A microchip-based enzyme assay for protein kinase A.

A microchip-based enzyme assay for protein kinase A is described. The microchips were prepared by standard photolithographic techniques. The assay reagents were placed in wells on the microchips, and electroosmosis was used to transport aliquots of these reagents into the network of etched channels, where the enzymatic reaction takes place. Protein kinase A catalyzes the transfer of a phosphate group from ATP to the serine residue of the heptapeptide LeuArgArgAlaSerLeuGly (Kemptide). The outcome of the enzymatic reaction was assessed by performing an on-chip electrophoretic separation of the fluorescently labeled peptide substrate and product. All liquid-handling steps were performed by controlling the electroosmotically driven flow from reagent and buffer wells using electrical current. On-chip dilutions of the peptide substrate, ATP and H-89, a known protein kinase A inhibitor, were performed and the kinetic constants (K(m), K(i)) of these compounds were determined. This prototype assay demonstrates the usefulness of the microchips for performing enzymatic assays for which fluorogenic substrates cannot easily be designed.     

Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody.

A competitive fluorescence polarization (FP) assay has been developed for the serine/threonine kinase, AKT. The FP assay has been formatted in a 384-well microtiter plate and automated using a pipeting workstation with performance suitable for high-throughput screening. The assay design utilizes a fluorescent phosphorylated peptide complexed to a product-specific anti-phospho-serine antibody. When unlabeled substrate is phosphorylated, by the kinase, the product competes with the fluorescent phosphorylated peptide for the antibody. The fluorescent phosphorylated peptide is then released from the antibody into solution resulting in a loss in polarization signal. Seven fluorescent phosphorylated peptides and 19 antibodies were evaluated for this assay. RARTSpSFAEPGK-Fl peptide and anti-phospho-GSK-3alpha Ser21 antibody gave the best affinity and change in polarization signal. The apparent kinetic constants were calculated for the FP assay and were consistent with reported values. The FP assay was validated with known inhibitors and the results compared to a radioactive Flashplate transfer assay, utilizing [(33)P]ATP and a biotinylated substrate, also developed in our laboratory. The IC(50) values generated were comparable between the two methods suggesting the competitive FP assay and Flashplate assay have similar sensitivities and abilities to identify inhibitors during screening.     

Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence

Two homogeneous proximity assays for tyrosine kinases, scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence (HTRF), have been developed and compared. In both formats, the kinase assay was performed using biotinylated peptide substrate, ATP ([33P]ATP in the case of SPA), and tyrosine kinase in a 96-well assay format. After the kinase reaction was stopped, streptavidin-coated SPA beads or europium cryptate-labeled anti-phosphotyrosine antibody and streptavidin-labeled allophycocyanin were added as detection reagents for SPA or HTRF assays, respectively. Since the assay signal was detected only when the energy donor (radioactivity for SPA, Eu for HTRF) and the energy acceptor molecules (SPA beads for SPA, allophycocyanin for HTRF) were in close proximity, both assays required no wash or liquid transfer steps. This homogeneous ("mix-and-measure") nature allows these assays to be much simpler, more robust, and easier to automate than traditional protein kinase assays, such as a filter binding assay or ELISA. Both assays have been miniaturized to a 384-well format to reduce the assay volume, thereby saving the valuable screening samples as well as assay reagents, and automated using automated pipetting stations to increase the assay throughput. Several advantages and disadvantages for each assay are described.     

A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates

New methods to quantify protein kinase activities directly from complex cellular mixtures are critical for understanding biological regulatory pathways. Herein, a fluorescence-based chemosensor strategy for the direct measurement of kinase activities in crude mammalian cell lysates is described. We first designed a new fluorescent peptide reporter substrate for each target kinase. These kinase chemosensors were readily phosphorylated by recombinant target enzyme and underwent a several-fold fluorescence increase upon phosphorylation. Then, using unfractionated cell lysates, a homogeneous kinase assay was developed that was reproducible, linear and highly preferential for monitoring changes in cellular activity of the target kinase. The general protocol was developed for the kinase Akt and then easily extended to measure protein kinase A (PKA) and mitogen-activated protein kinase-associated protein kinase 2 (MK2) activities. This assay platform is immediately useful for studying protein kinase signaling in crude cellular extracts.     

