Some estimates suggest that about 30 trillion cells make up a human body. Approximating each cell as 10 microns across—probably a conservative estimate—and lining up all 30 trillion cells would make a string that could wrap around the earth’s equator about 7.5 times. Put more bluntly, a human body consists of one heck of a lot of cells. Cellular signaling, often called signal transduction, keeps all of these cells operating as a team.

Signal transduction detects changes from outside and communicates these events to the interior of cells. Cell surface receptors—membrane-bound proteins, such as G protein-coupled, tyrosine-kinase, and ion-channel receptors—collect the external information through stimuli, or signals. Those signals usually consist of extracellular factors, like hormones or neurotransmitters. The signal-receptor complex sets off a cascade of protein interactions.

Much of today’s signal transduction research revolves around kinases. These proteins add a phosphate group to a target protein, and trigger kinase mediated signaling cascades. Nevertheless, proteins called phosphatases remove phosphate groups. Francine Fang, group leader for cell biology products at BD Biosciences Clontech, a unit of BD (Becton, Dickinson and Company) says, “Whenever we discuss kinases, we should also mention phosphatase. It is the yin-yang balance of signal transduction.”

Through studying this balance, today’s scientists gain a better understanding of how proteins work. In addition, they interpret the communication among a human being’s 30 trillion or so cells, and how they might be repaired when things go wrong.

Kinases as Keys
The pathways in cellular signaling include many kinases. A single mammalian cell might have more than a hundred different kinases. Consequently, a variety of companies—including Amersham Biosciences, BioSource International, Cell Signaling Technology, Promega, and Upstate Group—offer kinase receptors and related products.

Brian Conkle, president of Axxora, pays special attention to kinases. His company introduces European manufacturers, including Alexis Biochemicals, to the North American market. Conkle says, “Cataloging all of the protein kinases is a big issue in today’s signal transduction. We could use such a catalog to start looking at different pathways and see what a cell does in response to stimuli or to maintain viability.” He also points out the value of knowledge about specific kinases. “We need more selective inhibitors to specific kinases, as well as antibodies to look at the state of that protein or enzyme. Is it ‘on’ or ‘off’?” Conkle asks.

This expanded knowledge of kinases could supply new clinical tools. Christopher Bunker, key account manager at Cell Signaling Technology, says, “Tyrosine kinases are important in cancers, so they can be used in cancer drug discovery.” To simplify work with kinases, Cell Signaling Technology developed its Kinase Substrate Screening kit. Bunker says, “This product gets high throughput screening up and running rapidly.” This kit includes optimally paired peptide substrates and phosphorylation specific antibodies in a 96-well format. Bunker adds, “More than 50 percent of all published research using phospho-specific antibodies is done with Cell Signaling Technology antibodies. CST is also exceptional in having internal research programs in phosphoproteomics and cell signaling, which gives us a firsthand understanding and appreciation of the reagent needs and the important applications for studying signal transduction.”

BioSource offers antibodies to study individual phosphorylation sites on many receptor tyrosine kinases, including EGFR, ErbB2, c-MET, c-Kit, PDGFR, Insulin/IGF1R, and others. “Understanding the signaling pathways downstream of this class of receptors is a hot area of investigation, and these new tools provide considerably more insight as to how these receptors are regulated,” says Erik Schaefer, vice president for research and development, signal transduction at BioSource.

Chandra Mohan, director of technical services at Calbiochem-Novabiochem Corporation, points out the value of receptors in drug discovery. He says, “The VEGF, or vascular endothelial growth factor, receptor has tyrosine kinase activity and is involved in angiogenesis. If you could block this receptor and prevent growth of blood vessels to a tumor, it might stop growing or not metastasize.”

Signal transduction also involves two other classes of receptors. G protein-coupled receptors make up the largest family—over a thousand receptors. Biochemicals and reagents for the study of G protein-coupled receptors can be obtained from BioSource International and STI-Signal Transduction Products. Finally, ion channels make up the third class of receptors.

