Chapter 7: Cell Signaling

Introduction

Signal transduction is one of the most widely studied areas in biology. Extracellular information perceived at the surface of a cell must be translated into an intracellular response that involves a complex network of interwoven signaling cascades. These signaling events ultimately regulate such cellular responses as proliferation, differentiation, secretion and apoptosis. Signal transduction cascades are generally triggered by the binding of extracellular ligands, such as growth factors, cytokines, neurotransmitters or hormones, to a cell-surface receptor. These receptors transmit the stimulus to the interior of the cell, where the signal is amplified and directed a targeted signaling pathway.

The propagation and amplification of the primary signal involves a wide array of enzymes with specialized functions. Many of these signaling enzymes propagate the signal by post-translationally modifying other cellular proteins that are involved in the signaling cascade. Protein phosphorylation, one of the most common post-translational modifications, plays a dominant role in almost all signaling events and involves the transfer of a phosphate group from adenosine triphosphate (ATP) to the target protein (van der Geer et al. 1994). In general, phosphorylation either activates or inactivates a given protein to perform a certain function. Protein kinases and phosphatases are the enzymes responsible for determining the phosphorylation state of cellular proteins and, thus, whether a signal gets transduced within a cell. Changes in the level, subcellular localization and activity of kinases and phosphatases have consequences for normal cell function and maintenance of cellular homeostasis (De Meyts, 1995; Denton and Tavare, 1995).

The MAPK Pathways

The Mitogen-Activated Protein Kinase (MAPK) signaling pathways play an important role in signal transduction in eukaryotic cells, where they modulate many cellular events including: mitogen-induced cell cycle progression through the G1 phase, regulation of embryonic development, cell movement and apoptosis, and cell differentiation (Murray, 1998; Schaeffer and Weber, 1999). These evolutionarily conserved pathways are organized in three-kinase modules consisting of a MAP kinase, an activator of MAP kinase (MAP Kinase Kinase or MEK) and a MAP Kinase Kinase Kinase (MEK Kinase, MEKK, or MAPK Kinase Kinase). There are at least three distinct MAP kinase signal transduction pathways in mammalian cells, each named after the particular MAPK associated with it (Figure 7.1). These include the extracellular signal-regulated kinases, ERK 1/2 (also known as MAPKs), the c-JUN N-terminal kinases/stress-activated protein kinase (JNK/SAPK) and the p38 kinases. An animated presentation highlighting some of the events during MAPK signaling is available.

Signal transduction cascades involving ERK/MAPK enzymes are also regulated by the activities of a variety of protein phosphatases. Several dual-specificity protein phosphatases have been identified that can differentially dephosphorylate MAPK, JNK or p38 enzymes (Neel and Tonks, 1997; Ellinger-Ziegelbauer et al. 1997). In addition, individual Ser/Thr (e.g., PP2A) or Tyr (e.g., PTP1) phosphatases also appear to regulate the activity of the ERK/MAPK enzymes by dephosphorylating either core residue (Hunter, 1995; Keyse, 1995; Alessi, 1995; Doza, 1995). Thus, the cell can tightly regulate the activity of the ERK/MAPK enzymes by judicious use of different combination of MEKS, mono- and dual-specificity protein phosphatases and the subcellular localization of each enzyme to elicit the appropriate physiological response (Payne, 1991; Zhang, 2001).

The Phosphoinositol 3-Kinases (PI3-Ks)

Phosphoinositol 3-Kinases (PI3-Ks) catalyze the transfer of the gamma phosphate group from ATP to the 3-OH of three different substrates: phosphotidylinasitol (PI), phosphatidyl inositol 4 phosphate (PI4P), and phosphatidyl inositol 4,5 phosphate (PI(4,5)P₂; Vanhaesebroeck et al. 2001). PI3-Ks modulate the levels of PIs in cells to influence many cellular functions including cell growth, gluconeogenesis and glycolysis, motility, and cell development and differentiation. These enzymes are divided into three classes, largely based on substrate preferences (Rameh and Cantley, 1999; Okkenhaug and Vanhaesebroeck, 2003). This review will focus on the class I PI3-Ks. Class I PI3-Ks comprise a 110kDa catalytic subunit (p110) and a regulatory/adaptor subunit. The class I PI3-Ks can be divided into subclasses A and B based on their upstream signaling partners. Class IA PI3-Ks signal downstream of tyrosine kinases; class IB PI3-Ks signal downstream of G protein-coupled receptors (GPCRs; Vanhaesebroeck et al. 2001).

The adaptor/regulatory subunit of Class IA PI3-K contains two Src homology 2 (SH2) domains through which it can bind to activated receptor tyrosine kinases (RTKs) or to cytosolic tyrosine kinases such as Src family kinases or JAK kinases. Binding to phosphotyrosine in the RTKs is thought to bring the cytosolic PI3-Ks to the membrane where the PI substrates reside (Cooray, 2004; Vanhaesebroeck et al. 2001). All mammalian cells investigated to date express at least one Class IA PI3-K, and stimuli that result in tyrosine kinase activity generally lead to class IA PI3-K activation (Vanhaesebroeck et al. 2001).

