New tools to study in vivo functions of new genes and drug candidates
Li Xu * Tim Sanchez * ChaoFeng Zheng
Stratagene Cloning Systems, Inc.
Stratagene's PathDetect in vivo signal transduction pathway reporting systems are designed for specific, rapid and convenient assessment of the in vivo activation of signal transduction pathways. These reporting systems can be used to study the in vivo effects of new genes, growth factors and drug candidates on the activation of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK), cyclic AMP-dependent protein (PKA) and other signaling molecules leading to the activation of these kinases. When a fusion activator plasmid, reporter plasmid and uncharacterized gene are cotransfected into mammalian cells, either direct or indirect phosphorylation of the fusion activator protein by the uncharacterized gene product will activate transcription of the luciferase gene from the reporter plasmid. Similarly, the effects of extracellular stimuli can be studied with these systems. Very low background and as high as 200fold activation have been observed with the PathDetect c-Jun, Elk1 and CREB transreporting systems.
Signal transduction is essential for cellular growth, differentiation and the coordination of various cellular activities in multicellular organisms. Many human diseases are caused by disturbances in signal transduction. Through standard techniques in molecular biology, hundreds of genes encoding receptors, protein kinases, phosphatases and other regulatory proteins have been cloned, and many of these gene products are implicated in signal transduction pathways. Because most of the standard cloning methods do not yield information about the function of gene products, other methods are needed in order to assess the in vivo function or activity of such proteins.
Many extracellular signals modulate cellula r activities by inducing intracellular signal transduction pathways that converge at a protein kinase.1-4 Thus, the in vivo effects of a newly discovered gene, growth factor or drug candidate on the signaling molecules along these pathways can be assessed by measuring the activation or inactivation of key kinases. Many of these protein kinases, such as JNK, MAPK and PKA, can in turn phosphorylate and activate specific transcription factors. As a result, the in vivo activities of these kinases can be measured by their effect on specific transcription factors whose activity can be determined by reporter enzyme assays.
The PathDetect trans-reporting systems have been designed for rapid, easy and reliable in vivo assessment of the activation of certain signal transduction pathways. The PathDetect systems include a fusion trans-activator expression vector (figure 1, panel A) specific for the designated pathway, a reporter vector (figure 1, panel B), and plasmids that contain genes that encode proteins known to activate the trans-activator protein as a positive control (figure 1, panel C). All three fusion trans-activator plasmids share the GAL4 DNA binding domain5,6 but have different activation domains derived from c-Jun,7-9 Elk110-12 or CREB13 transcription factors, which are specifically phosphorylated and activated by JNK, MAPK or PKA, respectively. This specificity allows a protein or chemical to be assigned to a pathway. Since no transcription factor in mammalian cells can bind the GAL4 binding element efficiently, the use of GAL4 binding elements in the reporter vectors minimizes backgrou nd.
The reporter vector, the pFR-Luc plasmid (figure 1, panel B), is identical in all three PathDetect kits described here and consists of the firefly luciferase gene14 controlled by five repeats of GAL4 binding element followed by a basic transcriptional promoter (TATATA). When pFR-Luc is cotransfected into mammalian cells alone or in the presence of the PathDetect fusion activator plasmid, little or no luciferase is expressed. This level of luciferase activity will constitute the background expression level for the cell line used. When the PathDetect activator and reporter vectors are cotransfected into mammalian cells along with an uncharacterized gene, luciferase activity will either remain at the background level or will increase. If expression of the gene results in direct or indirect phosphorylation of the activator fusion protein, the luciferase gene will be expressed, which is a strong indication that the gene of interest is involved in the pathway being evaluated. If expression of the gene of interest neither directly nor indirectly results in the phosphorylation of the activator fusion protein, luciferase expression will not significantly exceed the background level.
