Study the signal transduction pathway of your choice
Li Xu Chao-Feng Zheng
Stratagene Cloning Systems, Inc.
The PathDetect in vivo signal transduction pathway reporting systems are designed for assessing the in vivo activation of signal transduction pathways. The PathDetect trans-reporting systems include a unique fusion trans-activator plasmid that expresses pathway-specific trans-activator proteins fused to the yeast GAL4 DNA binding domain. To facilitate the construction and expression of new fusion trans-activator proteins, Stratagene has designed the new pFA-CMV plasmid for expressing the activation domain of any transcription factor fused to the yeast GAL4 DNA binding domain. The pFA-CMV plasmid features a multiple cloning site and the cytomegalovirus (CMV) promoter for convenient construction and high-level expression of fusion trans-activator proteins in a variety of mammalian cell lines. We describe two new fusion trans-activator plasmids (constructed with the pFA-CMV plasmid) expressing GAL4 fusions with c-Fos (208-313) and ATF2 (1-96).
Recently, Stratagene introduced the PathDetect in vivo signal transduction pathway trans-reporting systems, which are designed for specific, rapid and convenient assessment of the in vivo activation of signal transduction pathways.1 These reporting systems are used for studying the in vivo effects of new genes, growth factors, drug candidates and extracellular stimuli on the activation of c-Jun N-terminal kinase (JNK),2,3 mitogen-activated protein kinase (MAPK),4,5 cyclic AMP-dependent protein (PKA)6 and other signaling molecules leading to the activation of these kinases. The PathDetect report ing systems include unique fusion trans-activator plasmids, a reporter plasmid that encodes the firefly luciferase gene and positive control plasmids. The fusion trans-activator plasmids express pathway-specific trans-activator proteins that consist of the yeast GAL4 DNA binding domain and the activation domains of various transcription factors. When a fusion trans-activator plasmid, reporter plasmid and uncharacterized gene are cotransfected into mammalian cells, either direct or indirect phosphorylation of the fusion trans-activator protein by the uncharacterized gene product will activate transcription of the luciferase gene from the reporter plasmid (figure 1).
To increase the utility of the PathDetect systems, Stratagene has designed the pFA-CMV plasmid for convenient insertion of the activation domain of any transcription factor fused to the yeast GAL4 DNA binding domain. The pFA-CMV plasmid was used to construct two new PathDetect fusion trans-activator plasmids, pFA-cFos and pFA-ATF2 plasmids, for studying certain signal transduction pathways converging at the transcription factors c-Fos and ATF2. These plasmids will expand our capacity to study various aspects of cellular signal transduction pathways. Each plasmid is highly purified, tested and available separately for use with the PathDetect systems.
Researchers can use the pFA-CMV plasmid (figure 2) for easy cloning of the activation domain of any transcription factor sequence and subsequent expression of GAL4 fusion proteins in a variety of mammalian cells. The pFA-CMV plasmid features (1) a multiple cloning site (MCS) that offers 10 unique, conveniently arranged restric tion sites for insertion of the transcription factor activation domain sequence, (2) expression of the fusion trans-activator protein driven by the CMV promoter, a strong promoter that allows high-level constitutive expression in a variety of cell lines and (3) the neomycin-resistance gene, which facilitates selection of stable cell lines that express the fusion trans-activator protein of interest.
The backbone of the pFA-CMV plasmid was derived from Stratagenes pcmv-script vector.7 This backbone, along with a polylinker encoding 10 unique cloning sites, was fused to the DNA binding domain of GAL4. To test the functionality of the pFA-CMV plasmid, we cloned several activation domains, including the CREB, c-Jun and Elk1 domains, into the multiple cloning site of the pFA-CMV plasmid. Expression of the fusion proteins was confirmed by Western blot analysis with an antibody to the GAL4 DNA binding domain. All constructs were tested and confirmed for specificity and activation levels in transient transfection assays in Chinese hamster ovary (CHO) and HeLa cells (unpublished data). We also cloned the activation domains of the ATF2 and c-Fos transcription factors into the pFA-CMV plasmid, creating two new fusion trans-activator plasmids.
c-Fos is a proto-oncogene and an immediate-early gene.8-10 Although the direct activator of c-Fos remains somewhat elusive, c-Fos is thought to be activated by Fos-regulating kinase (FRK), which is distinct from JNK and extracellular signal-regulated kinase (ERK). A portion of c-Fos (amino acid residues 208 to 313) that contains the FRK phosphorylation site (Thr-232) has been shown to be Ras-responsive.11 The pFA-cFos plasmid was constructed by inserting the DNA fragment encoding residues 208 to 313 of rat c-Fos12 into the BamH I and EcoR I sites of pFA-CMV. Our results (figure 3A) in transient transfection assays demonstrate that the MEKK protein activates the GAL4-c-Fos fusion protein expressed from the pFA-cFos plasmid. The mechanism of this activation is not yet characterized. PKA, MEK1 and other extracellular stimuli failed to activate the GAL4-c-Fos fusion protein (data not shown).
ATF2, or activating transcription factor 2, belongs to a large family of transcription factors that recognize the ATF2/CRE elements identified in the adenovirus early gene promoters.8-10,13 ATF2 forms homodimers as well as heterodimers with other members of the ATF, Jun and Fos transcription factors. The regulation of ATF2 is not fully characterized. However, phosphorylation of Thr-69 and Thr-81 in ATF2 by JNK is known to activate the ATF2 transcription factor.13 In order to construct pFA-ATF2 (1-96), the DNA fragment encoding amino acid residues 1-96 of human ATF214,15 was inserted into the BamH I and EcoR I sites of the pFA-CMV vector. Consistent with previous reports, the GAL4-ATF2 fusion protein expressed from the pFA-ATF2 plasmid was strongly activated by the MEKK protein in transient transfection assays (figure 3A).
Stratagene has constructed three new fusion trans-activator plasmids for use with the PathDetect in vivo signal transduction pathway reporting systems. All three of these plasmids can be used in a variety of mammalian cell lin es and feature high-level expression from the CMV promoter. Researchers can conveniently insert the activation domain of any transcription factor sequence into the MCS of the pFA-CMV plasmid, and the trans-activator proteins will be expressed as GAL4 fusion proteins. The pFA-ATF2 and pFA-cFos plasmids can be used to study the upstream signaling molecules for ATF2 and c-Fos, respectively. Each plasmid is available separately; all plasmids are highly purified and tested for use with the PathDetect systems.
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