After the PI3-K is activated, it can phosphorylate its phosphoinositide substrates. PI can be phosphorylated to produce PI3P, which appears to bind selectively proteins that contain an FYVE domain, a Zn²⁺ finger domain that has been found in a diverse group of proteins, many of which are involved in membrane trafficking (Vanhaesebroeck et al. 2001). PI(4,5)P₂ can be phosphorylated by activated PI3-K to produce phosphotidyl inositol 3,4,5 triphosphate (PIP₃). PIP₃ in turn interacts with a variety of molecules, such as Akt Kinase (also called PKB) via the pleckstrin homology (PH) domains of these downstream targets (Cooray, 2004). Interaction of PIP₃ with Akt allows phosphorylation of Akt by PDK 1 and subsequent activation of Akt (Rameh and Cantley, 1999). Akt is a serine/threonine kinase that phosphorylates many different target proteins. Many pro-apoptotic proteins are substrates of Akt that are inactivated by Akt phosphorylation, including Bad, caspase-9 and GSK-3 (Cooray, 2004). Akt also regulates transcription of many genes including forkhead transcription factors and NF-κB (Cooray, 2004; Sliva, 2004). An animated presentation that shows some events associated with the PI3-K pathway is available.

In Drosophila, PI3-K is implicated in regulating cell growth without affecting cell division rates. Studies of wing imaginal discs show that overexpression of the p110 subunit of PI3-K results in increased growth. Reducing activity reduces the size of the wing imaginal disc, producing adult flies with small wings (Vanhaesebroeck et al. 2001). This size effect does not appear to be tied to differences in cell division rates. Similar results have been observed in studies of PI3-K signaling in mouse heart, where cell growth is affected but cell division rates are not.

PI3-K signaling is also implicated in progression to S phase and DNA synthesis in cells. PI3-K activity is tied to the accumulation of cyclin D in cells and may act at a variety of levels, transcription, post transcription, and post translation, to affect cyclin D accumulation. PI3-Ks may also play roles in relieving inhibition of the cell cycle (Vanhaesebroeck et al. 2001).

PI3-Ks and Cancer

PI3-Ks are implicated in breast, colon, endometrial, head and neck, kidney, liver, lymphoma, melanoma, sarcoma and stomach cancers (Sliva, 2004), making them an important therapeutic target for human cancer therapy. In fact, PI3-K mutations found in human cancers have oncogenic activity (Kang et al. 2005), and PI3-K might mediate its activity through mTOR (Aoki et al. 2001). Cell motility is one of many cell functions influenced by PI3-K signaling. Invasive breast cancer MDA-MB-231 cells, have higher than normal PI3-K activity. Inhibition of PI3-K by dominant negative mutations of the PI3-K regulatory/adaptor subunits or treatment with LY 294002 or wortmannin (PI3-K-specific inhibitors) suppresses motility of these cells (Sliva, 2004). Studies indicate that PI3-K may play a role in actin cytoskeleton rearrangements, perhaps through guanosine nucleotide exchange factors and GTPase-activating proteins (Vanhaesebroeck et al. 2001).

PI3-Ks can also activate NF-κB through a variety of mechanisms in different cells. In HepG2 cells, IL-1 stimulates the phosphorylation and activation of NF-κB through a PI3-K-dependent pathway (Sliva, 2004). Expression of a dominant negative regulatory subunit of PI3-K or treatment with PI3-K inhibitors suppressed NF-κB activation as well as motility in the MDA-MB-231 cell line (Sliva, 2004).

A viral oncogene that encodes a variant PI3-K was isolated from a chicken retrovirus. Expression of this oncogene increases cellular PI, activates Akt and transforms chicken embryo fibroblasts (Rameh and Cantley, 1999). These oncogenic effects may be mediated through the pathways by which PI3-Ks normally influence cell growth, cell cycle progression and transcription.

PI3-K signaling is balanced by the activities of inositol lipid phosphatases. The most well studied PI phosphatase is PTEN, which was first described as a tumor suppressor that is deleted or mutated in several human cancers (Rameh and Cantley, 1999). Furthermore, physical interaction of PTEN with the MSP58 oncogene inhibits cellular transformation, thus validating the role of PTEN as a tumor suppressor (Okamura et al. 2005).

