A magnetic bead-based protein kinase assay with dual detection techniques

A novel magnetic bead-based protein kinase assay was developed using MALDI-TOF mass spectrometry (MALDI-TOF MS) and immunochemifluorescence as two independent detection techniques. Abltide substrate was immobilized onto magnetic beads via noncovalent biotin–streptavidin interactions. This noncovalent immobilization strategy facilitated peptide release and allowed MALDI-TOF MS analysis of substrate phosphorylation. The use of magnetic beads provided rapid sample handling and allowed secondary analysis by immunochemifluorescence to determine the degree of substrate phosphorylation. This dual detection technique was used to evaluate the inhibition of c-Abl kinase by imatinib and dasatinib. For each inhibitor, IC₅₀ (half-maximal inhibitory concentration) values determined by these two different detection methods were consistent and close to values reported in the literature. The high-throughput potential of this new approach to kinase assays was preliminarily demonstrated by screening a chemical library consisting of 31 compounds against c-Abl kinase using a 96-well plate. In this proof-of-principle experiment, both MALDI-TOF MS and immunochemifluorescence were able to compare inhibitor potencies with consistent values. Dual detection may significantly enhance the reliability of chemical library screening and identify false positives and negatives. Formatted for 96-well plates and with high-throughput potential, this dual detection kinase assay may provide a rapid, reliable, and inexpensive route to the discovery of small-molecule drug leads.     

Phosphorylation of a pancreatic zymogen granule membrane protein by endogenous calcium/phospholipid-dependent protein kinase

The occurrence of phospholipid-sensitive calcium-dependent protein kinase (referred to as C kinase) and its endogenous substrate proteins was examined in a membrane preparation from rat pancreatic zymogen granules. Using exogenous histone H1 as substrate, C kinase activity was found in the membrane fraction. The kinase was solubilized from membranes using Triton X-100 and partially purified using DEAE-cellulose chromatography. An endogenous membrane protein (Mr approximately equal to 18 000) was found to be specifically phosphorylated in the combined presence of Ca2+ and phosphatidylserine. Added diacylglycerol was effective in stimulating phosphorylation of exogenous histone by the partially purified C kinase, but had no effect upon phosphorylation of the endogenous 18 kDa protein by the membrane-associated C kinase. Phosphorylation of the 18 kDa protein was rapid (detectable within 30 s following exposure to Ca2+ and phosphatidylserine), and highly sensitive to Ca2+ (Ka = 4 microM in the presence of phosphatidylserine). These findings suggest a role for this Ca2+-dependent protein phosphorylation system in the regulation of pancreatic exocrine function.     

Determination of adenosine kinase activity by high-pressure liquid chromatography

High-pressure liquid chromatography (reverse-phase mode) was used to assay adenosine kinase in cell and tissue extracts. The method is optimized for a rapid and selective analysis using 6-methylthiopurine riboside as substrate, isocratic elution and detection at 300 nm. A complete separation of substrate and product is achieved in about 3 min with no interference by other UV-absorbing compounds; the limit of detection is 20 pmoles.     

A rapid purification of T4 polynucleotide kinase using Blue Dextran-Sepharose chromatography.

A rapid batch procedure is described for purification of T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to near homogeneity using Blue Dextran-Sepharose chromatography. The enzyme preparation is sufficiently free of contaminating endonuclease and alkaline phosphatase activities to be suitable for radioactively labeling nucleic acids in vitro. Kinetic measurements indicate that the chromophore of Blue Dextran, Cibacron Blue F3GA, inhibits the activity of T4 polynucleotide kinase competitively with respect to single stranded DNA substrate and non-competitively with respect to the rATP substrate.     

An assay for sphingosine kinase activity using biotinylated sphingosine and streptavidin-coated membranes

Sphingosine kinase catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, a lipid signaling molecule implicated in roles in a diverse range of mammalian cell processes through its action as both a ligand for G-protein-coupled cell-surface receptors and an apparent intracellular second messenger. This paper describes a rapid, sensitive, and reproducible assay for sphingosine kinase activity using biotinylated sphingosine (biotinyl-Sph) as a substrate and capturing the phosphorylated product with streptavidin-coated membranes. We have shown that both human sphingosine kinase 1 and 2 (hSK1 and hSK2) can efficiently phosphorylate biotinyl-Sph, with K(m) values similar to those of sphingosine. The assay utilizing this substrate has high sensitivity for hSK1 and hSK2, with detection limits in the low-femtomole range for both purified recombinant enzymes. Importantly, we have also demonstrated the capacity of this assay to measure endogenous sphingosine kinase activity in crude cell extracts and to follow changes in this activity following sphingosine kinase activation. Together, these results demonstrate the potential utility of this assay in both cell-based analysis of sphingosine kinase signaling pathways and high-throughput screens for agents affecting sphingosine kinase activity in vitro.     

