A microtiter-based assay for the detection of protein tyrosine kinase activity

A 96-well microtiter enzyme-linked immunosorbent assay (ELISA) for protein tyrosine kinases has been developed. This assay uses one of several substrates that are phosphorylated by tyrosine kinase, an antibody to phosphotyrosine, and a peroxidase-linked second antibody. Color development is monitored by a change in absorbance at 450 nm and is dependent upon time, enzyme, ATP, and substrate concentrations. Specificity of the ELISA for phosphotyrosine was shown by inhibition of binding of the anti-phosphotyrosine antibody with phenyl phosphate. Results obtained in the ELISA compared favorably with those obtained by direct phosphorylation of the substrate with [³²P]ATP. Staurosporine and K252a, protein kinase inhibitors, showed titratable inhibition of tyrosine kinase activity. This assay is a rapid, nonradioactive alternative to traditional methodology and is also amenable to automation.     

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.     

Interactions of the human red cell membrane tyrosine kinase with heparin

Heparin interacts with protein kinases in various ways; the different patterns of behavior of heparin towards protein kinases contributes to the characterization of these enzymes. We studied the interactions between heparin and a new type of tyrosine kinase extracted from the normal human red cell membrane. We found that heparin inhibited kinase activity by competition with ATP. Furthermore the interaction of heparin with the red cell membrane tyrosine kinase allowed us to use heparin-agarose chromatography as a step towards tyrosine kinase purification.     

Rapid protein kinase assay using phosphocellulose-paper absorption.

Protein kinase (EC 2.7.1.37) catalyzes the phosphorylation of serine and threonine residues of a number of proteins. Histone is widely used as an acceptor substrate in measuring the activity of this enzyme isolated from a variety of sources. We have devised a rapid procedure for resolving phosphohistone from ATP and its metabolites based on the specific absorption of phosphorylated histone onto phosphocellulose paper. Using [γ-³²P]ATP as the phosphoryl donor, aliquots of the protein kinase assay mixture are applied to phosphocellulose-paper disks that are then immersed in water which elutes [γ-³²P]ATP and metabolites. After brief organic solvent extraction and drying, bound radioactivity is measured by liquid scintillation spectrometry.     

Endogenous phosphorylation of proteins and phosphatidylinositol in the plasma membranes of a human astrocytoma

Incubation of plasma membrane preparations from several tissues with [gamma-32P]ATP resulted in the phosphorylation of phosphatidylinositol as well as of proteins. The presence of an active phosphatidylinositol kinase in these membranes was indicated by equal or greater incorporation of 32P into phosphatidylinositol phosphate than into proteins. Phosphorylation of endogenous protein and lipid substrates by protein and phosphatidylinositol kinases in the plasma membranes of a human astrocytoma was investigated in detail. Maximal protein phosphorylation required the presence of Nonidet-P40 and phosphatase inhibitors (vanadate or fluoride). The rate of protein phosphorylation was greater with Mg2+ than with Mn2+, and phosphoserine accounted for 60% of the radioactivity incorporated into proteins. In the presence of Mn2+, phosphorylation of tyrosine was increased and was equal to that of serine phosphorylation (40%). With one exception, the overall pattern of phosphorylated proteins was similar with either Mg2+ or Mn2+. Maximal phosphatidylinositol phosphorylation of the astrocytoma plasma membranes also required detergent and phosphatase inhibitors. However, the enzymatic characteristics of lipid phosphorylation differed from those of protein phosphorylation with respect to divalent cation activation, ATP dependence, and sensitivity to inhibition by p-chloromercuriphenyl sulfonate, quercetin, and nucleoside derivatives. These results suggest that phosphorylation of plasma membrane proteins and phosphatidylinositol is catalyzed by different enzymes. The fact that membrane preparations exhibited phosphatidylinositol kinase activity almost 100,000 times greater than that exhibited by the purified tyrosine kinase of ros gene would exclude this and similar oncogene proteins from making a significant contribution to the overall phosphatidylinositol phosphorylation of cell membranes.     