Unambiguous Antibodies
Tracking signal transduction often requires phosphorylation-specific antibodies. Schaefer says, “A protein can have 20 phosphorylation sites, and scientists need to look at key sites that work together or against each other to create the appropriate phenotype. To do that, you need selective antibodies to get fingerprints of what the protein is supposed to do.” Other companies—including BD Biosciences Pharmingen, Cell Sciences, Chemicon, Upstate, and Zymed Laboratories—also provide antibodies used to study the role of kinases.

Cyber Signals Science’s STKE (Signal Transduction Knowledge Environment) provides additional resources for cell signaling. This site supplies links to the latest literature, a forum, job postings, and the Connections Map database, which contains information on signaling components and relations between them. Users can even personalize this site to meet individual needs. www.stke.org

Phosphorylation specific antibodies unveil many characteristics of a protein. Schaefer says, “Phosphorylation determines how a protein interacts with other proteins.” He adds, “It can predict where a protein will go. Maybe a resting protein stays in the cytoplasm, but then it gets phosphorylated and goes to the nucleus or the plasma membrane.” In some cases, fully activating a protein requires multiple phosphorylation events. To keep track of all of this, BioSource offers more than 180 phosphorylation specific antibodies. Still, Schaefer says this covers only a small portion of the targets in signal transduction, so BioSource keeps coming out with more antibodies. For example, Schaefer points out that signaling involves more than 500 protein kinases and each can come in four or five variants, which further increases the complexity.

BioSource takes advantage of its own phosphorylation-specific antibodies in a variety of products. For instance, this company recently released 20 phosphorylation-specific ELISAs in 96-well formats. In addition, BioSource also created Luminex bead assays that reveal the phosphorylation of multiple cytokines as well as three kinases: AKT (also known as protein kinase B, which is encoded by the akt gene), JNK (c-Jun amino-terminal kinase), and p38. Schaefer says, “The ability to perform quantitative, higher throughput assessment of extracellular ligands as well as intracellular signaling proteins is providing considerably more insight for basic research and drug development efforts.” Upstate offers similar Luminex bead assays for a variety of kinases.

Signaling Specialists
Some companies jumped into specialized lines for signal transduction research. Biomol and Tocris Cookson, for instance, got into cell signaling from the start. These companies provide purified biochemicals and reagents for cell signaling research. Other companies—including Alexis Biochemicals, Calbiochem-Novabiochem, and PanVera—also offer a wide range of kits and reagents for cell signaling studies.

Alexis, for example, tries to cover every aspect of signal transduction research: biologicals, including kinases and phosphatases; modulators, including selective inhibitors and substrates; and antibodies for detecting interactions. Conkle of Axxora says, “If there’s a hole in the line, we try to fill that with another original manufacturer. We want to provide a complete package of products for studying function.”

In the past year, Alexis introduced several new products. For example, this company’s PI(3) Kinases help researchers determine the function of this protein’s various isotypes. Alexis also put out the first selective JNK inhibitor. External cell stress often activates this kinase, so studying its function could unravel various disease processes. To complete the newest selections, Alexis also created an antibody to the receptor interacting protein kinase 3, or RIP3.

Other companies help scientists explore the gene expression side of signal transduction. These experiments usually involve blotting techniques or gel-shift assays, but new approaches change that. Stratagene, for example, makes its PathDetect system for studying the activation of signal transduction pathways. This system provides a quantitative measurement of transcription, and reporter enzymes replace cellular genes. Consequently, PathDetect creates transcription data from enzyme assays.

Putting Together the Parts
Today’s experiments in signal transduction require biological and technological pieces. K. Gregory Moore, director of business development for drug discovery at Upstate, says that his company takes an “integrated systems approach to setting up cell signaling assays plus detection systems.” So, Upstate provides enzymes, substrates, and antibodies. “You can build your own assay,” Moore says.

Sometimes, a scientist might want more help than a do-it-yourself assay. So, Upstate provides kinase specificity testing. Moore says, “We can test an inhibitor against 85 kinases to see if the inhibition is general or specific.” Companies send Upstate groups of 20 or more compounds, and Upstate determines what kinases the compounds affect.