An improved method to unravel phosphoacceptors in Ser/Thr protein kinase-phosphorylated substrates

Identification of the phosphorylated residues of bacterial Ser/Thr protein kinase (STPK) substrates still represents a challenging task. Herein, we present a new strategy allowing the rapid determination of phosphoacceptors in kinase substrates, essentially based on the dual expression of the kinase with its substrate in the surrogate E. coli, followed by MS analysis in a single-step procedure. The performance of this strategy is illustrated using two distinct proteins from Mycobacterium tuberculosis as model substrates, the GroEL2 and HspX chaperones. A comparative analysis with a standard method that includes mass spectrometry analysis of in vitro phosphorylated substrates is also addressed.     

A green fluorescent protein kinase substrate allowing detection and localization of intracellular ERK/MAP kinase activity

We describe a versatile intracellular reporter of ERK/MAP kinase activity: a cDNA construct, pGFP.MBP, encoding amino acids 85-144 of the human myelin basic protein fused to the C-terminus of an enhanced green fluorescent protein (GFP). The fused fragment of myelin basic protein contains a single consensus ERK/MAP kinase phosphorylation motif (PRTP, where the threonine is phosphorylated). Phosphorylation of the specific motif can be detected via immunoblotting or immunofluorescence with a commercially available phospho-specific monoclonal antibody. When expressed in mammalian cells by either transient or stable transfection, the fusion protein acts as a bona fide kinase substrate, as demonstrated by rapid serum-induced phosphorylation that is blocked by a specific MEK inhibitor. Moreover, the localization of the total substrate pool is easily visualized by GFP autofluorescence and the extent of its phosphorylation simultaneously detected within intact fixed cells by immunofluorescence using the commercially available phospho-specific antibody. The approach described should be generally applicable to the intracellular analysis of many specific protein kinase substrates for which phospho-specific antibodies have been produced.     

Novel destabilization of nucleotide binding by the gamma phosphate of ATP in the yeast SR protein kinase Sky1p

SR protein kinases (SRPKs) regulate the temporal and cell-specific selection of alternative splice sites. These enzymes are highly unique members of the protein kinase family. SRPKs contain a large domain insert (approximately 200 residues) within the kinase core, do not require phosphorylation for regulation, have an extended helix insert near the nucleotide pocket, and possess unusual substrate specificity determinants. The yeast SRPK, Sky1p, rapidly phosphorylates its natural substrate Npl3 but binds ATP with a high K(m), suggesting that some of these distinctive structural features may be correlated with nucleotide binding [Aubol et al. (2002) Biochemistry 41, 10002-10009]. To address this issue, the nucleotide binding properties of Sky1p were studied using fluorescence spectroscopy. The affinities of several nucleotides (ATP, ADP, AMP, adenosine, and AMPPNP) to Sky1p and the prototype kinase, cAMP-dependent protein kinase, were compared in the absence and presence of the metal activator, Mg(2+), using a fluorescence-based displacement assay. The data indicate that Sky1p, unlike cAMP-dependent protein kinase, potently destabilizes the gamma phosphate of ATP. This novel finding suggests that rapid phosphoryl transfer may be facilitated by unique mechanisms in both protein kinases.     

Nerve growth factor stimulates the tyrosine phosphorylation of MAP2 kinase in PC12 cells.

NGF treatment of PC12 cells results in the rapid activation of MAP2 kinase. We report here that the induction of enzyme activity was correlated with the phosphorylation of MAP2 kinase, detected by metabolic labeling of the enzyme and with anti-phosphotyrosine antibodies. NGF stimulated the phosphorylation of MAP2 kinase on tyrosine, as well as serine and threonine residues. Western blot analysis using a polyclonal anti-phosphotyrosine antibody demonstrated that the tyrosine phosphorylation of MAP2 kinase was maximal within 2 min following NGF exposure and preceded the induction of MAP2 kinase activity. The NGF-stimulated tyrosine phosphorylation of an identified substrate provides direct evidence for the participation of a tyrosine kinase in the mechanism of action of NGF.     