Investigating Phosphatases and Kinases as Potential Therapeutic Targets

The human genome is reported to contain 518 protein kinases that are involved in phosphorylation of 30% all cellular proteins (Manning et al. 2002). Taken together, genes for protein kinases and phosphatases represent five percent of the human genome (Cohen, 2001). Many other phosphotransferases play equally important roles in cellular reactions that use ATP as substrate but are not classified as protein kinases. These include PI3-kinases (Shears, 2004), lipid kinases such as sphingosine kinases (French et al. 2003) and sugar kinases such as glucokinase (Grimsby et al. 2003). Changes in the level, activity or localization of these kinases, phosphotransferases and phosphatases greatly influence the regulation of key cellular processes. Because of the role that these enzymes play in cellular functions and in various pathologies, they represent important drug targets (Cohen, 2002). By 2002, more than twenty-six small molecule inhibitors of protein kinases alone were either approved for clinical use or in phase I, II or III clinical trials (Cohen, 2002; Pearson and Fabbro, 2004).

This chapter describes the tools available for investigating the activities of kinases and phosphatases that are involved in signaling cascades. We describe a variety of technologies including luminescent and fluorescent assays for kinase and phosphatases. The phosphorylation state of the substrates of kinases can also be informative when studying cell signaling. We describe a variety of antibodies for detecting the phosphorylated forms of some kinase substrates as well as kinase substrates and inhibitors that can be used as tools to analyze kinase activities in samples.

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Kinase Activity Assays

Luminescent Kinase Assays

Kinases are enzymes that catalyze the transfer of a phosphate group from ATP to a substrate. The depletion of ATP as a result of kinase activity can be monitored in a highly sensitive manner through the use of Kinase-Glo^® or Kinase-Glo^® Plus Reagent, which uses luciferin, oxygen and ATP as substrates in a reaction that produces oxyluciferin and light (Figure 7.2).

The Kinase-Glo^® and Kinase-Glo^® Plus Reagents rely on the properties of a proprietary thermostable luciferase (Ultra-Glo™ Recombinant Luciferase) that is formulated to generate a stable “glow-type” luminescent signal. The reagents are prepared by combining the Kinase-Glo^® or Kinase-Glo^® Plus Buffer with the lyophilized substrate provided with each system.

The protocol for both systems involves a single addition of an equal volume of Reagent to a completed kinase reaction that contains ATP, purified kinase and substrate. The plate is mixed and luminescence read. The luminescence is directly proportional to the ATP present in the kinase reaction, and kinase activity is inversely correlated with luminescent output.

The Kinase-Glo^® Luminescent Kinase Assay (Cat.# V6711) and the Kinase-Glo^® Plus Luminescent Kinase Assay (Cat.# V3771) can be used with virtually any kinase and substrate combination. The Kinase-Glo^® Assay is extremely sensitive and is linear from 0 to 10μM ATP. It routinely provides Z´-factor values greater than 0.8 in both 96-well and 384-well formats (Figure 7.3). Z´-factor is a statistical measure of assay dynamic range and variability; a Z´-factor greater than 0.5 is indicative of a robust assay (Zhang et al. 1999).

We have demonstrated the utility of the Kinase-Glo^® Assay for high-throughput screening (Somberg et al. 2003; Goueli et al. 2004a). We tested the Kinase-Glo^® Assay using a commercially available Library of Pharmacologically Active Compounds (LOPAC) to determine if the assay could score true kinase hits in that library. When we screened the LOPAC collection for inhibitors of PKA using the manual protocol, we found six wells in which we could detect kinase inhibition (Somberg et al. 2003). The same six wells also showed detectable kinase inhibition when we tested the Kinase-Glo^® Assay in low-volume 384 and 1536-well formats (Goueli et al. 2004b; Figure 7.4). The Kinase-Glo^® Assay can also be used to determine IC₅₀ values for kinase inhibitors. The IC₅₀ values for one of the six hits from the LOPAC library were determined using the Kinase-Glo^® Assay. The Kinase-Glo^® Assay gave values similar to values reported in the literature, further establishing the utility of the Kinase-Glo^® Assay for high-throughput screening (Goueli et al. 2004b).

The Kinase-Glo^® Plus Assay not only allows users to detect kinase inhibitors, but also to distinguish between ATP competitive and noncompetitive inhibitors. Because the concentration of ATP in cells is fairly high, inhibitors of protein kinases that are not ATP-competitive are more desirable as therapeutic agents than ATP-competitive kinase inhibitors. Because the catalytic domains and active sites of protein kinases have been evolutionarily conserved, inhibitors that are not only ATP non-competitive, but also selective toward the target kinase are most desireable. The Kinase-Glo^® Plus Assay is optimized to work at ATP concentrations that more closely reflect cellular ATP concentrations and is linear up to 100μM ATP.

Materials Required:

• Kinase-Glo^® Assay System (Cat.# V6711, V6712, V6713, V6714) or Kinase-Glo^® Plus Assay System (Cat.# V3771, V3772, V3773, V3774 ) and Protocol (Technical Bulletin #TB318 or #TB343).
• solid white multiwell plates
• multichannel pipet or automated pipetting station
• plate shaker
• luminometer capable of reading multiwell plates
• ATP
• appropriate kinase substrate
• appropriate kinase reaction buffer

Figure 7.5 provides an overview of the Kinase-Glo^® Assay Protocol. The Kinase-Glo^® Plus Assay follows the same format.