Pre-steady-state kinetics of the activation of rabbit skeletal muscle myosin light chain kinase by Ca2+/calmodulin.

Myosin light chain kinase is activated by Ca2+/calmodulin. Insights into the kinetic mechanism of this activation by Ca2+/calmodulin have now been obtained using extrinsically labeled fluorescent calmodulin, a fluorescent peptide substrate, and a stopped-flow spectrophotofluorimeter. We employed spinach calmodulin labeled with the sulfhydryl-selective probe, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid, to measure changes in the fluorescence intensity of the 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin upon binding to rabbit skeletal muscle myosin light chain kinase. The fluorescent peptide substrate KKRAARAC(sulfobenzo-furazan)SNVFS-amide was used to measure kinase activity. Our results showed that the binding interaction could be modeled as a two-step process: a bimolecular reaction with an association rate of 4.6 x 10(7) M-1 s-1 followed by an isomerization with a rate of 2.2 s-1. Phosphorylation of the peptide during stopped-flow experiments could be modeled by a two-step process with a catalytic association rate of 6.5 x 10(6) M-1 s-1 and a turnover rate of 10-20 s-1. Our results also indicated that kinase activity occurred too rapidly for the slower isomerization rate of 2.2 s-1 to be linked specifically to the activation process.     

A Nonradioactive Fluorescent Gel-Shift Assay for the Analysis of Protein Phosphatase and Kinase Activities toward Protein-Specific Peptide Substrates

Synthetic peptides are important tools with which to study the activities of protein kinases and phosphatases toward specific substrate sequences which are present within selected regions of a protein. Most existing assays for the phosphorylation or dephosphorylation of such peptides utilize ³²P and either affinity chromatography or HPLC separation and require extensive characterization and validation. Here, we describe a method for monitoring the phosphorylation or dephosphorylation of almost any peptide of interest which does not require the use of radioactivity, making its reagents stable for a prolonged period, and which can be performed in any standard laboratory. For this, after performance of kinase or phosphatase reactions with the peptide of interest, products are derivatized with fluorescamine and are separated according to charge by agarose gel electrophoresis. Phosphorylated and nonphosphorylated peptides are readily separated and can be both identified and quantified by uv detection. The lower limit for detection of peptide in the agarose gel was 0.02 nmol using the gel-shift kinase assay with cAMP-dependent kinase and Kemptide as substrate. This had sensitivity and reproducibility similar to those of a standard assay using [γ-³²P]ATP with this substrate. Dephosphorylation of a synthetic phosphopeptide corresponding to a segment of the cholecystokinin receptor was tested in an analogous assay with known amounts of protein phosphatase 2A. Phosphopeptide and dephosphopeptide were easily detected and quantified with as little as 0.03 mU/mI protein phosphatase 2A activity. Therefore, with this assay, most synthetic peptides and phosphopeptides can be used as substrates without further modification. This will be of particular interest for monitoring the purification of highly specific protein kinase and phosphatase activities.     

Label-free and real-time cell-based kinase assay for screening selective and potent receptor tyrosine kinase inhibitors using microelectronic sensor array

Kinases are the 2nd largest group of therapeutic targets in the human genome. In this article, a label-free and real-time cell-based receptor tyrosine kinase (RTK) assay that addresses limitation of existing kinase assays and can be used for high-throughput screening and lead optimization studies was validated and characterized. Using impedance, growth factor-induced morphological changes were quantitatively assessed in real time and used as a measure of RTK activity. COS7 cells treated with epidermal growth factor (EGF) and insulin results in a rapid increase in cell impedance. Assessment of these growth factor-induced morphological changes and levels of receptor autophosphorylation using fluorescent microscopy and enzyme-linked immunosorbent assay, respectively, demonstrates that these changes correlate with changes in impedance. This assay was used to screen, identify, and characterize a potent EGF receptor inhibitor from a compound library. This report describes an assay that is simple in that it does not require intensive optimization or special reagents such as peptides, antibodies, or probes. More important, because the assay is cell based, the studies are done in a physiologically relevant environment, allowing for concurrent assessment of a compound's solubility, stability, membrane permeability, cytotoxicity, and off-target interaction effects.     

