Cell-Signaling A Cascade of Kinases, Phosphatases, and Cytokines

Date: July 5, 1999Table of Cell Signaling Tools At today's research pace, new signaling mechanisms within and between cells are emerging not one by one but in a chain reaction. Each new discovery has strong implications for previously established models, sometimes overturning several assumptions at once. With such a large number of interacting systems--from cell adhesion to differentiation and apoptosis--and receptor pathways, keeping up with the wealth of cell-signaling research tools can be like hunting for nails in a hardware superstore. The problem isn't that the store doesn't have them, it's that there are so many different kinds to choose from, it's hard to know where to look first. Cell-signaling diagram supplied by Oncogene Research Cell signaling starts when a hormone, neurotransmitter, cytokine, drug, or other external signaling molecule reaches the cell membrane from outside and binds to a receptor protein on the cell surface. The ligand activates the receptor, often through a conformational change and coupling or uncoupling to other proteins in or near the cell membrane, and sets off a cascade of signaling actions within the cell. Eventually, the signaling pathway crosses the nuclear membrane to control specific events in DNA transcription and translation. Some of these processes also produce proteins, peptides, and other substances for secretion and signaling to other cells. Typical cascades involve a succession of phosphorylations and dephosphorylations of cytosolic enzymes, as in the mitogen-activated protein kinase (MAPK) pathway, but recent research also reveals signaling roles for other chemical moieties in turning key regulatory proteins on and off. A recent LabConsumer profile examined the wealth of antibody reagents and kits available for monitoring phosphorylation states and targeting specific phosphoproteins in the MAPK/ERK1, JAK/STAT, and related cell cycle regulatory pathways.1 Inhibitors, substrates, and some purified proteins are also available to study many of the kinases and phosphatases in these pathways. These cascades often generate cytosolic "second messengers" such as cyclic AMP (cAMP), cGMP, inositol phosphates, and lipids through coupling and uncoupling interactions with membrane-associated G proteins, adenylate cyclase, protein kinases, and phospholipases. Where second-messenger generation occurs in the newer signaling cascades is a question that will be answered using a combination of traditional methods and reagents developed in response to recent discoveries. Previous LabConsumer articles have reviewed assays for tyrosine kinases and phosphatases, cytokines, and growth factors, as well as tools for exploring regulatory mechanisms in transcription and apoptosis. This article will focus on a handful of relatively recent developments in cell-signaling research. MAP kinase signaling diagram supplied by Promega Because many of the same cell-signaling products are involved in so many different contexts, it pays to search under more than one category when looking them up in catalogs or company Web sites. Large commercial data-mining Web sites such as BioSupplyNet (www. biosupplynet.com) list cell signaling tools variously under immunology, inflammation, neuroscience, cy-tokine and growth factor research, apoptosis, cell cy-cle regulation, signal transduction, and cancer research. However they are listed, the most important reagents for direct cell-signaling analysis are antibodies, recombinant receptors and enzymes, activators and inhibitors, and substrates. More and more often, these reagents are being offered in fluorescence-labeled and fluorogenic forms in addition to the more traditional radiolabeled ones. Some of the proteins are also modified with histdine tags and similar ligands for affinity chromatography. Many cell-surface receptors, particularly those in tyrosine kinase and adrenergic pathways, send their signals to the inside world of the cell through their association with membrane-bound G proteins. Cholera toxin, which permanently activates the alpha-subunit, and pertussis toxin, which uncouples G proteins from their receptors, are still popular, as are labeled cyclic nucleotide substrates and analogs. Modified and heterotrimeric recombinant proteins are also widely available, as are antibodies and antibody pairs for them. Since the structures of the G-protein subunits were determined, posttranslational modification has emerged as an important area of G-protein regulation. G proteins require modification with isoprenyl groups and palmitoyl, myristoyl, and other lipid adducts for proper insertion into the membrane and functionality. Many companies offer fluorescence- or radiolabeled lipid substrates, analogs, and inhibitors for prenylation and lipid adduction. What's new about G proteins? With the shift in cancer research focus from oncogenes to their translation products has come the discovery that the wild-type