Screening for the optimal specificity profile of protein kinase C using electrospray mass-spectrometry

A peptide library approach based on electrospray mass-spectrometric (ESI-MS) detection of phosphopeptides was designed for rapid and quantitative characterization of protein kinase specificity. The k(cat)/K(m) values for the protein kinase Cbeta (PKCbeta) were determined for a systematically varied set of individual substrate peptides in library mixtures by the ESI-MS method. The analysis revealed a complex structural specificity profile in positions around the phosphorylated serine with hydrophobic and/or basic residues being mostly preferred. On the basis of the kinetic parameters, a highly efficient peptide substrate for PKCbeta (K(m)value below 100 nM) FRRRRSFRRR and its alanine substituted pseudosubstrate-analog inhibitor (K(i) value of 76 nM) were designed. The quantitative specificity profiles obtained by the new approach contained more information about kinase specificity than the conventional substrate consensus motifs. The new method presents a promising basis for design of substrate-site directed peptide or peptidomimetic inhibitors of protein kinases. Second, highly specific substrates could be designed for novel applications such as high-throughput protein kinase activity screens on protein kinase chips.     

Insulin stimulates a novel Mn2+-dependent cytosolic serine kinase in rat adipocytes

The cytosolic fraction of insulin-treated adipocytes exhibits a 2-fold increase in protein kinase activity when Kemptide is used as a substrate. The detection of insulin-stimulated kinase activity is critically dependent on the presence of phosphatase inhibitors such as fluoride and vanadate in the cell homogenization buffer. The cytosolic protein kinase activity exhibits high sensitivity (ED50 = 2 X 10(-10) M) and a rapid response (maximal after 2 min) to insulin. Kinetic analyses of the cytosolic kinase indicate that insulin increases the Vmax of Kemptide phosphorylation and ATP utilization without affecting the affinities of this enzyme toward the substrate or nucleotide. Upon chromatography on anion-exchange and gel filtration columns, the insulin-stimulated cytosolic kinase activity is resolved from the cAMP-dependent protein kinase and migrates as a single peak with an apparent Mr = 50,000-60,000. The partially purified kinase preferentially utilizes histones, Kemptide, multifunctional calmodulin-dependent protein kinase substrate peptide, ATP citrate-lyase, and acetyl-coenzyme A carboxylase as substrates but does not catalyze phosphorylation of ribosomal protein S6, casein, phosvitin, phosphorylase b, glycogen synthase, inhibitor II, and substrate peptides for casein kinase II, protein kinase C, and cGMP-dependent protein kinase. Phosphoamino acid analyses of the 32P-labeled substrates reveal that the insulin-stimulated cytosolic kinase is primarily serine-specific. The insulin-activated cytosolic kinase prefers Mn2+ to Mg2+ and is independent of Ca2+. Unlike ribosomal protein S6 kinase and protease-activated kinase II, the insulin-sensitive cytosolic kinase is fluoride-insensitive. Taken together, these results indicate that a novel cytosolic protein kinase activity is activated by insulin.     

Mechanism of phosphorylation of the epidermal growth factor receptor at threonine 669

The major site of phosphorylation of the epidermal growth factor (EGF) receptor after treatment of cells with EGF is threonine 669. Phosphorylation of this site is also associated with the transmodulation of the EGF receptor caused by platelet-derived growth factor and phorbol ester. A distinctive feature of the primary sequence surrounding threonine 669 is the proximity of 2 proline residues (-Pro-Leu-Thr669-Pro-). This site is not a substrate for phosphorylation by protein kinase C. To investigate the mechanism of the increased phosphorylation of the EGF receptor at threonine 669, in vitro assays were used to measure protein kinase and protein phosphatase activities present in homogenates prepared from cells treated with and without EGF. No evidence for the regulation of protein phosphatase activity was obtained in experiments using the [32P]phosphate-labeled EGF receptor as a substrate. A synthetic peptide corresponding to residues 663-681 of the EGF receptor was used as a substrate for protein kinase assays. Incubation of murine 3T3 L1 pre-adipocytes and human WI-38 fibroblasts with EGF caused a rapid increase (3-10-fold) in the level of threonine protein kinase activity detected in cell homogenates. Similar results were obtained after EGF treatment of Chinese hamster ovary cells expressing wild-type (Thr669) and mutated (Ala669) human EGF receptors. Activation of the threonine protein kinase activity was also observed in cells treated with platelet-derived growth factor, serum, and phorbol ester. Insulin-like growth factor-1 caused no significant change in protein kinase activity. Together these data indicate a role for the regulation of the activity of a threonine protein kinase in the control of the phosphorylation state of the EGF receptor at threonine 669. The significance of the identification of a growth factor-stimulated threonine protein kinase to the mechanism of signal transduction is discussed.     