Rational design of homogenous protein kinase assay platforms that allow both fluorometric and colorimetric signal readouts

Protein kinases play important roles in signaling pathways that regulate many cellular biological processes, including apoptosis, cell growth, and differentiation in response to extracellular stimuli. Design of homogenous protein kinase assay platforms including design of potent protein kinase substrates is essential for exploration of the phosphoproteome. Here, we describe a unique chromism-based assay (CHROBA) technique for the direct measurement of protein kinase activities. The CHROBA is a novel chemosensor system that produces signals based on the photochromic and thermodynamic properties of a spiropyran derivative incorporated into peptide substrates. The CHROBA technique for detecting protein kinase activities involves the following five steps: (i) phosphorylation, (ii) photobleaching of the reaction mixture, (iii) addition of ionic polymer(s), (iv) incubation in the dark, and (v) signal readout. This simple 'end-point' assay method allows quantitative measurements of protein kinase A, Src protein tyrosine kinase, c-Abl protein tyrosine kinase, and protein kinase Calpha activities even with excess ATP. Our results showed that spiropyran-containing peptide substrates with net charges between +2 and 0 are suitable for the present CHROBA method. This information should aid in the rational design of diverse protein kinase assay platforms. The present CHROBA technique can be adapted to a microplate format with both fluorometric and colorimetric readouts and would be useful for high-throughput drug discovery and analysis of the phosphoproteome.     

Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye

Ultrasensitive detection of minute amounts of phosphorylated proteins and peptides is a key requirement for unraveling many of the most important signal transduction pathways in mammalian systems. Protein microarrays are potentially useful tools for sensitive screening of global protein expression and post-translational modifications, such as phosphorylation. However, the analysis of signaling pathways has been hampered by a lack of reagents capable of conveniently detecting the targets of protein kinases. Historically, phosphorylation detection methods have relied upon either radioisotopes ((gamma-(32)P)ATP(gamma-(33)P)ATP labeling) or phosphoamino acid-selective antibodies. Both of these methods suffer from relatively well-known shortcomings. In this study, a small molecule fluorophore phosphosensor technology is described, referred to as Pro-Q Diamond dye, which is capable of ultrasensitive global detection and quantitation of phosphorylated amino acid residues in peptides and proteins displayed on microarrays. The utility of the fluorescent Pro-Q Diamond phosphosensor dye technology is demonstrated using phosphoproteins and phosphopeptides as well as with protein kinase reactions performed in miniaturized microarray assay format. Instead of applying a phosphoamino acid-selective antibody labeled with a fluorescent or enzymatic tag for detection, a small, fluorescent probe is employed as a universal sensor of phosphorylation status. The detection limit for phosphoproteins on a variety of different commercially available protein array substrates was found to be 312-625 fg, depending upon the number of phosphate residues. Characterization of the enzymatic phosphorylation of immobilized peptide targets with Pro-Q Diamond dye readily permits differentiation between specific and non-specific peptide labeling at picogram to subpicogram levels of detection sensitivity.     

Fluorescein isothiocyanate-hapten immunoassay for determination of peptide-cell interactions

We have developed a fluorescein isothiocyanate (FITC)-hapten immunoassay, where a FITC-labeled peptide binding to a cell is assayed as the amount of immunoreactive fluorescein present in a cell lysate. An antifluorescein-horseradish peroxidase conjugate binds to either a fluoresceinated peptide in the lysate or a fluorescein attached to the wells of a microtiter plate in a competitive fashion. After washing, solid-phase peroxidase activity is measured and inversely related to the amount of FITC-labeled peptide present. To demonstrate the assay, the interaction of a FITC-labeled bombesin-like peptide with the gastrin-releasing peptide receptor on PC-3 and HT-29 cells was investigated. Using PC-3 cells, we obtained similar displacement curves and numbers of binding sites per cell by both the FITC-hapten immunoassay and a reference radioreceptor assay. The FITC-hapten immunoassay is a sensitive and versatile method, since the same commercially available reagents can be used to assess interactions between any peptide and any receptor. In addition, the FITC-labeled peptide can be used to visualize receptors in fluorescent-activated cell sorting or fluorescent microscopy.     