Use of hydrophilic interaction chromatography for the study of tyrosine protein kinase specificity

A new HPLC method has been developed to assay tyrosine protein kinase activity. Using hydrophilic interaction chromatography, it is possible to resolve the four components of the incubation medium: substrate peptide, [³²P]phosphorylated peptide, unreacted [γ-³²P]ATP, and ³²P-labelled inorganic phosphate. ATP interacts so strongly with the stationary phase material that it can be removed selectively from the incubation medium with solid-phase extraction cartridges packed with the same type of material. The three remaining components of interest can then be resolved by reversed-phase or hydrophilic interaction HPLC. This procedure permits the evaluation of almost every type of peptide as a substrate of tyrosine protein kinase.     

Rapid measurement of protein kinase and phosphatase activities by slot-filtration.

A slot-filtration method has been developed for the detection and quantitation of protein kinase and phosphatase activities. In this technique, after kinase-dependent phosphorylation or phosphatase-dependent dephosphorylation of different substrates, samples are transferred under vacuum onto nitrocellulose using a slot-blotting apparatus. Non-incorporated or released radioactivity is then removed by filtration and washing under vacuum. Quantitation is performed by scintillation or Cerenkov counting of the excised membrane slots. Application of the method to the assay of four different protein kinases (protein kinase N, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinases type I and type III) and one phosphatase is presented. A number of protein substrates with varying molecular masses and isoelectric points were found suitable for the slot-filtration technique. The method is applicable to impure as well as purified kinase and phosphatase preparations, can be used over a wide range of concentrations of substrates, has a very low background of nonspecific ATP binding and provides highly reproducible data. The slot-filtration method can also be adapted for use with ion-exchange paper, particularly for assays using peptides as substrates. The technique, with either nitrocellulose or ion-exchange paper, can be used to rapidly process large numbers of samples and can be simultaneously applied to direct comparison of different kinases, phosphatases and/or substrates in the same experiment.     

Effects of kinetin on phosphorylation of leaf membrane proteins.

Isolated Chinese cabbage leaf membranes were phosphorylated by membrane-associated protein kinase(s) in the presence or [gamma-32P]ATP. Membrane-associated 32P radioactivity appeared to be bound to membrane proteins. Both smooth cell membranes and chloroplast lamellae reacted with ATP. Phosphorylation of the membranes was inhibited by Ca2+ and partially inhibited by kinetin or 6-benzyladenine. The possibility that cytokinin effects on membrane phosphorylation might increase ion availability was investigated in vivo. It was found that Ca2+ could substitute for kinetin in the leaf disc expansion assay.     

Tyrosine phosphorylation of a membrane protein from Pseudomonas solanacearum.

We have investigated a tyrosine kinase activity from Pseudomonas solanacearum, an economically important plant pathogen. In vitro incubation of membrane fractions with [gamma-32P]ATP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an 85-kDa phosphoprotein. Phosphorylation of this protein on tyrosine residues was demonstrated by phosphoamino acid analysis of base hydrolysis products and by immunoanalysis of Western blots (immunoblots) with antiphosphotyrosine monoclonal antibody. In vitro incubation of membranes with ATP was not required for recognition by the antibody, indicating that the 85-kDa protein is phosphorylated in vivo. These results demonstrate that membranes from P. solanacearum exhibit a tyrosine kinase activity toward an endogenous membrane protein. This bacterium provides an opportunity to study the structure and function of a prokaryotic tyrosine kinase.     

Photocleavable peptide-oligonucleotide conjugates for protein kinase assays by MALDI-TOF MS

Robust methods for highly parallel, quantitative analysis of cellular protein tyrosine kinase activities may provide tools critically needed to decipher oncogenic signaling, discover new targeted drugs, diagnose cancer and monitor patients. Here, we describe proof-of-principle for a novel protein kinase assay with the potential to help overcome these challenges. MALDI-TOF mass spectrometry provides an ideal tool for label-free multiplexed analysis of peptide phosphorylation, but is poorly matched to homogeneous assays and complex samples. Thus, we conjugated a common oligonucleotide tag to multiple peptide substrates, offering efficient capture from solution-phase kinase reactions by annealing to the complementary sequence tethered to PEG-passivated superparamagnetic microparticles. To enable reversible conjugation, we developed a novel bifunctional cross-linker allowing simple and efficient preparation of photocleavable peptide-oligonucleotide conjugates. After washing away contaminants and following photorelease, MALDI-TOF analysis yielded relative phosphorylation of each peptide with high sensitivity and specificity. Validating the hybridization-mediated multiplexed kinase assay, when three peptide substrate-oligonucleotide conjugates were mixed with the tyrosine kinase c-Abl and ATP, we readily observed their differential phosphorylation yet measured a common IC(50) for the Abl kinase inhibitor imatinib. This new assay enables analysis of protein kinase activities in a multiplexed format amenable to screening inhibitors against multiple kinases in parallel, an important capability for drug discovery and predictive diagnostics.     