Metal and metal-ATP interactions with brain and cardiac adenylate cyclases.

Metal (Me) and MeATP interactions with adenylate cyclases associated with rabbit ventricular particles and with a detergent-dispersed preparation from rat cerebellum have been studied. data were simulated to fit kinetic models in which an inhibitor (HATP or ATP) is added in constant proportion to the variable substrate (MeATP). The specific models considered were that the enzyme binds (a) MeATP as the substrate; (b) MeATP as the substrate and HATP or ATP as an inhibitor; (c) MeATP as the substrate and free Me as an activator; and (d) MeATP as the substrate, free Me as an activator, and HATP or ATP as an inhibitor. Both equilibrium-ordered and random (rapid equilibrium assumption) types of sequential kinetic models were considered. The various models were tested using cardiac particulate adenylate cyclase in the presence of either a phosphoenolpyruvate-pyruvate kinase or a creatine phosphate-creatine kinase ATP-regeneration system. Although the enzyme with either system appeared to bind Mg2+ as an activator, one or both ATP-regeneration systems also seemed to interact directly with adenylate cyclase, making clear interpretations difficult. With the phosphoenolpyruvate-pyruvate kinase system, kinetic patterns on double reciprocal plots were linear as a function of MgATP, but with creatine phosphate-creatine kinase, kinetic patterns were concave downward. The kinetic models were further tested using the detergent-dispersed cerebellar enzyme, a preparation with low adenosine triphosphatase activity and not requiring the addition of an ATP-regeneration system. Reciprocal plots were linear and intersecting as a function of either MeATP or Me (Me = Mg2+ or Mn2+), and secondary replots of slopes and intersecting as function of either MeATP or Me (Me = Mg2+ or Mn2+), and secondary replots of slopes and intercepts also were linear. These data indicate that the brain detergent-dispersed enzyme conforms to a bireactant, sequential mechanism where free cation is a required activator and free ATP is not a potent inhibitor.     

An electrostatic engine model for autoinhibition and activation of the epidermal growth factor receptor (EGFR/ErbB) family

We propose a new mechanism to explain autoinhibition of the epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases based on a structural model that postulates both their juxtamembrane and protein tyrosine kinase domains bind electrostatically to acidic lipids in the plasma membrane, restricting access of the kinase domain to substrate tyrosines. Ligand-induced dimerization promotes partial trans autophosphorylation of ErbB1, leading to a rapid rise in intracellular [Ca(2+)] that can activate calmodulin. We postulate the Ca(2+)/calmodulin complex binds rapidly to residues 645--660 of the juxtamembrane domain, reversing its net charge from +8 to -8 and repelling it from the negatively charged inner leaflet of the membrane. The repulsion has two consequences: it releases electrostatically sequestered phosphatidylinositol 4,5-bisphosphate (PIP(2)), and it disengages the kinase domain from the membrane, allowing it to become fully active and phosphorylate an adjacent ErbB molecule or other substrate. We tested various aspects of the model by measuring ErbB juxtamembrane peptide binding to phospholipid vesicles using both a centrifugation assay and fluorescence correlation spectroscopy; analyzing the kinetics of interactions between ErbB peptides, membranes, and Ca(2+)/calmodulin using fluorescence stop flow; assessing ErbB1 activation in Cos1 cells; measuring fluorescence resonance energy transfer between ErbB peptides and PIP(2); and making theoretical electrostatic calculations on atomic models of membranes and ErbB juxtamembrane and kinase domains.     

Salicylic acid activates a 48-kD MAP kinase in tobacco.