Fluorescent Kinase Assays

The ProFluor^® Kinase Assays measure PKA (Cat.# V1240, V1241) or PTK (Cat.# V1270, V1271) activity using purified kinase in a multiwell plate format and involve “add, mix, read” steps only. The user performs a standard kinase reaction with the provided bisamide rhodamine 110 substrate. The provided substrate is nonfluorescent. After the kinase reaction is complete, the user adds a Termination Buffer containing a Protease Reagent. This simultaneously stops the reaction and removes amino acids specifically from the nonphosphorylated R110 Substrate, producing highly fluorescent rhodamine 110. Phosphorylated substrate is resistant to protease digestion and remains nonfluorescent. Thus, fluorescence is inversely correlated with kinase activity (Figure 7.6).

We tested the ability of several tyrosine kinases to phosphorylate the peptide substrate provided in the ProFluor^® Src-Family Kinase Assay using protease cleavage and fluorescence output as an indicator of enzyme activity. The PTK peptide substrate served as an excellent substrate for all of the Src-family PTKs such as Src, Lck, Fyn, Lyn, Jak and Hck and the recombinant epidermal growth factor receptor (EGFR) and insulin receptor (IR). The fluorescence decreases with increasing concentrations for four Src family enzymes tested (Goueli et al. 2004a). The amount of enzyme required to phosphorylate 50% of the peptide (EC₅₀) was quite low (EC₅₀ for Src, Lck, Fyn, Lyn A and Hck were 14.0, 1.38, 4.0, 4.13 and 1.43ng, respectively). As low as a few nanograms of Lck could be detected using this system.

Generalized Protocol for the ProFluor^® Kinase Assays

Materials Required:

• ProFluor^® PKA Assay (Cat.# V1240, V1241) or ProFluor^® Src-Family Kinase Assay (Cat.# V1270, V1271) and protocol (Technical Bulletin #TB315 or #TB331, respectively)
• black-walled multiwell plates (e.g., Microfluor 2, black 96-well plate; ThermoElectron Cat.# 7805)
• multichannel pipet or automated pipetting station
• plate shaker (e.g., DYNEX MICRO-SHAKER^® II)
• plate-reading fluorometer with filters capable of reading R110 and AMC fluorescence
• protein kinase

General Assay Protocol and Format for ProFluor^® Kinase Assays

1. Dilute kinase and R110 Substrate in 1X Reaction Buffer.

2. Dilute ATP in 1X Reaction Buffer.

3. Mix contents in wells of plate and incubate at room temperature (20 minutes for PKA Assay, 60 minutes for Src-Family Kinase Assay).

4. Dilute Protease Reagent in 1X Termination Buffer A.

5. Mix plate and incubate at room temperature (30 minutes for PKA Assay, 60 minutes for Src-Family Kinase Assay).

6. Dilute Stabilizer Reagent in 1X Termination Buffer A.

7. Mix plate and R110 and AMC fluorescence.

We highly recommend performing a kinase titration to determine the optimal amount of kinase to use for screening and to determine whether or not the enzyme preparation contains components that negatively affect the performance of the assay. Please see Technical Bulletins #TB315 or #TB331 for additional information.

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Radioactive Kinase Assays

SAM^2® Biotin Capture Membrane and Biotin Capture Plate

The SAM^2® Biotin Capture Membrane (Cat.# V2861, Cat.# V7861; Figure 7.8) is a proprietary technology that relies on the high-affinity streptavidin:biotin interaction for the capture and detection of biotinylated molecules regardless of their sequence. The unique features of the SAM^2® Membrane compared to other membranes or substrates (e.g., P81 phosphocellulose or streptavidin-coated plates), is the high density of covalently linked streptavidin per square centimeter and the selective mode of capture. This high-density streptavidin matrix efficiently captures biotinylated molecules or substrates, providing high signal-to-noise ratios even in assays using low enzyme concentrations or crude cell extracts. The SAM^2® Biotin Capture Membrane offers superior assay performance by providing high binding capacity, low nonspecific binding, sequence-independent capture and the flexibility of multiple format configurations. The SAM^2® Membrane is available as a sheet containing 96 numbered and partially cut squares. This format is used in the SignaTECT^® Kinase Assay Systems. The SAM^2® Membrane is also available as a 7.6 × 10.9cm solid sheet, which can be used for high-throughput applications. The membrane can be analyzed by autoradiography, PhosphofImager^® analysis, or scintillation counting.

The SAM^2® 96 Biotin Capture Plate (Cat.# V7541, V7542) contains the SAM^2® Biotin Capture Membrane in the wells of a microfiltration plate. The 96-well plate configuration allows users to perform washes with a vacuum manifold or a plate washer. The plate is supplied with a transparent top seal and opaque bottom seal for adding scintillation fluid to perform quantitation using a microplate liquid scintillation counter.