A series of experiments (figure 3) tested the level of activity and the specificity of the PathDetect systems. For each fusion activator plasmid, known activators and nonactivators were cotransfected into HeLa cells with the pFR-Luc reporter plasmid. Cotransfections were performed using standard lipofection methods, and lysates were prepared and assayed for luciferase activity. The pFR-Luc plasmid was cotransfected into HeLa cells together with the fusion activator plasmid specific for the JNK signaling pathway ((pFA-cJun) and the pFC-MEKK control plasmid (figure 3, panel A). The MEKK protein, a known JNK activator, increased the expression of the luciferase gene by more than 100 fold (figure 3, panel A), indicating activation of the c-Jun transactivator fusion protein.7,15 The pFC-dbd plasmid, which expresses the GAL4 DNA binding domain only and no activation domain, could not be activated by overexpression of the MEKK protein. A mutated form of the c-Jun transactivator fusion protein, which had alanine residues substituted for serine 63 and 73 in c-Jun, could not be activated by MEKK (data not shown).
In a similar fashion, the specificity of the pFA-Elk1 fusion activator plasmid was also demonstrated (figure 3, panel B). The ternary complex factor Elk1 is phosphorylated and activated by MAPK.10,11 The MEKK and MEK1 proteins are both known activators of the Elk1 protein. Cotransfection of the pFA-Elk1 vector with pFC-MEK1 vector resulted in 50-fold increased expression of luciferase, whereas cotransfection with pFC-MEKK vector resulted in 200-fold increased expression of luciferase. These data are consistent with reports that both MEK1 and MEKK proteins are known activators of the MAPK pathway. 16-18
The specificity of the pFA-CREB fusion activato r plasmid is shown in figure 3, panel C. The PKA protein is a known activator of the CREB protein. When the pFA-CREB plasmid was cotransfected with the pFC-PKA plasmid and the reporter plasmid, a 100-fold increase in luciferase expression was seen. Further experiments (data not shown) using a construct with a mutation in the PKA phosphorylation site of the pFA-CREB plasmid showed that the mutated fusion activator could not be activated by the PKA protein.
In addition to activation by uncharacterized gene products, the PathDetect trans-reporting systems can be used to study the effects of physiological stimuli and drug candidates. Figure 4 shows the activation of the pFA-Elk1 vector by 10% serum, indicating activation of the MAPK signal transduction pathway. The amount of pFA-Elk1 vector used in this experiment seems limiting because the activation obtained from 10 ng of pFA-Elk1 is 10-fold greater than the activation obtained with 1 ng. Similarly, the PathDetect c-Jun trans-reporting system can be activated by ultraviolet irradiation, and the PathDetect CREB system was activated by known PKA-activating agents such as the adenylate cyclase toxin from Bordetella pertussis and forskolin (data not shown).
The PathDetect c-Jun, Elk1 and CREB trans-reporting systems give specific assessment of JNK, MAPK or PKA activity. Such specificity allows the effects of uncharacterized proteins on the JNK, MAPK and PKA pathways to be determined. These reporting systems show minimal background activity because mammalian transcripti on factors are not able to bind efficiently to the yeast-derived GAL4 UAS sequence. The high specificity and low background of the PathDetect systems have additional applications, including mapping a protein's position in the signal transduction pathway by introducing known inhibitors of the pathway and screening for drugs that promote or inhibit the activity of JNK, MAPK, PKA or other upstream signaling molecules (such as Ras, raf and JNKK).
The PathDetect in vivo signal transduction pathway reporting systems can be used to determine and map the signal transduction pathway for a gene product and to study the effects of extracellular stimuli on the JNK, MAPK or PKA signal transduction pathways. Researchers can use these systems to evaluate the in vivo role of a new gene product by the activation, inhibition or regulation of the selected signal transduction pathway. The firefly luciferase gene is used as the reporter gene in the PathDetect reporter plasmid. Therefore, the activation of these signaling pathways can be readily measured by the luciferase assay, which is convenient, sensitive and quantitative. The kits include fusion transactivator plasmids, reporter plasmids and positive and negative control plasmids that are highly purified and transfection ready.