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Assess the In Vivo Activation of Signal Transduction Pathways with,,,PathDetect Reporting Systems


A review of products for signal transduction research

Assess the In Vivo Activation of Signal Transduction Pathways with PathDetect Reporting Systems

Li Xu Tim Sanchez Mary Buchanan Chao-Feng Zheng
Stratagene

Signal transduction is essential to many cellular processes, such as growth, differentiation, and coordination of cellular activities. One approach for determining the in vivo function of newly identified gene products, growth factors, and drug candidates is to identify the changes they exert on various signal transduction pathways. Since many signal transduction pathways converge at a trans-acting transcription factor or a cis-acting enhancer element, Stratagene offers the PathDetect in vivo signal transduction pathway reporting systems. The PathDetect trans-reporting systems, with specific fusion trans-activators, assess the activation of specific kinases and the corresponding signal transduction pathways by a gene product or extracellular stimuli. The PathDetect cis-reporting systems assess the activation of transcriptional factors acting on the cis-elements and provide preliminary clues on the function of the gene product or the effects of the stimulus. Together, these systems provide researchers with comprehensive and unique tools for studying the complex interactions of signal transduction.

Cells respond to environmental cues or other cells through numerous proteins or modifications of these proteins. These responses are the basis of signal transduction, whereby extracellular or intercellular signals are conveyed between cells by delicate networks of signaling molecules. By studying the intricacies of signal transduction reactions, we will learn about many aspects of cellular growth, differentiation, and coordination of cellular functions. In fact, the cause of many human diseases is attributed to disturbances in signal transduction.

With advances in molecular biology techniques and cell biology research, hundreds of genes encoding receptors, protein kinases, phosphatases, and other regulatory proteins have been cloned. Many of these gene products are implicated in signal transduction pathways. Unfortunately, standard cloning methods do not offer functional information about these newly identified proteins. Methods are required that will allow the in vivo function or activity of these proteins to be determined.

Stratagenes PathDetect in vivo signal transduction pathway reporting systems are designed to specifically, rapidly, and conveniently assess the in vivo activation of certain signal transduction pathways. At this time, Stratagene offers two types of pathway reporting systems, the PathDetect trans- and cis-reporting systems, each applicable to specific research needs. Together, these systems give researchers comprehensive tools to evaluate the role or involvement of an uncharacterized gene product, growth factor, or drug candidate in complex signal transduction interactions.

Trans-Reporting Systems: Assess Activation of Specific Pathways

Figure 1

Many extracellular signals modulate cellular activities by inducing intracellular signal transduction pathways that lead to activation of specific protein kinases.1,2,3,4 In turn, these kinases phosphorylate and activate specific transcription factors. Measuring the activation or inactivation of key kinases is one method for assessing the in vivo effects of a newly discovered gene product or drug candidate on the signaling molecules along these pathways. Each PathDetect trans-reporting system5 (Figure 1) is designed for studying the activation of specific pathways and includes the following plasmids: a pathway-specific fusion trans-activator plasmid, which is an in-frame fusion of the activation domain of a specific transcription factor, and the DNA binding domain of the yeast transcriptional activator GAL46,7; the pFR-Luc reporter plasmid for expression of the Photinus pyralis (firefly) luciferase8 gene controlled by a synthetic promoter that contains the yeast GAL4 binding sites; a positive control plasmid that constitutively expresses an activated kinase that is known to activate the trans-activator protein; and a negative control plasmid that contains only the DNA binding domain of the yeast GAL4.

The PathDetect trans-reporting systems are easy to use.5 When the pFR-Luc plasmid is transfected into mammalian cells alone or together with the pFC2-dbd plasmid, which contains only the GAL4 DNA binding domain, little or no luciferase is expressed, establishing the background level of luciferase expression for the cell line used. When a fusion trans-activator plasmid, the pFR-Luc reporter plasmid and a plasmid encoding an uncharacterized gene are cotransfected into mammalian cells, either direct or indirect phosphorylation of the fusion trans-activator protein by the uncharacterized gene product will cause increased transcription of the luciferase gene from the reporter plasmid. Thus, increased luciferase activity indicates that the uncharacterized gene product is involved in a specific pathway. In contrast, 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. Similarly, if cells transfected with pFR-Luc and a fusion trans-activator plasmid are treated with a compound or other extracellular stimulus, increased luciferase expression indicates that the signal pathway converging at the fusion trans-activator is affected by this compound or stimulus.