SignaTECT^® Protein Kinase Assay Systems

The SignaTECT^® Protein Kinase Assay Systems use biotinylated peptide substrates in conjunction with the streptavidin-coated SAM^2® Biotin Capture Membrane. The binding of biotin to the streptavidin is rapid and strong, and the association is unaffected by rigorous washing procedures, denaturing agents, wide extremes in pH, temperature and salt concentration. High signal-to-noise ratios are generated even with complex samples, while the high substrate capacity allows optimum reaction kinetics. The systems can be used to measure protein kinase activities using low femtomole levels of purified enzyme or crude cellular extracts. SignaTECT^® Assays are available to measure protein tyrosine kinases (Cat.# V6480), cdc2 kinase (Cat.# V6430), cAMP-dependent protein kinase (Cat.# V7480), protein kinase C (Cat.# V7470), DNA-dependent protein kinase (Cat.# V7870) and calmodulin-dependent protein kinase (Cat.# V8161).

As outlined in Figure 7.10, the assay steps and analysis of results are straightforward and require only common laboratory equipment. Following phosphorylation and binding of the biotinylated substrate to the numbered and partially cut squares of SAM^2® Biotin Capture Membrane, unincorporated [γ-³²P]ATP is removed by a simple washing procedure. This procedure also removes nonbiotinylated proteins that have been phosphorylated by other kinases in the sample. The bound, labeled substrate is then quantitated by scintillation counting or PhosphorImager^® analysis. Typical results generated using the SignaTECT^® Assays are presented in Figure 7.11.

Other Kinase Assay Formats (non-radioactive)

The PepTag^® Protein Kinase Assays are fast and quantitative non-radioactive alternatives to [γ-³²P]ATP-based assays for measuring protein kinase C (Cat.# V5330) and cAMP-dependent protein kinase (Cat.# V5340) activity. The assays use fluorescently-tagged peptide substrates with a net positive charge. Phosphorylation changes the charge of the peptide to a net negative, which influences the migration of the peptide in an agarose gel. This is the basis for detecting changes in phosphorylation via a rapid, 15-minute agarose gel separation (Figure 7.12).

General PepTag^® Assay Protocol

Materials Required:

• PepTag^® Non-Radioactive PKC Assay (Cat.# V5330) or PepTag^® Non-Radioactive cAMP-Dependent Protein Kinase Assay (Cat.# V5340) and protocol (#TB132)
• PKA or PKC dilution buffer
• horizontal agarose gel apparatus
• glycerol, 80%
• Tris-HCl, 50mM (pH 8.0)
• agarose, 0.8% in 50mM Tris-HCl (pH 8.0)
• probe sonicator

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Phosphorylation-Specific Antibodies

The Anti-ACTIVE^® phosphorylation-specific antibodies were developed to provide an accurate measure of enzyme activation. These antibodies specifically recognize the active, phosphorylated form of a given kinase. The Anti-ACTIVE^® Antibodies are raised against phosphorylated peptide sequences present in the activating loop of a number of protein kinases. Whether used in Western analysis, immunocytochemistry or immunohistochemical staining, the Anti-ACTIVE^® MAPK, JNK, p38 and CaM KII Antibodies will recognize only the active form of the enzyme.

Phosphorylation-Specific Antibodies in MAPK Signaling Pathways

Anti-ACTIVE^® MAPK, pAb, Rabbit, (pTEpY)

This antibody is an affinity purified polyclonal antibody that specifically recognizes the dually phosphorylated, active form of MAPK. The antibody is raised against a dually phosphorylated peptide sequence representing the catalytic core of the active ERK enzyme and recognizes the active forms of ERK1, ERK2 and ERK7.

Anti-ACTIVE^® JNK pAb, Rabbit, (pTPpY)

Anti-ACTIVE^® JNK pAb is an affinity purified polyclonal antibody that recognizes the dually phosphorylated, active form of cJun N-terminal protein Kinase (JNK). Anti-ACTIVE^® JNK pAb is raised against a dually phosphorylated peptide sequence representing the catalytic core of the active JNK enzyme. The antibody recognizes the active forms of JNK1, JNK2, and JNK3 isoforms.

Anti-ACTIVE^® p38 pAb, Rabbit, (pTGpY)

Anti-ACTIVE^® p38 Ab, Rabbit, is an affinity purified polyclonal antibody that recognizes the active form of p38 kinase. The Anti-ACTIVE^® p38 pAb is raised against the dually phosphorylated peptide sequence representing the catalytic core of the active p38 enzyme. The Anti-ACTIVE^® p38 pAb recognizes the active forms of p38α, γ, and δ isoforms.

