JAB1) was first identified as an interaction partner and coactivator of c-Jun. Subsequently, it was found to be a subunit of the COP9 signalosome (CSN) and termed CSN subunit 5 (CSN5). This complex regulates light-mediated development in plants and plays an essential role in a variety of organisms. A striking feature of JAB1/CSN5 is its reported interaction with a wide range of proteins and its modulation of their activity or stability. We applied the yeast two-hybrid system to screen for proteins interacting with the DNA-binding domain of the transcription factor c-Myb and found JAB1/CSN5 among the double-positive clones. To our surprise JAB1/CSN5 was shown to interact with the DNA-binding domain of GAL4 alone and had to be rejected as a false positive in the GAL4-based two-hybrid system. This finding emphasizes the necessity of particular caution when JAB1/CSN5 is found in two-hybrid screenings.

Introduction

The Jun activation domain binding protein 1 (JAB1) was first identified by Claret et al. as a c-Jun interactant and coactivator 〚1〛. Subsequently, an Arabidopsis homologue of JAB1 was characterized as a component of the COP9 signalosome (CSN) 〚2〛 and has recently been termed COP9 signalosome subunit 5 (CSN5) 〚3〛. The CSN complex was initially isolated from plants as a repressor of photomorphogenesis 〚4〛, but has later been found in a wide range of organisms, suggesting a more general function. In Schizosaccharomyces pombe the CSN complex has been demonstrated to play a role during S-phase progression 〚5〛. Furthermore, Drosophila melanogaster CSN seems to be essential for development 〚6〛. A human CSN complex has also been isolated 〚7〛, 〚8〛 and shown to harbor a kinase activity that phosphorylates IκBα, p105, c-Jun and p53 〚8〛, 〚9〛, 〚10〛. These observations indicate that the CSN complex has a key role in signal transduction. Interestingly, the subunits of CSN possess high homology with the subunits of the 19S regulatory complex of the 26S proteasome and the eIF3 translation-initiation factor complex 〚11〛, 〚12〛, 〚13〛.

Both a free form and a CSN-complexed form of JAB1/CSN5 has been detected in Arabidopsis thaliana and Drosophila melanogaster . The CSN has been observed predominantly in the nucleus, while JAB1/CSN is localized both in the nucleus and the cytoplasm 〚2〛, 〚6〛.

In mammalian cells JAB1/CSN5 has been shown to interact with an exceptionally large variety of proteins in addition to the other subunits of the CSN complex. These interaction partners include the transcription factors c-Jun and JunD 〚1〛, the cyclin-dependent kinase inhibitor p27^Kip1 〚14〛, the IκB protein Bcl-3 〚15〛, the progesterone receptor and the steroid receptor coactivator 1 (SRC-1) 〚16〛, the adhesion receptor LFA-1 〚17〛, the intracellular precursor of the lutropin/choriogonadotropin receptor (rLHR) 〚18〛, the cytoplasmic part of the cytokine macrophage migration inhibitory factor (MIF) 〚19〛, and very recently also the tumor suppressor and transcription factor p53 〚10〛. Several of these studies indicate that JAB1/CSN5 is involved in the regulation of protein stability and degradation 〚10〛, 〚14〛, 〚18〛. There is also some evidence to suggest that JAB1/CSN5 regulates the cell cycle 〚14〛, 〚19〛, 〚20〛 and transcription 〚1〛, 〚16〛, 〚17〛. For recent reviews on the CSN complex and JAB1/CSN5, see 〚21〛 and 〚22〛.

Here we report that JAB1/CSN5 interacts with the GAL4 DNA binding domain with sufficient affinity to produce false positives in GAL4-based two-hybrid screenings.

Section snippets

Two-hybrid screening

The DNA-binding domain of human c-Myb (aa 1–192) was fused in frame with the GAL4 DNA-binding domain (DBD) in the pDBT vector 〚23〛. A cDNA library from human bone marrow, fused to the GAL4 transactivation domain, was purchased from Clontech (pACT2 vector) and screened with the c-Myb DBD bait in the PJ69-4A yeast reporter strain 〚24〛. Yeast was transformed according to 〚25〛 and selected for growth on Trp^– Leu^– His^– medium with 5 mM 3-aminotriazole. Colonies growing on this medium were checked

Results and discussion

The c-Myb oncoprotein is a transcription factor, primarily expressed in hematopoietic tissues, where it regulates genes important for cell proliferation, differentiation and survival 〚29〛, 〚30〛. We applied a GAL4-based two-hybrid strategy to search for protein interaction partners of c-Myb. A cDNA library from human bone marrow was screened with the DNA binding domain (DBD) of c-Myb fused to GAL4 DBD as bait. From approximately 3 million yeast transformants screened, eight reproducibly

Acknowledgements

This work was supported by The Norwegian Research Council, The Norwegian Cancer Society and the Anders Jahres Foundation.