Expression, purification, and inhibition of human RET tyrosine kinase

Tyrosine kinases are emerging as frequent targets of primary oncogenic events and therefore represent an optimal focus of therapeutical intervention. Genetic alterations that cause dysregulated activation of the RET tyrosine kinase are responsible for a significant fraction of thyroid carcinomas. In an effort towards therapeutic RET inactivation, we have developed a method for expression and purification of recombinant RET catalytic domain for structural purposes and for use in the screening of potential inhibitors of RET kinase activity. His-tagged RET kinase domain was purified from Sf9 insect cell lysate using a two-step chromatographic protocol and characterised. Purified recombinant RET phosphorylated itself and exogenous substrates at physiological pH. A specific peptide substrate, derived from RET activation loop, was identified and experimentally validated. These reagents were used to develop a rapid ELISA-based kinase assay for screening potential inhibitors. Novel RET inhibitors were identified using this assay.     

Cattle tick Boophilus microplus salivary gland contains a thiol-activated metalloendopeptidase displaying kininase activity

This work reports on the characterization of a metalloendopeptidase kininase present in Boophilus microplus salivary glands. Using the guinea pig ileum assay, salivary gland whole extracts (SGE) were found to have a potent kininase activity. Ion-exchange chromatography separated two kininase activities from SGE. The major enzymatic component, eluted at lower ionic strength, was named BooKase (Boophilus Kininase). Analysis of the hydrolysis products by capillary electrophoresis identified Phe5-Ser6 as the only hydrolyzable peptide bond in bradykinin after BooKase treatment. This is the same specificity as the mammalian thimet oligoendopeptidase (EC 3.4.24.15). Like this enzyme, BooKase is also a metallo-peptidase (requires Mn2+) and is activated by-SH protecting reagents. In addition, BooKase was partially inhibited by cFP-AAF-pAB, a specific inhibitor of thimet oligopeptidase. Contrary to other kininases, BooKase had no activity upon angiontensin I. Our results show that BooKase behaves as a typical peptidase with kinase activity.     

Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction

A novel fluorescent substrate was devised for the sirtuin (SIRT) class of human protein deacetylases comprised of a peptide sequence containing a single acetyl-lysine residue, with a fluorescent group (tetramethylrhodamine-6-carboxylic acid, 6-TAMRA) near the carboxyl terminus and a nonfluorescent quenching group (QSY-7) near the amino terminus. The peptide sequence is modeled after the p53 acetylation site but is unreactive toward trypsin because all other lysine and arginine residues have been replaced by serine. However, the SIRT-deacetylated peptide is readily cleaved by trypsin, resulting in a maximal 30-fold enhancement of the 6-TAMRA fluorescence. Nicotinamide at millimolar concentrations stops the deacetylation but does not inhibit trypsin, and a microtiter plate assay of the SIRTs has been devised using the fluorescent substrate and these reagents. Using this method, the kinetics of the reaction of the cosubstrate nicotinamide adenine dinucleotide and the competitive inhibitor nicotinamide with SIRT1 and SIRT2 has been analyzed. Several nicotinamide analogs have also been tested as inhibitors and found to have much lower affinity for these enzymes than does the parent compound.     

Real-time fluorescence monitoring of GSK3β-catalyzed phosphoryation by use of a BODIPY-based Zn(II)–Dpa chemosensor

We developed a new fluorescence assay system for GSK3β-catalyzed kinase reaction using the BODIPY-based fluorescent chemosensor. This system exploits the selective sensing property of the chemosensor for a (i, i+4) bis-phosphorylated peptide, which allows us to conveniently detect the phosphorylation reaction with a fluorescence increase in a real-time fashion.     