BD Biosciences Clontech also provides integrated kits. Tiina Sepp, this BD unit’s product manager, says, “The BD TransFactor glass array is part of our line of signal transduction products. These are miniature ELISAs on a glass array format.” These arrays consist of DNA bound to glass, and a user adds a protein extract from normal or diseased tissue. Then, antibodies reveal which DNA on the array binds to a transcription factor. Moreover, the DNA is printed in duplicate sets, so a scientist can do control and experimental runs simultaneously. Sepp adds, “With these arrays, scientists can monitor the profile of what is up-regulated and down-regulated in a specific event, say, oncogenesis.”

In addition, BD Biosciences Pharmingen offers the beginning of a line of phospho-specific antibodies for multicolor flow cytometry—BD PhosFlow reagents. These reagents use technology from Garry Nolan at Stanford University and enable qualitative and quantitative studies of the signaling pathways within single cells or distinct cell subpopulations in normal or disease states.

Combating Cancer
“About 80 percent of human cancers apparently involve oncogenic protein kinases,” says Tomi Sawyer, vice president of drug discovery at ARIAD Pharmaceuticals. To battle cancer and other diseases, Sawyer adds, “Scientists correlate a disease to specific pathways and then to specific targets within such pathways.” In other words, researchers focus on signal transduction to associate specific molecular and cellular activities with a particular disease.

“How cells communicate is also very important,” Sawyer says, “because cell-to-cell communication provides a basis for cancer metastasis.” For example, scientists at ARIAD believe that an inhibitor to Src—a protein kinase—may block cancer metastasis. Sawyer says, “We have developed Src inhibitors that are extraordinarily potent—picomolar range—and may provide novel small-molecule therapeutics treating certain cancers, including cancer metastases. Furthermore, we have identified dual selective inhibitors of both Src and Abl, the latter protein kinase being the key target of Gleevec for the treatment of leukemia.” Recently, ARIAD started clinical testing of AP23573, an inhibitor of the protein kinase mTOR, which is involved in cellular proliferation and nutrient state. A blocker of mTOR could starve and inhibit the growth of cancer cells.

Many other diseases could also come under attack through signal transduction techniques. But first, scientists must continue to explore the interactions between the tens of trillions of cells. Much remains unknown about the communication that keeps human bodies in step, but efforts at unveiling these secrets promise advances in basic research and medicine.

WEBLINKS ADVERTISERS Affymetrix DNA microarrays, based on the principles of semiconductor technology 408-731-5000 www.affymetrix.com BioSource International, Inc. reagents for functional proteomics and drug discovery 805-987-0086 www.biosource.com MDL Information Systems discovery informatics and software solutions for the life sciences 510-895-1313 www.mdl.com Takeda Chemical Industries producer of pharmaceuticals www.takeda.com/index-e.html Upstate Biotechnologies products, technology platforms, and services based on cell signaling for life science research and drug discovery 781-890-8845 www.upstatebiotech.com FEATURED COMPANIES Alexis Biochemicals signal transduction kits and reagents www.alexis-corp.com Amersham Biosciences research instruments, kits, and reagents www.amershambiosciences.com ARIAD Pharmaceuticals, Inc. drug discovery www.ariad.com Axxora, LLC signal transduction reagents www.axxora.com BD Biosciences Clontech signal transduction kits and reagents www.clontech.com BD Biosciences Pharmingen antibodies for signal transduction research www.pharmingen.com Biomol Research Laboratories, Inc. signal transduction kits and reagents www.biomol.com BioSource International, Inc. signal transduction reagents www.biosource.com Calbiochem-Novabiochem Corp. signal transduction reagents www.calbiochem.com Cell Sciences, Inc. antibodies for signal transduction research www.cellsciences.com Cell Signaling Technology signal transduction reagents, www.cellsignal.com Chemicon International, Inc. antibodies for signal transduction research www.chemicon.com PanVera, LLC kits and reagents for genomic research www.panvera.com Promega Corporation signal transduction kits and reagents www.promega.com Signal Transduction Knowledge Environment cell signaling resource site www.stke.org Stanford University university www.stanford.edu STI-Signal Transduction Laboratories signal transduction reagents www.stisignals.com Stratagene signal transduction kits and reagents www.stratagene.com Tocris Cookson, Inc. signal transduction reagents www.tocris.com Upstate USA, Inc. signal transduction kits and reagents www.upstate.com Zymed Laboratories, Inc. antibodies for signal transduction research www.zymed.com

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