A simple assay method for determination of the specific radioactivity of the gamma-phosphate group of 32P-labelled ATP.

A simple and rapid assay method is described for determining the specific radioactivity of the gamma-phosphate group of 32P-labelled ATP. Labelled ATP is incubated with cyclic AMP, cyclic AMP-dependent protein kinase and histone H2A under conditions leading to maximum phosphorylation of the histone. The specific radioactivity of the gamma-phosphate group of the 32P-labelled ATP is calculated from the total amount of [32P]Pi incorporated into a standard amount of histone. This assay method uses inexpensive commercially available materials, and it yields an accurate specific radioactivity with as little as 0.25 nmol of 32P-labelled ATP.     

In vitro phosphorylation of bovine adrenal tyrosine hydroxylase by adenosine 3':5'-monophosphate-dependent protein kinase.

We have studied the effects of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase on the phosphorylative and functional modification of bovine adrenal tyrosine hydroxylase. Incubation of partially purified tyrosine hydroxylase with cAMP-dependent protein kinase in the presence of [gamma32P]ATP and 5 micron cAMP led to a 3- to 5-fold activation of tyrosine hydroxylase and to incorporation of [32P]phosphate into protein. When tyrosine hydroxylase preparations activated by exposure to enzymatic phosphorylating conditions were analyzed by sucrose density gradient centrifugation, polyacrylamide gel electrophoresis, and gel electrofocusing, the radioactivity of 32P was coincident with the activity of tyrosine hydroxylase, suggesting incorporation of 32P from [gamma-32P]ATP into tyrosine hydroxylase. Polyacrylamide gel electrophoresis of the phosphorylated tyrosine hydroxylase preparation in the presence of 0.1% sodium dodecyl sulfate revealed that the 60,000-dalton polypeptide subunit of tyrosine hydroxylase served as the phosphate acceptor.     

Functional characterization of peanut serine/threonine/tyrosine protein kinase: molecular docking and inhibition kinetics with tyrosine kinase inhibitors

Serine/threonine/tyrosine (STY) protein kinase from peanut is developmentally regulated and is induced by abiotic stresses. In addition, STY protein kinase activity is regulated by tyrosine phosphorylation. Kinetic mechanism of plant dual specificity protein kinases is not studied so far. Recombinant STY protein kinase occurs as a monomer in solution as shown by gel filtration chromatography. The relative phosphorylation rate of kinase against increasing enzyme concentrations follows a first-order kinetics indicating an intramolecular phosphorylation mechanism. Moreover, the active recombinant STY protein kinase could not transphosphorylate a kinase-deficient mutant of STY protein kinase. Molecular docking studies revealed that the tyrosine kinase inhibitors bind the protein kinase at the same region as ATP. STY protein kinase activity was inhibited by the tyrosine kinase inhibitors, and the inhibitor potency series against the recombinant STY protein kinase was tyrphostin > genistein > staurosporine. The inhibition constant (K(i)), and the IC(50) value of STY protein kinase for tyrosine kinase inhibitors with ATP and histone are discussed. All the inhibitors competed with ATP. Genistein was an uncompetitive inhibitor with histone, whereas staurosporine and tyrphostin were linear mixed type noncompetitive inhibitors with histone. Molecular docking and kinetic analysis revealed that Y148F mutant of the "ATP-binding loop" and Y297F mutant of the "activation loop" showed a dramatic increase in K(i) values for genistein and tyrphostin with respect to wild-type STY protein kinase. Data presented here provide the direct evidence on the mechanism of inhibition of plant protein kinases by tyrosine kinase inhibitors. This study also suggests that tyrosine kinase inhibitors may be useful in unraveling the plant tyrosine phosphorylation signaling cascades.     