As with all transfection experiments, various conditions for experiments using the PathDetect systems should be optimized to achieve the best possible results. Some variables to consider for optimization include the choice of cell line, transfection method, and protocol. In particular, the amounts of the fusion activator plasmid and the expression vector for the gene of interest are the easiest variables to optimize and will have a dramatic effect on transfection results.9

Make New Trans-Activator Plasmids

To offer further research options, Stratagene has designed the pFA-CMV vector15 for use with the trans-reporting systems. Similar to all trans-activator plasmids, the pFA-CMV vector provides expression driven by the CMV promoter### and the capability for G418 selection of stable cell lines. However, the pFA-CMV vector features a multiple cloning site with 10 unique, conveniently arranged restriction sites for insertion of any activation domain sequence. By cloning a transcription factor of interest into the multiple cloning site of the pFA-CMV vector, researchers will have their own fusion trans-activator vectors for studying signaling pathways converging on transcriptional factors of interest.

Specificity of PathDetect Trans-Reporting Systems

A series of experiments (Figure 2) tested the activity and specificity of the trans-reporting systems. For each experiment, the fusion trans-activator plasmid and a known activator were cotransfected with the pFR-Luc reporter plasmid into 1.5 x 105 HeLa cells in 35-mm culture dishes. The total amount of DNA in each reaction was kept constant by adding quantities as needed of an unrelated plasmid (pBluescript plasmid). The pFC2-dbd plasmid, which expresses the GAL4 DNA binding domain but lacks an activation domain, was used as the negative control. Transfection procedure, maintenance of transfected cells, cell lysate preparation, and luciferase assay have been described previously.5

The pFR-Luc plasmid, the pFA2-cJun fusion trans-activator plasmid, and the pFC-MEKK control plasmid were cotransfected into HeLa cells. The pFA2-Jun fusion trans-activator plasmid is specific for the c-Jun N-terminal kinase10,16 (JNK) signaling pathway and the pFC-MEKK control plasmid constitutively expresses MEKK kinase17. The MEKK protein, a known JNK activator, increased the expression of the luciferase gene by more than 100-fold (Figure 2A), indicating activation of the GAL4-cJun fusion protein.5,10 The pFC2-dbd plasmid could not be activated by overexpression of the MEKK protein.

The specificity of the pFA2-CREB fusion activator plasmid is shown in Figure 2B. Cyclic AMP-dependent protein kinase (PKA) is a known activator of the CREB protein, and the pFC-PKA plasmid expresses this active kinase.14,18 When the pFA2-CREB plasmid was cotransfected with the pFC-PKA plasmid and the pFR-Luc reporter plasmid, a 100-fold increase in luciferase expression was seen. As expected, cotransfection of the pFR-Luc and pFC-PKA plasmids with the pFC2-dbd plasmid yielded only background levels of luciferase activity.

In a similar fashion, the specificity of the pFA2-Elk1 fusion activator plasmid is demonstrated (Figure 2C). Since the MEK1 protein is a known upstream activator of the Elk1 protein12 the pFC-MEK1 plasmid, constitutively expressing this kinase, was used to demonstrate the specificity of the pFA2-Elk1 plasmid. Cotransfection of the pFA2-Elk1 plasmid with the pFR-Luc plasmid and pFC-MEK1 plasmid showed a dramatic increase in luciferase activity. The parallel, control cotransfection of the pFC2-dbd, pFR-Luc, and pFC-MEK1 plasmids showed negligible luciferase activity. Similar experiments have also been performed for the pFA-ATF-cFos and pFA-CHOP plasmids (data not shown).

Cis-Reporting Systems: Looking for Initial Clues

Cis-acting enhancer elements usually bind more than one transcriptional factor and, hence, can interact with a wide spectrum of signals. For this reason, Stratagene designed the PathDetect cis-reporting systems as another approach to study signal transduction pathways. By using these systems, it is possible to assess the effects of a gene product or extracellular stimulus with specific enhancer elements. Binding of an activated transcription factor to specific enhancer elements, which are found in the promoters of various cellular genes, is often the final step in many activational series. The activation of involved signaling events is reflected by the transcriptional level of the cellular genes. For Stratagenes cis-reporting systems, activation of the luciferase reporter gene, rather than a cellular gene, is used to indicate activation status.