Western Blot Analysis with Anti-ACTIVE^® MAPK, JNK and p38 pAbs

Materials Required:

• Anti-ACTIVE^® MAPK (Cat.# V8031), JNK (Cat.# V7931), or p38 (Cat.# V1211) pAb
• Anti-ACTIVE^® Qualified Donkey Anti-Rabbit IgG (H+L), HRP (Cat.# V7951) or Donkey Anti-Rabbit IgG (H+L) AP (Cat.# V7971) Secondary Antibodies
• protein sample transfered to nitrocellulose or PVDF membrane
• bovine serum albumin, 1%
• TBS buffer
• TBST or PVDF buffer
• shaking platform

Immunocytochemistry with Anti-ACTIVE^® MAPK, JNK and p38 pAbs

The following method is for preparing and immunostaining PC12 cells stimulated by either nerve growth factor to activate MAP kianses or soribitol to activate JNK and p38 kinases. For additional information see Technical Bulletin #TB262

Materials Required:

• Anti-ACTIVE^® Qualified Donkey Anti-Rabbit IgG (H+L), HRP (Cat.# V7951) or Donkey Anti-Rabbit IgG (H+L), AP (Cat.# V7971) Secondary Antibodies
• LabTek^® 4-chambered slides (Fisher Cat.# 12-565-21)
• rat-tail collagen (Collaborative BioScience Products)
• RPMI 1640 with 25mM HEPES, 300mg/l l-glutamine, 10% horse serum, 5% fetal bovine serum and 0.5mM EGTA
• NGF (Cat.# G5141) or sorbitol
• PBS
• 10% paraformaldehyde
• methanol, –20°C
• blocking buffer
• donkey anti-rabbit Cy^®3 conjugate (Jackson ImmunoResearch Cat.# 741-165-152)

Preparation and Activation of PC12 Cells

1. Coat 4-chambered slides with rat tail collagen (6μg/cm² in sterile PBS) for one hour.

2. Grow PC12 cells in chambers at 37° in 5% CO₂ in medium containing RPMI 1640 with 25mM HEPES, 300mg/L l-glutamine, 10% horse serum, 5% fetal bovine serum and 0.5mM EGTA. The medium should be changed every other day until the cells reach 80% confluence.

3. Activate the cells in 2 chambers as described below. Use the cells in the remaining 2 chambers as untreated controls.

   NGF: The day before immunocytochemistry, add fresh medium with serum. The next day add 200ng/ml NGF in RPMI. Incubate for 5 minutes at 37°C.

   Sorbitol: The day before immunocytochemistry, add fresh medium without serum. The next day add sorbitol to a final concentration of 1M. Incubate for 30 minutes at 37°C.

4. Proceed with staining as outlined in Figure 7.14.

Phosphorylation-Specfic CaM KII Antibody

This antibody recognizes calcium/calmodulin-dependent protein kinase CaM KII that is phosphorylated on threonine 286. The Anti-ACTIVE^® CaM KII pAb (Cat.# V1111) was raised against the phosphothreonine-containing peptide derived from this region.

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Kinase Inhibitors

MEK Inhibitor U0126

MEK Inhibitor U0126 (Cat.# V1121) inhibits the activity of MAP Kinase Kinase (MEK 1/2) and thus prevents the activation of MAPK. U0126 inhibits MEK 1 with an IC₅₀ of 0.5µM in vitro (Favata et al. 1998). U0126 inhibits phosphorylation activated MEK 1 and MEK 2 as well as constitutively active MEK 1 and MEK 2 mutants (Favata et al. 1998; Goueli et al. 1998). U0126 is noncompetitive with respect to the MEK substrates ATP and ERK (Favata et al. 1998; Tolwinski et al. 1999).

PD 98059

PD 98059 (Cat.# V1191) inhibits MEK activation (Alessi et al. 1995; Dudley et al. 1995; Favata et al. 1998). PD 98059 inhibits MEK 1 but is an inefficient inhibitor of MEK 2. (Alessi et al. 1995; Dudley et al. 1995). It inhibits activation of MEK 1 by Raf with an IC₅₀ of 5μM and of the active MEK 1 mutant with an IC₅₀ of 10µM (Alessi et al. 1995; Dudley et al. 1995).

SB 203580

SB 203580 (Cat.# V1161) is a specific, cell-permeant inhibitor of the stress and inflammatory cytokine-activate MAP kinase homologues p38α, β and β2. It acts as a competitive inhibitor of ATP binding to the kinase. Reported IC₅₀ values range from 21nM to 1µM. SB 203580 has no significant effect on the activities of ERKs, JNKs, p38γ or p38δ.