versions of some oncogenes code for small functional G proteins such as Rac, Rab, Arf1, and Ras, an upstream component of the MAPK signaling pathway. Like the larger G proteins, Ras also requires posttranslational lipid modification at the C terminus--characteristically by addition of a farnesyl lipid moiety. This reaction is catalyzed by the enzyme farnesyl tranferase (FTase), identified by researchers at Squibb in 1990 and assigned a crystallographic structure by H.W. Park of Duke University in 1997. Antibodies and purified enzymes are offered by a number of companies for research. Apoptosis diagram supplied by Alexis Biochemicals More recently, the fact that Ras mutants are present in about a third of all cancers has led to a race by drug companies such as Merck to develop FTase inhibitors as candidate anticancer agents. While it is not clear how effective FTase inhibition will be as chemotherapy, these inhibitors, along with substrate analogs for farnesyl pyrophosphate and inhibitors and substrates for the closely related geranylgeranyltransferase, are likely to be handy tools in the lab. Inositol phosphate and phospholipase pathways are being studied with new inhibitors including wortmannin, quercetin, and hypericin. Wortmannin, first identified in 1974, is a fungal "hemorrhagic factor" that blocks phosphatidyl inositol-3 kinase (PI3K) and phospholipases D and A2. At 100-fold higher concentrations it also inhibits MAPK, MLCK, and PI4K. Its phospholipase-inhibiting properties were first reported in 1990, and its role as a PI3K inhibitor emerged a few years later. Since then, its popularity has accelerated--more than half of all the wortmannin papers listed by Grateful Med since 1990 have been published in the past two years. But don't forget your work gloves (and goggles and ventilation) with this inhibitor--every catalog includes the warning "Highly Toxic." Two other phospholipase system inhibitors gaining ground in cell-signaling labs are hypericin and quercetin. Hypericin, the active ingredient in St. John's wort of antidepressant fame, has been explored both as a chemotherapeutic drug and as an antiretroviral agent. One unusual feature of this plant pigment is its photoactivity: The drug makes some animals hypersensitive to light, and its nanomolar inhibitory effect on PI3K is achieved by illuminating treated cells. Quercetin is a flavone that inhibits PI3K and PLA2. Quercetins, found in wine and grape juice, have been touted by the medical news media as an ingredient that might be responsible for the lower heart disease rates seen in wine drinkers. That shouldn't stop you from using them in your laboratory; just remember to do so in moderation--and don't operate heavy machinery. Phospholipase and inositol phosphate pathways oil the gears for an even greasier pathway. Both churn out arachidonic acid, the starting material for bioactive eicosanoid lipids. Cyclooxygenases process arachidonic acid to produce prostaglandins and thromboxanes, important mediators of pain, inflammatory response, and platelet activation. Arachidonic acid, diacyl glycerol, and a host of eicosanoid lipid analogs are nonsteroidal anti-inflammatory drugs (NSAIDs) that suppress prostaglandin production, and depending on which prostaglandin they hit first, sometimes wipe out more than one down the line. Other cyclooxygenase inhibitors include caffeic acid, which has antitumor and anti-inflammatory activity and inhibits nitric oxide synthase induction in activated macrophages. Lipoxygenases turn arachidonic acid into leukotrienes, lipoxins, and other bioactive lipids. Caffeic acid and baicalein are two of the inhibitors offered for laboratory work, but a new class of asthma drugs may be good grease cutters as well. In the past few years, leukotrienes have emerged as signaling molecules in a newly discovered pathway to chronic airways. Inflammation associated with asthma, and antileukotriene drugs such as zafirlukast have recently been shown to work for a subset of patients. Nitric oxide (NO) is one molecule that seems to be everywhere at once: opening the floodgates for blood vessels and tissues through phosphodiesterase action (hence, Viagra); controlling oxidation and reduction of lipids, peroxides, and other molecules; mediating inflammatory and immune functions; and tying together many signaling pathways through interactions with Ca2+ and calmodulin, inositol phosphates, and cGMP. Three isoforms of NO synthase (NOS) and one receptor, soluble guanylate cyclase, have been identified so far. NOS generates NO in vivo by converting L-arginine to L-citrulline plus NO, using NADPH as a cofactor. Products for NO research include NOS isoforms and guanylate cyclase and antibodies to them, colorimetric and fluorescent indicator molecules oxidized by intracellular NO, NO donors and precursors, and activators and inhibitors of NOS and guanylate cyclase. T-cell signaling diagram supplied by Santa Cruz Biotechnology Much of the known regulatory