No More Mixed Signals

from Stratagene Signal transduction seems like such a simple concept: transmitting messages within and between cells. But when you consider the hundreds of genes that are involved in signal transduction-receptors, kinases, phosphatases, to mention but a few-achieving an understanding seems like an onerous task. To make matters worse, what these signaling molecules do in a test tube, more often than not, is not what they do in a living cell. Stratagene's novel reporting systems for signal transduction pathways, PathDetect in Vivo Signal Transduction Pathway trans-Reporting Systems, offer new insights into the process by providing the opportunity to detect the activation of several signal transduction pathways in vivo, using a dual reporting/activating system. The pFR-Luc plasmid was cotransfected into the HeLa cells with the JNK activator plasmid (panel A), the Elk-1 activator plasmid (panel B) and the CREB activator plasmid (Panel C). Panel A shows a greater than 100x activation of JNK and concommitant increase in luciferase activity with MIKK; panel B shows induction of Elk 1 by both MEKK and MED1; panel C shows induction of CREB by PKA. The pFC-dbd is a negative control for the fusion activator vectors. These reporting systems contain two key elements: a fusion activator plasmid, which contains the activation domain of a signal transduction pathway-specific transactivator-joined with the DNA binding domain of the GAL4 gene of yeast; and a reporter vector, which contains the firefly luciferase gene under the control of the GAL4 binding element. Using these vectors in combination with a plasmid containing a gene of interest, it is possible to determine whether the unknown gene is part of a particular signal transduction pathway by assaying for the expression of the luciferase gene. Luciferase will be expressed only when the unknown gene directly or indirectly phosphorylates the particular activation domain contained on the activation plasmid. Three different fusion activation plasmids are currently available: c-Jun, Elk1, and CREB, which are phosphorylated by c-Jun N-terminal kinase, mitogen-activated protein kinase, and cyclic AMP dependent protein kinase, respectively. The phosphorylation of the activation domain will in turn, through its association with the GAL4 binding element, induce the expression of luciferase on the reporter plasmid. The choice of a yeast gene (GAL4) is designed to keep backgrounds low, as there are no proteins in mammalian cells that will bind to and activate the GAL4 binding element on its own. Luciferase expression is therefore dependent on the activation of the domain on the transactivator plasmid. As part of the system, Stratagene also provides positive control plasmids, which contain genes known to activate the particular domains on the transactivator plasmids hooked up to a strong promoter (CMV). For example, the protein kinase MEKK is provided as a positive control for the c-Jun activation domain, MEK1 for the Elk1 activation domain, and PKA for the CREB domain. This system is not restricted to use with cloned genes. It is also possible to assay in vivo effects of physiological stimuli or drugs on these signal transduction pathways. Company literature provides examples of the activation of the pFA-Elk1 vector by serum stimulation or the activation of c-Jun by ultraviolet irradiation. If this is a little too complicated to contemplate, a similar but simpler version of this system is contained in Stratagene's PathDetect in Vivo Signal Tranduction Pathway cis-Reporting Systems. In these systems, a single plasmid contains the luciferase reporter gene hooked up to one of five transcription enhancer elements that are activated through the signal transduction pathway. The five elements are cyclic AMP response element, serum response element, nuclear factor kB activator protein 1, and serum response factor. As each of these elements is activated by more than one signal transduction pathway, a single vector can provide information on more than a single pathway. In this system, as above, a plasmid containing a gene of unknown activity can be co-transfected with the reporter plasmid, and if the gene is involved with signal transduction of the pathway represented on the reporter vector, luciferase expression will ensue. While the cis-reporting system is useful for initial characterization of an unknown gene product, the trans system can be used to provide more specific information. Anning Lin, Assistant Professor of Pathology at University of Alabama, Birmingham, uses signal transduction reporting systems routinely in his research. Of the Stratagene systems, he said "They work very well. For people without strong molecular biology training, they provide the opportunity to address difficult and interesting questions." For more information, contact Stratagene at 800-424-5444 or visit its web site at www.stratagene.com.

No More Mixed Signals

The Scientist Staff

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