Promega Publications

CN001 Frequently asked questions: Kinase inhibitors and activators

BR095 Signal Transduction Resource

Citations

PI3 Kinase Inhibitor LY 294002

LY 294002 (Cat.# V1201) is a potent and specific cell-permeant inhibitor of phosphatidylinositol 3-kinases (PI3-K) with an IC₅₀ value in the 1–50μM range. LY 294002 competitively inhibits ATP binding to the catalytic subunit of PI3-Ks and does not inhibit PI4-Kinase, DAG-kinase, PKC, PKA, MAPK, S6 kinase, EGFR or c-src tyrosine kinases and rabbit kidney ATPase (Rameh and Cantley, 1999; Fruman et al. 1998). LY 294002 has improved stability and specificity compared to Wortmannin, which is an irreversible inhibitor that covalently interacts with PI3-Ks.

cAMP-Dependent Protein Kinase (PKA) Peptide Inhibitor

The cAMP-Dependent Protein Kinase Inhibitor (Cat.# V5681), also known as PKI, TTYADFIASGRRNAIHD, inhibits phosphorylation of target proteins by binding to the protein-substrate site of the catalytic subunit of PKA. It corresponds to the region 5–24 of the naturally occurring PKI.

InCELLect^® AKAP St-Ht31 Inhibitor Peptide

The InCELLect^® AKAP St-Ht31 Inhibitor Peptide (Cat.# V8211) and the InCELLect^® Control Peptide (Cat.# V8221) can be used for in vivo studies of PKA activation. The Inhibitor Peptide is a stearated (St) form of the peptide Ht31 derived from the human thyroid AKAP (A-kinase anchoring protein). The presence of the hydrophobic stearated moiety enhances the cellular uptake of the peptides through the lipophilic microenvironment of the plasma membrane.

Myristoylated Protein Kinase C Peptide Inhibitor

Myristoylated Protein Kinase C Peptide Inhibitor (Cat.# V5691) specifically inhibits calcium- and phospholipid-dependent protein kinase C. It is based on the pseudosubstrate region of PKC-α and PKC-β (Eicholtz, 1993).

Olomoucine cdc2 Protein Kinase Inhibitor

Olomoucine is a chemically synthesized inhibitor that is specific for p34^cdc2 and related protein kinases. Its molecular weight is 298, and its molecular formula is C1₅H₁₈N₆O.

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Phosphatase Assays

Protein phosphorylation plays a key role in signal transduction, and genes for protein kinases and phosphatases represent a large portion of the human genome (Goueli et al. 2004b; Cohen, 2001). They are the opposing partners to the kinases in the cell, catalyzing the dephosphorylation of molecules involved in cellular pathways. Protein phosphatases can be divided into three general categories: a) protein tyrosine phosphatases, which remove phosphate from phosphotyrosine-containing proteins, b) protein serine/threonine phosphatases, which remove phosphate from phosphoserine- or phosphothreonine-containing proteins, and c) dual-specificity phosphatases, which can remove phosphate from phosphotyrosine, phosphothreonine, and phosphoserine (Hunter, 1995).

Fluorescent Phosphatase Assays

We have developed the ProFluor^® Phosphatase Assays to overcome safety issues associated with radioactive assays while maintaining sensitivity and specificity. The ProFluor^® Phosphatase Assays use bisamide R110-linked phosphopeptides that serve as substrates for PTPases. Phosphorylation of the peptide substrate renders it resistant to cleavage by the Protease Reagent that is included with these assay systems, reducing the fluorescence generated. However, when the phosphoryl moiety is removed by a phosphatase, the peptides become cleavable by the protease, releasing the highly fluorescent, free R110 molecule (Figure 7.15).

The ProFluor^® PPase Assays offer the simplicity, sensitivity and specificity required for screening chemical libraries for novel inhibitors of protein phosphatases. These assays are robust with Z´ factor values routinely greater than 0.8 (Figure 16; Goueli et al. 2004b)

Z´ factor is a statistical description of the dynamic range and variability of an assay. Z´ factor values >0.5 are indicative of a robust assay (Zhang et al. 1999). These fluorescent assays can be performed in single tubes, 96-well plates or 384-well plates, giving the user flexibility in format. The signal-to-noise ratio is very high, and the generated signal is stable for hours.

General Protocol for the ProFluor^® Phosphatase Assays

Materials Required:

• ProFluor^® Ser/Thr Phosphatase Assay (Cat.# V1260, V1261) or ProFluor^® Tyrosine Phosphatase Assay (Cat.# V1280, V1281) and protocol (Technical Bulletin #TB324 or TB334, respectively)
• opaque-walled multiwell plates
• multichannel pipet or automated pipetting station
• plate shaker (DYNEX MICRO-SHAKER^® or equivalent)
• plate-reading fluorometer with filters for reading R110 and AMC fluorescence
• protein tyrosine phosphatase or S/T protein phosphatase
• okadaic acid (for PP1 and PP2A)
• calmodulin (for PP2B)

1. Dilute the phosphatase in Reaction Buffer and add to wells.

2. Dilute the PTPase R110 Substrate and the Control AMC Substrate in Reaction Buffer and add to wells.

3. Mix the contents of the plate for 15 seconds and incubate at room temperature (10 minutes for PP1 and PP2A; 30 minutes for PP2B; 60 minutes for tyrosine PPase).