machinery in the cell cycle involves phosphorylation of key enzymes and receptors. Recently, another adduct system has been gaining greater attention as a cell cycle regulatory mechanism: Covalent modification of selected proteins with multiple ubiquitin polypeptides marks them for degradation by proteasomes in an ATP-dependent manner. Proteasomal digestion was identified in the late 1980s as an important new nonlysosomal protein bre-akdown pathway. In the past five years it has become clear that proteasomes play an important role in cell cycle regulation by breaking down short-lived cell-cyle regulatory proteins such as cyclins, NF-alpha-B, and a variety of transcription factors. These large barrel-shaped protease complexes consist of a ring of multicatalytic protein-chewing subunits that form an internal chamber for the ubiquitinylated substrate. A smaller complex at each end contains several ATPases and a ubiquitin binding site. These smaller complexes recognize and unfold ubiquitinylated proteins for entry into the central proteolytic chamber so that only ubiquitin-labeled proteins are digested. The cytosolic proteasome-ubiquitin pathway has been linked in recent papers to a wide variety of cell cycle and cell-signaling processes, including activation of MHC-class I presentation, ligand-induced ubiquitinylation of receptor-tyrosine kinases, interaction with CFTR and alpha-1-antitrypsin, and degradation of secretory proteins. Products for studying proteasome and ubiquitin activity include the usual array of antibodies against ubiquitin and ubiquitin chains, ubiquitin-activating enzymes and hydrolases, proteasome subunits, and more. Recently, a new class of inhibitory peptides derived from streptomyces has been discovered. Lactacystin and lactacystin analogs were developed and patented two years ago and were quickly commercialized. Adhesion molecules and extracellular matrix proteins such as selectins and integrins are known as key players in tissue formation and cell migration. They also play an important role in cell signaling and are involved in a variety of immune, inflammatory, and other signaling events, notably in vascular damage, asthma, and fibrotic lung diseases. One adhesion protein that has been identified for signal transduction is integrin. When integrin reaches the cell membrane from outside, paxillin and focal adhesion kinase (FAK) approach the adhesion junction from the cytosolic side, and FAK undergoes autophosphorylation and kinase activation, thereby setting off a signaling cascade inside the cell. Other adhesion molecules now appear to have roles in regenerating sensory receptors after they shut down in response to overstimulation. Many companies today offer a broad selection of matrix and adhesion proteins and their antibodies. Other important tools include peptide inhibitors and substrates, ligands, matrix metalloproteinases and their inhibitors, plasma proteins and inhibitors, and as always, antibodies to just about everything. As more connections are discovered between pathways previously thought to be unrelated, the list of cell-signaling reagents is bound to grow, and so are the possibilities for new assay kits. Oncogenes and oncogene products are just beginning to find a place in our understanding of normal cell regulation and signaling mechanisms. Anticancer, antiviral, anti-inflammatory, and immunosuppressant drugs will likely have an increasing role in cell-signaling research tools as well. One promising area of development is the generation of new inhibitors from culture banks or combinatorial chemistry protocols. Many of the recent entries in this area, including lactacystin, were first identified as cytotoxic through mass screenings of fungal metabolites from a large bank of streptomyces strains collected and maintained at the University of Tokyo. Mass screening of natural products is still more effective than combinatorial chemistry as a way to generate candidate drugs and toxins. But partial combinatorial chemistry schemes that build on a known functional group with the desired inhibitory effect may open the door to targeted inhibitor generation. Finally, proteins aren't everything. The tools discussed in this article have focused on protein-substrate interactions, but DNA transcription represents another important focus of cell signaling. Regulatory protein- and peptide-specific oligonucleotide probes, plasmids, and antisense kits are just some of the gene tools for following the regulatory effects of signaling pathways inside the nucleus and the feedback loops controlling gene expression of the signal transducing receptors and enzymes themselves. Table of Cell Signaling Tools D. Wilkinson, "Tyr'd & true: immunochemical reagents and kits for studying tyrosine phophorylation," The Scientist, 13[10]: 21, May 10, 1999. The author can be contacted at djnoble@access1.net.

Cell-Signaling A Cascade of Kinases, Phosphatases, and Cytokines

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