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Many jobs for one good cop - The COP9 signalosome guards development and defense
2012, Plant Science
Citation Excerpt :
CSN5-effector interactions were identified using a yeast two-hybrid approach [134]. Even though the authors took great care with the design of yeast experiments, it should be noted that we (Hind and Stratmann, unpublished results) and others [136,137] found that CSN5 interacts unspecifically with many proteins. Therefore, it will be important to independently confirm such experiments.
The COP9 signalosome (CSN) is a multiprotein complex that regulates the activity of CULLIN-RING E3 ubiquitin ligases (CRLs). CRLs ubiquitinate substrate proteins and thus target them for proteasomal degradation. This post-translational modification of proteins is arguably as important as reversible protein phosphorylation. The number of putative CRLs that recognize specific substrate proteins is vast, and known CRL substrates are involved in many cellular plant processes such as hormone signaling, the cell cycle, and regulation of growth, development, and defenses. By controlling the activity of CRLs, the CSN may integrate and fine-tune all of these processes. Recent research has unraveled in great mechanistic detail how the two multiprotein complexes CSN and CRL interact. As a consequence of CSN pleiotropy, complete loss of CSN function results in seedling lethality. However, recent work on plants that exhibit a partial loss of CSN function, has uncovered a role of the CSN during later life stages in processes such as development and defenses against pathogens and herbivorous insects. Not all aspects of development and defense are affected equally by CSN silencing, probably due to the differential participation and importance of CSN-regulated CRLs in these processes. This review will provide an overview of the highly complex regulation of CRL activity by CSN, and the many roles of the CSN in plant development and defense.
Symmetrical Modularity of the COP9 Signalosome Complex Suggests its Multifunctionality
2009, Structure
Citation Excerpt :
A second potential problem is that endogenous proteins might facilitate two-hybrid interactions that are not direct, and the presence of endogenous proteins might confuse the analysis. For instance, Csn5 binds to Gal4 and this might turn up as false positive in two hybrid assays (Nordgard et al., 2001). Moreover, it is noteworthy that yeast comprises endogenous PCI and MPN, proteins that might act as a bridge and interfere with the assay; specifically, a C-terminal fragment of human Csn1 has been shown to interact with yeast signaling molecules (Spain et al., 1996).
The COP9 signalosome (CSN) is an eight-subunit protein complex that is found in all eukaryotes. Accumulating evidence indicates its diverse biological functions that are often linked to ubiquitin-mediated proteolysis. Here we applied an emerging mass spectrometry approach to gain insight into the structure of the CSN complex. Our results indicate that the catalytically active human complex, reconstituted in vitro, is composed of a single copy of each of the eight subunits. By forming a total of 35 subcomplexes, we are able to build a comprehensive interaction map that shows two symmetrical modules, Csn1/2/3/8 and Csn4/5/6/7, connected by interactions between Csn1-Csn6. Overall the stable modules and multiple subcomplexes observed here are in agreement with the “mini-CSN” complexes reported previously. This suggests that the propensity of the CSN complex to change and adapt its subunit composition might underlie its ability to perform multiple functions in vivo.
The plant CDK inhibitor NtKIS1a interferes with dedifferentiation, is specifically down regulated during development and interacts with a JAB1 homolog
2008, Plant Science
CKIs from plant and animal share functional similarities although their roles have evolved in each kingdom to become adapted to the specific requirement of developmental programs. In plants, two types of CKI have been described so far and only one is related to a limited extend to the animal p27^KIP1 a fact essentially based on sequence similarities and interacting partners. To decipher the role of plant CKIs, a gain of function approach was mainly applied in Arabidopsis thaliana and showed that overexpressed CKIs inhibit cell division in planta. Here, Arabidopsis overexpressing NtKIS1a, a tobacco CKI, was used to address the question of how accumulation of the CDK/cyclin complex inhibitor NtKIS1a still allowed the development of a mature plant. We analysed the re-induction of division from differentiated cells and showed that NtKIS1a overexpression interfered with dedifferentiation. In order to test whether some discrete cells could get depleted of the inhibitory NtKIS1a protein, we analysed the presence of NtKIS1a protein in the various plant meristems. Surprisingly, its absence was revealed in the vegetative apical meristem. Characterisation of an interaction between NtKIS1a and AtAJH1, a plant JAB1 homolog, suggested that like p27^KIP1, AtAJH1 might target NtKIS1a degradation. Furthermore, NtKIS1a sub-cellular localisation was modified in the presence of AtAJH1.