A chromism-based assay (CHROBA) technique for in situ detection of protein kinase activity

A unique chromism-based assay technique (CHROBA) using photochromic spiropyran-containing peptides has been firstly established for detection of protein kinase A-catalyzed phosphorylation. The alternative method has advantages that avoid isolation and/or immobilization of kinase substrates to remove excess reagents including nonreactive isotope-labeled ATP or fluorescently-labeled anti-phosphoamino acid antibodies from the reaction mixture. Such a novel protocol based on thermocoloration of the spiropyran moiety in the peptide can offer not only an efficient screening method of potent kinase substrates but also a versatile analytical tool for monitoring other post-translational modification activities.     

Fluorescence polarization competition immunoassay for tyrosine kinases

To increase the sensitivity and throughput of protein tyrosine kinase (PTK), simple, homogeneous, nonradioactive, direct and indirect fluorescence polarization (FP) protein tyrosine kinase immunoassays have been developed that are compatible with high-throughput and ultrahigh-throughput screening for developing drugs. In the direct method, a fluorescinylated peptide substrate is incubated with the kinase, ATP, and antiphosphotyrosine antibody. The phosphorylated peptide product is immunocomplexed with the antiphosphotyrosine antibody, resulting in an increase in the polarization signal. Since the direct method can be used only with a peptide substrate and requires large amounts of antiphosphotyrosine antibody, a modified indirect method, wherein a phosphorylated peptide or protein produced by kinase reaction will compete with a fluorescent phosphopeptide used as a tracer for immunocomplex formation with phosphotyrosine antibody, was developed. In this format kinase activity will result in loss of the polarization signal. Both the direct and indirect FP-PTK immunoassays have been compared with a more commonly used (32)PO(4) transfer assay and validated using lymphoid T-cell protein tyrosine kinase (Lck). In both assays, Lck activity showed a similar dependence on ATP, Lck enzyme, and peptide substrate concentration, comparable to the (32)PO(4) transfer assay. Inhibition by staurosporine and the Lck inhibitor 4-amino-5-(methylphenyl)-7-(tert-butyl)pyrazolo[3, 4-d]pyrimidine in these two FP assays was similar to that obtained in the (32)PO(4) transfer assay. The advantages of these FP-PTK assays over the other kinase assays, besides high sensitivity, are use of inexpensive nonisotropic substrate; environmental safety; homogeneous nature of FP kinase assays that are done in the same tube (or in a well of 96- or 384-well microtiter plates), without separation, precipitation, or washing; and increase of throughput.     

Fluorescent Biosensor of CDK5 Kinase Activity in Glioblastoma Cell Extracts and Living Cells

CDK5 plays a major role in neuronal functions, and is hyperactivated in neurodegenerative pathologies as well as in glioblastoma and neuroblastoma. Although this kinase constitutes an established biomarker and pharmacological target, there are few means of probing its activity in cell extracts or in living cells. To this aim a fluorescent peptide reporter of CDK5 kinase activity, derived from a library of CDK5‐specific substrates, is engineered and its ability to respond to recombinant CDK5/p25 is established and CDK5 activity in glioblastoma cell extracts is reported on through sensitive changes in fluorescence intensity. A cell‐penetrating variant of this biosensor which can be implemented to image CDK5 activation dynamics in space and in time is further implemented. This original biosensor constitutes a potent tool for quantifying differences in CDK5 activity following treatment with selective inhibitors and for monitoring CDK5 activation, following inhibition or stimulation, in a physiologically relevant environment. As such it offers attractive opportunities to develop a diagnostic assay for neuronal pathologies associated with hyperactivated CDK5, as well as a companion assay to evaluate response to new therapies targeting this kinase.     