A generic, homogenous method for measuring kinase and inhibitor activity via adenosine 5'-diphosphate accumulation

The authors describe an assay to measure the generation of adenosine 5'-diphosphate (ADP) resulting from phosphorylation of a substrate by a kinase. ADP accumulation is detected by conversion to a fluorescent signal via a coupled enzyme system. The technology has potential applications for the assessment of inhibitor potency and mode of action as well as kinetic analysis of enzyme activity. The assay has a wide dynamic range (0.25-75 microM) and has been validated with several kinases including the highly active cyclic adenosine monophosphate-dependent protein kinase (PKAalpha), casein kinase 1 (CK1), and the weakly active kinase Jun N-terminal kinase 2 (Jnk2alpha2). Kinase activity can be measured either in an end point or continuous mode. Assay performance in end point mode was compared with an adenosine 5'-triphosphate (ATP) depletion assay and in continuous mode with a pyruvate kinase/lactate dehydrogenase coupled assay. The ability to characterize kinase kinetics was demonstrated by deriving ATP/substrate affinity (Michaelis-Menten constant; K(m)) values for PKAalpha, CK1, and Jnk2alpha2. The assay readily measured activity with kinase reactions using protein substrates, indicating the suitability for use with large macromolecules. A wide range of inhibitor activities could be determined even in the presence of high ATP concentrations, making the assay highly suitable to characterize the mode of action of the inhibitor in question. Collectively, this assay provides a homogenous, generic method for a number of applications in kinase drug discovery.     

An assay for acidic peptide substrates of protein kinases

The assay of acidic peptides as substrates for protein kinases has not been as easy to perform as testing basic peptides or polypeptides. We have developed a simple, rapid, and cost-effective procedure that allows the design and testing of potential peptide substrates without the constraints imposed by the phosphocellulose filter paper method (the need to incorporate positively charged residues into the peptide sequence). The technique combines the chelation of 32Pi by acid molybdate with PEI-cellulose chromatography. In this way the migration of 32P-labeled Pi, ATP, and protein are impeded while phosphopeptide is eluted in 1.5 ml from a 0.25-ml disposable column. In order to validate the assay we used two angiotensin II analogues as peptide substrates for the protein tyrosine kinase pp60c-src. The assay results using the new procedure were compared to those of the phosphocellulose filter paper technique. We also demonstrated the use of this method to test linear and cyclic peptides that could not be assayed with the phosphocellulose paper technique. This assay will aid those who are attempting to determine the substrate specificity of protein kinases.     

Purification and characterization of a protein tyrosine kinase from bovine spleen

Protein tyrosine kinase was purified extensively from a 30,000 X g particulate fraction of bovine spleen by a procedure involving four column chromatographies: DEAE-Sepharose, polyamino acids affinity, hydroxylapatite, and Sephacryl S-200 molecular sieving. The purification resulted in more than 3,000-fold enrichment in [Val5]angiotensin II phosphorylation activity (specific activity 202 nmol/min/mg). All column chromatography profiles showed single protein tyrosine kinase activity peaks with the exception of that of affinity chromatography, where about 50% of the enzyme activity appeared with the breakthrough fraction; only the bound enzyme was further purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of a purified sample phosphorylated in the presence of [gamma-32P]ATP revealed the presence of a single phosphorylated polypeptide of molecular weight 50,000 which represents about 40% of total protein. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions showed that protein tyrosine kinase activity co-migrated with the phosphoprotein. Stoichiometry of the phosphorylation of the 50-kDa polypeptide was found to be 1.0 mol/mol. The purified sample did not appear to contain phosphotyrosine protein phosphatase activity. Both casein and histone could be phosphorylated by the purified sample, and the phosphorylation occurred only at tyrosine residue, suggesting that there was no protein serine and threonine kinase contamination.     