COPing with hypoxia
2005, Seminars in Cell and Developmental Biology
To understand how cells respond to altered oxygenation, a frequent experimental paradigm is to isolate known components of bona fide oxygen responsive proteins. Recent studies have shown that a protein known as CSN5 or JAB1 interacts with both the HIF-1α oxygen-responsive transcription factor and its oxygen-dependent regulator, the Von Hippel-Lindau (pVHL) tumor suppressor. CSN5 is a component of the COP9 Signalosome (CSN) which is a multi-subunit protein that has high homology to the lid of the 19S lid of 26S proteasome. The exact function of the CSN5 interaction with pVHL and HIF-1α remains to be fully elucidated, but it is clear that the interaction is both oxygen dependent and that CSN5 may play different roles under oxic and hypoxic responses. Further, evidence has also been published indicating that pVHL can be potentially post-translationally modified by CSN5 (de-neddylation) and that CSN5 transcription is regulated by hypoxia as are many of the key pVHL/HIF-1α regulatory genes such as the PHDs and OS-9. This review will give a broad overview of known CSN5 and COP9 Signalosome functions and how these functions impact the pVHL/HIF-1α signaling complex and potentially other oxygen-sensitive response networks.
Ubiquitin-dependent degradation of Id1 and Id3 is mediated by the COP9 signalosome
2004, Journal of Molecular Biology
Recently, evidence is accumulating pointing to a function of the COP9 signalosome (CSN) in regulation of ubiquitination by specific ubiquitin ligases. Here, we demonstrate by mammalian two-hybrid analysis that the transcriptional regulators and substrates of the ubiquitin system Id1 and Id3, but not Id2 and Id4, bind to the CSN subunit CSN5. Pull-down experiments revealed that Id3 physically interacts with the CSN complex. Additional far Western and pull-down studies with Id3 support our two-hybrid data and show that the transcription regulator can bind to CSN5 and CSN7.
Recombinant Id3 is not phosphorylated by the CSN-associated kinases CK2 and PKD. However, it inhibits c-Jun and CSN2 phosphorylation by the isolated CSN complex and by the recombinant CK2. The inhibitors of CSN associated kinases, curcumin and emodin, significantly induce ubiquitination and proteasome-dependent degradation of transiently expressed Id3 in HeLa cells. Proteasome-dependent degradation of endogenous Id1 in HeLa cells is also stimulated by treatment with curcumin or emodin. Ubiquitination of Id3 is shown directly by cotransfection of HeLa cells with Id3 and His-ubiquitin cDNA. Curcumin increased Id3-ubiquitin conjugate formation, as shown by Western blotting and His-pull-downs. In addition, overexpression of CSN2 leads to stabilization of Id3 protein. On the basis of these data, it is speculated that CSN-mediated phosphorylation inhibits ubiquitination of Id1 and Id3.
COP9 signalosome: A multifunctional regulator of SCF and other cullin-based ubiquitin ligases
2003, Cell
COP9 Signalosome (CSN) is a fascinating protein complex whose biochemical and physiological functions are only beginning to be understood. It is conserved throughout eukaryotes and is critical to the proper development of all multicellular organisms in which its function has been explored. Recent work suggests that CSN plays a key role in sustaining the activity of SCF and other cullin-based ubiquitin ligases, which may account for its essential roles in development. Here, we summarize what is known about CSN, and discuss hypotheses for how CSN promotes the activity of SCF ubiquitin ligases.

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JAB1/CSN5 interacts with the GAL4 DNA binding domain: A note of caution about two-hybrid interactions

Abstract

Introduction

Section snippets

Two-hybrid screening

Results and discussion

Acknowledgements

Cell

J. Biol. Chem.

Cell

Trends Biochem. Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Curr. Opin. Plant Biol.

Biochim. Biophys. Acta

J. Biol. Chem.

FEMS Microbiol. Lett.

Adv. Cancer Res.

A new group of conserved coactivators that increase the specificity of AP-1 transcription factors

Nature

Arabidopsis homologs of a c-Jun coactivator are present both in monomeric form and in the COP9 complex, and their abundance is differentially affected by the pleiotropic cop/det/fus mutations

Plant Cell

Unified nomenclature for the COP9 signalosome and its subunits: an essential regulator of development

Trends Genet.