A method to identify cDNAs based on localization of green fluorescent protein fusion products

Misawa, Kazuhisa; Nosaka, Tetsuya; Morita, Sumiyo; Kaneko, Akimichi; Nakahata, Tatsutoshi; Asano, Shigetaka; Kitamura, Toshio

doi:10.1073/pnas.97.7.3062

Cited by 63 publications

(39 citation statements)

References 21 publications

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“…CD28 wild type (WT), ICOS WT, and their mutant constructs were subcloned into the mammalian expression vector pcDNA3.1/Zeo (Invitrogen Life Technologies) and a pMX-GFP vector (24). The CD28RE/AP-1 Luc reporter construct was obtained from A. Weiss (University of California, San Francisco, CA).…”

Section: Plasmidsmentioning

confidence: 99%

Grb2 and Gads Exhibit Different Interactions with CD28 and Play Distinct Roles in CD28-Mediated Costimulation

Watanabe

Harada

Takeda

et al. 2006

The Journal of Immunology

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Although both CD28 and ICOS bind PI3K and provide stimulatory signal for T cell activation, unlike CD28, ICOS does not costimulate IL-2 secretion. CD28 binds both PI3K and Grb2, whereas ICOS binds only PI3K. We have generated an ICOS mutant, which can bind Grb2 by replacement of its PI3K binding motif YMFM with the CD28 YMNM motif, and shown that it induces significant activation of the IL-2 promoter. However, this mutant ICOS was insufficient to activate the NF-κB pathway. In this study, we show that Gads, but not Grb2, is essential for CD28-mediated NF-κB activation, and its binding to CD28 requires the whole CD28 cytoplasmic domain in addition to the YMNM motif. Mutagenesis experiments have indicated that mutations in the N-terminal and/or C-terminal PXXP motif(s) of CD28 significantly reduce their association with Gads, whereas their associations with Grb2 are maintained. They induced strong activity of the NFAT/AP-1 reporter comparable with the CD28 wild type, but weak activity of the NF-κB reporter. Grb2- and Gads-dominant-negative mutants had a strong effect on NFAT/AP-1 reporter, but only Gads-dominant-negative significantly inhibited NF-κB reporter. Our data suggest that, in addition to the PI3K binding motif, the PXXP motif in the CD28 cytoplasmic domain may also define a functional difference between the CD28- and ICOS-mediated costimulatory signals by binding to Gads.

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Section: Plasmidsmentioning

confidence: 99%

Grb2 and Gads Exhibit Different Interactions with CD28 and Play Distinct Roles in CD28-Mediated Costimulation

Watanabe

Harada

Takeda

et al. 2006

The Journal of Immunology

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“…In this paper, we report the identification and characterization of a novel SNARE, D12, from murine hematopoietic stem cells using a modified FL-REX (fluorescence localization-based retrovirus-mediated expression cloning) method (17). D12 shows significant homology to Use1p/Slt1p and p31.…”

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confidence: 99%

Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System

Okumura

Hatsuzawa

Tamura

et al. 2006

Journal of Biological Chemistry

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The cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to ␣-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomallysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes.

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“…Several groups have reported the generation of cDNA-GFP fusion libraries and their ectopic expression in cultured mammalian cells and plants (2,3), allowing the generation of information about protein localization on a large scale. These systems use ubiquitous promoters and do not provide any information about endogenous transcriptional regulations during cell cycle or developmental stages.…”

mentioning

confidence: 99%

A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila

Morin

Daneman

Zavortink

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

764

771

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In Drosophila, enhancer trap strategies allow rapid access to expression patterns, molecular data, and mutations in trapped genes. However, they do not give any information at the protein level, e.g., about the protein subcellular localization. Using the green fluorescent protein (GFP) as a mobile artificial exon carried by a transposable P-element, we have developed a protein trap system. We screened for individual flies, in which GFP tags fulllength endogenous proteins expressed from their endogenous locus, allowing us to observe their cellular and subcellular distribution. GFP fusions are targeted to virtually any compartment of the cell. In the case of insertions in previously known genes, we observe that the subcellular localization of the fusion protein corresponds to the described distribution of the endogenous protein. The artificial GFP exon does not disturb upstream and downstream splicing events. Many insertions correspond to genes not predicted by the Drosophila Genome Project. Our results show the feasibility of a protein trap in Drosophila. GFP reveals in real time the dynamics of protein's distribution in the whole, live organism and provides useful markers for a number of cellular structures and compartments.

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A method to identify cDNAs based on localization of green fluorescent protein fusion products

Cited by 63 publications

References 21 publications

Grb2 and Gads Exhibit Different Interactions with CD28 and Play Distinct Roles in CD28-Mediated Costimulation

Grb2 and Gads Exhibit Different Interactions with CD28 and Play Distinct Roles in CD28-Mediated Costimulation

Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System

A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila

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