Tissue distribution of the AMP-activated protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive peptide assay

1. We have synthesized two peptides, one based on the exact sequence around the unique site (Ser79) for the AMP-activated protein kinase on rat acetyl-CoA carboxylase (SSMS peptide) and another in which the serine residue corresponding to the site for cyclic-AMP-dependent protein kinase (Ser77) was replaced by alanine (SAMS peptide). 2. Both peptides were phosphorylated with similar kinetics by the AMP-activated protein kinase, but only the SSMS peptide was a substrate for cyclic-AMP-dependent protein kinase. The SAMS peptide was not phosphorylated by any of five other purified protein kinases tested. 3. The Km of AMP-activated protein kinase for the SAMS peptide is higher than that for acetyl-CoA carboxylase, but the Vmax for peptide phosphorylation is 2.5 times higher than that of its parent protein. This peptide therefore gives a convenient and sensitive assay for the AMP-activated protein kinase. 4. Acetyl-CoA-carboxylase kinase and peptide kinase activities copurify through six steps from a post-mitochondrial supernatant of rat liver, showing that the SAMS peptide is a specific substrate for the AMP-activated protein kinase in this tissue. We could not demonstrate AMP-dependence of the kinase activity in crude preparations, apparently due to endogenous AMP remaining bound to the enzyme. However, 8-bromoadenosine 5-monophosphate (Br8AMP) is a partial agonist at the allosteric (AMP) site, and inhibition by 2 mM Br8AMP can be used to test that one is measuring the AMP-stimulated form of the kinase. 5. Using this approach, we have examined the kinase activity in nine different rat tissues, plus a mouse macrophage cell line, and find that there is a correlation between tissues expressing significant levels of peptide kinase activity and those active in the synthesis or storage of lipids. 6. We also use the peptide assay to show that cyclic AMP-dependent protein kinase does not activate purified AMP-activated protein kinase, and does not affect the activation of partially purified AMP-activated protein kinase by endogenous kinase kinase.     

Mechanistic studies of cAMP-dependent protein kinase action

The details of the process by which protein kinase catalyzes phosphoryl group transfers are beginning to be understood. Early work that explored the primary specificity of cAMP-dependent protein kinase action enabled the synthesis of small peptide substrates for the enzyme. Enzyme-peptide interactions seem simpler to understand than protein-protein interactions, so peptide substrates have been used in most protein kinase studies. In most investigations the kinetics for the phosphorylation of small peptides have been interpreted as being consistent with mechanisms which do not invoke phospho-enzyme intermediates (see, for example, Bolen et al.). Protein kinase has been shown to bind two metal ions in the presence of a nucleotide. Using magnetic resonance techniques the binding of these ions has been utilized to elucidate the conformation of nucleotide and peptide substrates or inhibitors when bound in the enzymic active site. Also, two new peptides with the form Leu-Arg-Arg-Ala-Ser-Y-Gly, where Y was either Pro or (N-methyl)Leu, were synthesized and found not to be substrates, within the limits of detection, for protein kinase. The striking lack of affinity that protein kinase has for such peptides which are unlikely to form a beta 3-6 turn has not been reported before. Our results may indicate that this type of turn is a requirement for protein kinase catalyzed phosphorylation or that these peptides lack the ability to form a particular hydrogen bond with the enzyme. Magnetic resonance techniques have indicated that the distance between the phosphorous in the gamma-phosphoryl group of MgATP and the hydroxyl oxygen of serine in the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly is 5.3 +/- 0.7 A. This, together with certain kinetic evidence, suggests that the mechanism by which protein kinase catalyzes phosphoryl group transfer has considerable dissociative character. Chemical modifications, including one using a peptide-based affinity label, have identified two residues at or near the active site, lysine-72 and cysteine 199. While neither of these groups has been shown to be catalytically essential, similar studies may help to identify groups that are directly involved in the catalytic process. Finally, a spectrophotometric assay for cAMP-dependent protein kinase has been described. Using this assay the preliminary results of an in-depth study of the pH dependence of protein kinase catalyzed phosphoryl group transfer have been obtained. This study shall aid in the identification of active site residues and should contribute to the elucidation of the enzyme's catalytic mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)