Vascular endothelial growth factor receptor KDR tyrosine kinase activity is increased by autophosphorylation of two activation loop tyrosine residues

Vascular endothelial growth factor is an important physiological regulator of angiogenesis. The function of this endothelial cell selective growth factor is mediated by two homologous tyrosine kinase receptors, fms-like tyrosine kinase 1 (Flt-1) and kinase domain receptor (KDR). Although the functional consequence of vascular endothelial growth factor binding to the Flt-1 receptor is not fully understood, it is well established that mitogenic signaling is mediated by KDR. Upon sequencing several independent cDNA clones spanning the cytoplasmic region of human KDR, we identified and confirmed the identity of a functionally required valine at position 848 in the ATP binding site, rather than the previously reported glutamic acid residue, which corresponds to an inactive tyrosine kinase. The cytoplasmic domain of recombinant native KDR, expressed as a glutathione S-transferase fusion protein, can undergo autophosphorylation in the presence of ATP. In addition, the kinase activity can be substantially increased by autophosphorylation at physiologic ATP concentrations. Mutation analysis indicates that both tyrosine residues 1054 and 1059 are required for activation, which is a consequence of an increased affinity for both ATP and the peptide substrate and has no effect on kcat, the intrinsic catalytic activity of the enzyme. KDR kinase catalyzes phosphotransfer by formation of a ternary complex with ATP and the peptide substrate. We demonstrate that tyrosine kinase antagonists can preferentially inhibit either the unactivated or activated form of the enzyme.     

Properties of a membrane-bound tyrosine kinase phosphorylating the cytosolic fragment of the red cell membrane band 3 protein

Band 3 protein of human erythrocyte membrane is phosphorylated on a tyrosine residue located near the NH2 terminal by an endogenous tyrosine kinase activity (Dekowski, S., Rybicki, A. and Drickamer, K. (1983) J. Biol. Chem. 258, 2750-2753). A tyrosine kinase phosphorylating the band 3 protein in situ has been extracted from ghosts by non-ionic detergent and partially characterized (Phan-Dinh-Tuy, F., Henry, J. and Kahn, A. (1985) Biochem. Biophys. Res. Commun. 126, 304-312). We have studied the properties of the tyrosine kinase activity which remains bound to the ghosts after detergent extraction using the 43 kDa fragment of protein 3 as substrate. This activity, solubilized from the detergent-resistant material at 0.25 M NaCl and concentrated by phosphocellulose and tyrosine-agarose chromatographies, remains linked to high molecular weight complexes. It is specific for tyrosine. Assayed with the purified 43 kDa fragment it requires the presence of Mn2+ which cannot be replaced by Mg2+. Its affinity for 43 kDa fragment is very high with a Km of 3.3 microM. ATP acts as a phosphoryl donor with a Km of 0.55 microM. The tyrosine kinase activity was not modified by insulin, DMSO, phorbol ester and epidermal growth factor, vanadate and xanthine derivatives. Polyamines spermidine and the polylysine are inhibitors in the presence of Mn2+ but not in the presence of Mg2+. Heparin is a competitive inhibitor of ATP. 2,3-Diphosphoglycerate is an inhibitor at physiological concentrations (Ki = 2 mM). Purified red cell actin is not phosphorylated by the tyrosine kinase. These properties distinguish the red cell membrane-bound tyrosine kinase from other tyrosine kinases extracted from normal cells.     

Mitochondrial AKAP121 links cAMP and src signaling to oxidative metabolism

AKAP121 focuses distinct signaling events from membrane to mitochondria by binding and targeting cAMP-dependent protein kinase (PKA), protein tyrosine phosphatase (PTPD1), and mRNA. We find that AKAP121 also targets src tyrosine kinase to mitochondria via PTPD1. AKAP121 increased src-dependent phosphorylation of mitochondrial substrates and enhanced the activity of cytochrome c oxidase, a component of the mitochondrial respiratory chain. Mitochondrial membrane potential and ATP oxidative synthesis were enhanced by AKAP121 in an src- and PKA-dependent manner. Finally, siRNA-mediated silencing of endogenous AKAP121 drastically impaired synthesis and accumulation of mitochondrial ATP. These findings indicate that AKAP121, through its role in enhancing cAMP and tyrosine kinase signaling to distal organelles, is an important regulator in mitochondrial metabolism.     

An easy and rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with 32Pi

A rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts containing other ³²P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of ³²P into casein from [γ-³²P]ATP is measured by perchloric acid precipitation of the protein on filter paper. ³²P-Casein formation is linearly related to the specific radioactivity of the [γ-³²P]ATP. Separation of ATP from other ³²P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-³²P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to ³²P_i is demonstrated.