4. Add Protease Solution.

5. Mix the contents of the plate briefly and incubate at room temperature (90 minutes for PP2A, PP2B or PP1; 30 minutes for tyrosine PPase).

6. Add Stabilizer Solution.

7. Mix the contents of the plate and read fluorescence.

Colorimetric Phosphatase Assays

Both the Tyrosine Phosphatase (Cat.# V2471) and the Serine/Threonine Phosphatase (Cat.# V2460) Assay Systems detect the release of phosphate from specific peptide substrates by measuring the appearance of a phosphate complex of molybdate:malachite green. For assays of crude extracts, endogenous phosphate and other inhibitory molecules are first removed by a simple 20-minute procedure using Spin Columns that are supplied with each system. This step is unnecessary for assays using pure or partially purified enzyme preparations. Each system includes ready-to-use, specific substrates: the Tyrosine Phosphatase System provides two phosphotyrosine-containing peptides; the Serine/Threonine Phosphatase Assay System provides a phosphothreonine-containing peptide. Other phosphopeptides or phosphoproteins can be used as substrates to increase specificity or to use natural substrates. The simple assay procedure is outlined in Figure 7.17.

Materials Required:

• Serine/Threonine Phosphatase Assay System (Cat.# V2460) or Tyrosine Phosphatase Assay System (Cat.# V2471) and protocol (Technical Bulletin # TB218 or #TB212, respectively)
• 50ml disposable conical centrifuge tubes (e.g., Corning Cat.# 25330-50)
• appropriate storage buffer ( see TB212 or TB218)
• Sephadex^® G-25 storage buffer (for storing column)

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References

1. Alessi, A. et al. (1995) PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo J. Biol. Chem. 270, 27489–94.
2. Aoki, M. et al. (2001) A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3K and Akt. Proc. Natl. Acad. Sci. USA 98, 136–41.
3. Cohen, P. (2001) The role of protein phosphorylation in human health and disease: Delivered on June 30, 2001 at the FEBS meeting in Lisbon. Eur. J. Biochem 268, 5001–10.
4. Cohen, P. (2002) Protein kinases—the major drug targets of the 21st century? Nat. Rev. Drug Disc. 1, 309–15.
5. Cooray, S. (2004) The pivotal role of phosphatidylinositol-3-kinase-Akt signal transduction in virus survival J. Gen. Virol. 85, 1065–76.
6. De Meyts, P. et al. (1995) Role of the time factor in signaling specificity: Application to mitogenic and metabolic signaling by the insulin and insulin-like growth factor-1 receptor tyrosine kinases. Metabolism 44, 2–11.
7. Denton, R.M. and Tavare, J.M. (1995) Does mitogen-activated protein kinase have a role in insulin action? The cases for and against. Eur. J. Biochem. 227, 597–611.
8. Doza, Y.N. et al. (1995) Activation of the MAP kinase homologue RK requires the phosphorylation of Thr-180 and Tyr-182 and both residues are phosphorylated in chemically streaked KB cells. FEBS Letters 364, 223–8.
9. Dudley, D.T. et al. (1995) A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA 92, 7686–9.
10. Eicholtz, T. et al. (1993) A myristoylated psuedosubstrate peptide, a novel protein kinase C inhibitor. J. Biol. Chem. 268, 1982–6.
11. Ellinger-Ziegelbauer, H. et al. (1997) Direct activation of the stress-activated protein kinase (SAPK) and extracellular signal-regulated protein kinase (ERK) pathways by an inducible mitogen-activated protein kinase/ERK kinase kinase (MEKK) derivative. J. Biol. Chem. 272, 2668–74.
12. Favata, M. et al. (1998) Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273, 18623–32.
13. French, K.J. et al. (2003) Discovery and evaluation of inhibitors of human sphingosine kinase. Can. Res. 63, 5962–9.
14. Fruman, D.A. et al. (1998) Phosphoinositide kinases. Annu. Rev. Bioch. 67, 481–507.
15. Goueli, S.A. et al. (2004b) Assay protein tyrosine kinase and protein tyrosine phosphatase activity in a homogeneous, non-radioactive high-throughput format. Cell Notes 8, 15–20.
16. Goueli, S.A. et al. (1998) U0126: A novel, selective and potent inhibitor of MAP kinase kinase (MEK). Promega Notes 69, 6–8.
17. Goueli, S.A. et al. (2004a) High-throughput kinase screening using a universal, luminescent kinase assay. Cell Notes 10, 20–23.
18. Grimsby, J. et al. (2003) Allosteric activators of glucokinase: Potential role in diabetes therapy. Science 301, 370–3.
19. Hunter, T. (1995) Protein kinases and phosphatases: The yin and yang of protein phosphorylation and signaling. Cell 80, 225