Figure 3

Each of the PathDetect in vivo signal transduction pathway cis-reporting systems includes one of six cis-reporter vectors and a positive control plasmid. Positive control plasmids express gene products known to activate the signaling pathway converging at the corresponding enhancer elements. The six reporter plasmids (Figure 3) of the PathDetect cis-reporting systems contain tandem repeats of one of the following enhancer elements: cyclic AMP response element (CRE), serum response element (SRE), nuclear factor B (NF-kB), activator protein 1 (AP-1), serum response factor (SRF), or p53 binding sites. These enhancer elements, together with a TATA box, control the expression of the downstream luciferase reporter gene. When a cis-reporter vector and an experimental mammalian expression construct are cotransfected into mammalian cells, increased luciferase activity indicates transcription activation and the involvement of the gene product in signaling pathways converging at the cis-acting element. Because most enhancer elements are regulated by more than one transcription activator, each enhancer element of the cis-reporting systems can be used to monitor more than one signal transduction pathway. As a consequence, the PathDetect cis-reporting systems assess only the potential involvement of a gene product in a pathway. A drawback of cis-reporting systems is the higher background of luciferase expression, which is caused by the binding of endogenous transcription factors to the enhancer elements in the reporter vectors. For a more specific assessment, the PathDetect trans-reporting systems can be used.5

Specificity of Cis-Reporting Systems

Figure 4

Experiments demonstrating the activity of the cis-reporter plasmids are shown in Figure 4. For each experiment, 1 g of a cis-reporter vector was cotransfected into 1.5 x 105 HeLa cells together with an expression vector of a known activator, either MEK kinase (MEKK) or the catalytic subunit of cAMP-dependent protein kinase (PKA). The vector without an insert was used as a negative control.17,18 Transfection procedure, maintenance of transfected cells, cell lysate preparation, and luciferase assay are previously described.5

Expression of both MEKK and PKA increased the luciferase expression from the pAP1-Luc and pSRE-Luc plasmids. However, the pCRE-Luc plasmid responded only to PKA and the pNFkB-Luc plasmid only to MEKK. These results are consistent with previous reports that both MEKK and PKA are capable of activating the expression of genes containing SRE and AP-1 or NF-kB binding elements in their promoter, while the CRE element found in somatostatin promoter is very specific to PKA.19-21 MEKK, a protein kinase known to activate the stress signaling pathways and the MAP kinase pathway, also activates signaling molecules converging at NF-kB, as measured with the pNFkB-Luc plasmid.

Conclusions

The PathDetect reporting systems offer a unique way to study signal transduction, a cellular process that is key for understanding mechanisms of both normal cellular function and disease. With the PathDetect trans-reporting systems, assess the involvement of gene products, extracellular stimuli, or drug candidates in a particular pathway using c-Jun, Elk1, CREB, ATF2, CHOP, and c-Fos activation domains. Any transcription factor of interest can be cloned and subsequently studied using the pFA-CMV fusion trans-activator cloning vector. The PathDetect cis-reporting systems provide a simple format for studying the interaction of a gene product or extracellular stimulus with the CRE, SRE, AP-1, NF-kB, p53, or SRF enhancer elements. Activation of a signaling pathway or interaction with an enhancer element is readily measured by the luciferase assay, which is convenient, sensitive, and quantitative. The plasmids included in the PathDetect reporting systems are highly purified, functionally tested, and transfection ready; all plasmids can be purchased separately.

Pathway

Transcriptional Activator

PathDetect System

Plasmids available

JNK (c-Jun N-Terminal Kinase)

c-Jun

PathDetect c-Jun Trans-Reporting System

pFA2-cJun plasmid
pFC-MEKK plasmid

MAPK (Mitogen-Activated Protein Kinase)
and JNK

Elk1

PathDetect Elk1 Trans-Reporting System

pFA2-Elk1 plasmid
pFC-MEK1 plasmid

PKA
(Cyclic AMP-Dependent Protein Kinase)

CREB

PathDetect CREB Trans-Reporting System

pFA2-CREB plasmid
pFC-PKA plasmid

p38 MAP Kinase

CHOP

PathDetect CHOP Trans-Reporting System

pFA-CHOP
pFC-MEK3

Uncharacterized pathway

c-Fos

pFA-cFos

Uncharacterized pathway

ATF2

pFA-ATF2

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