The involvement of phosphorylation/dephosphorylation in the salicylic acid (SA) signal transduction pathway leading to pathogenesis-related gene induction has previously been demonstrated using kinase and phosphatase inhibitors. Here, we show that in tobacco suspension cells, SA induced a rapid and transient activation of a 48-kD kinase that uses myelin basic protein as a substrate. This kinase is called the p48 SIP kinase (for SA-Induced Protein kinase). Biologically active analogs of SA, which induce pathogenesis-related genes and enhanced resistance, also activated this kinase, whereas inactive analogs did not. Phosphorylation of a tyrosine residue(s) in the SIP kinase was associated with its activation. The SIP kinase was purified to homogeneity from SA-treated tobacco suspension culture cells. The purified SIP kinase is strongly phosphorylated on a tyrosine residue(s), and treatment with either protein tyrosine or serine/threonine phosphatases abolished its activity. Using primers corresponding to the sequences of internal tryptic peptides, we cloned the SIP kinase gene. Analysis of the SIP kinase sequence indicates that it belongs to the MAP kinase family and that it is distinct from the other plant MAP kinases previously implicated in stress responses, suggesting that different members of the MAP kinase family are activated by different stresses.     

The synthetic peptide RPRAATF allows specific assay of Akt activity in cell lysates

The Akt protein kinase is a critical signaling molecule in a range of cellular processes. A key to identifying the role of this pleiotropic kinase in any particular process is the ability to quantitate its activity. In this study we show that the synthetic peptide RPRAATF is a specific substrate for the kinase in crude cell extracts, thus enabling rapid, convenient, and sensitive assay of Akt activity. Peptide kinase activity was confined to a single peak upon sequential ion-exchange chromatography of whole-cell extracts of Balb/c 3T3 fibroblasts. This activity was stimulated by both platelet-derived growth factor and pervanadate, phosphatidyl inositol 3-kinase dependent, and inhibited by specific immunodepletion with anti-Akt antisera. Furthermore, direct assays of crude extracts from a range of cell types using this peptide were consistent with the results obtained using specific immunoprecipitation assays.     

Application of multiplexed capillary electrophoresis with laser-induced fluorescence (MCE-LIF) detection for the rapid measurement of endogenous extracellular signal-regulated protein kinase (ERK) levels in cell extracts

Multiplexed (96-lane) capillary electrophoresis with laser-induced fluorescence (MCE-LIF) detection was used for the rapid analysis of extracellular signal-regulated protein kinase (ERK) levels from in vitro cell extracts. The levels of ERK enzyme in cell extracts were determined by monitoring the conversion of a fluorescent-labeled peptide substrate to a phosphorylated fluorescent-labeled peptide product using MCE-LIF. The incorporation of a fluorescent internal standard was found to improve the precision of the analysis. The enzyme assay conditions including substrate concentration, reaction time and enzyme linear range were rapidly optimized using the MCE-LIF approach for both direct and immunoprecipitation-based ERK assays. The levels of ERK from in vitro cell extracts stimulated with angiopoietin 1 (Ang1*) were determined using the MCE-LIF approach. The advantages of MCE-LIF for developing and applying enzyme assays, as well as the figures of merit for the direct and immunoprecipitation ERK assays, are discussed.     

A direct radioassay for pyruvate kinase activity

Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate. A direct radioassay for this enzyme using [14C]PEP as substrate has been developed. The product, [14C]pyruvate, can be separated from the substrate rapidly and easily by applying the mixture to a hydroxyapatite column, and eluting the [14C]pyruvate directly into a scintillation vial. The [14C]PEP is bound to the column which can be regenerated and used indefinitely. The assay is sensitive, rapid, and particularly well suited for the simultaneous assay of large numbers of samples.     

IKK-i and TBK-1 are enzymatically distinct from the homologous enzyme IKK-2: comparative analysis of recombinant human IKK-i, TBK-1, and IKK-2

NF-kappaB is sequestered in the cytoplasm by the inhibitory IkappaB proteins. Stimulation of cells by agonists leads to the rapid phosphorylation of IkappaBs leading to their degradation that results in NF-kappaB activation. IKK-1 and IKK-2 are two direct IkappaB kinases. Two recently identified novel IKKs are IKK-i and TBK-1. We have cloned, expressed, and purified to homogeneity recombinant human (rh)IKK-i and rhTBK-1 and compared their enzymatic properties with those of rhIKK-2. We show that rhIKK-i and rhTBK-1 are enzymatically similar to each other. We demonstrate by phosphopeptide mapping and site-specific mutagenesis that rhIKK-i and rhTBK-1 are phosphorylated on serine 172 in the mitogen-activated protein kinase kinase activation loop and that this phosphorylation is necessary for kinase activity. Also, rhIKK-i and rhTBK-1 have differential peptide substrate specificities compared with rhIKK-2, the mitogen-activated protein kinase kinase activation loop of IKK-2 being a more favorable substrate than the IkappaBalpha peptide. Finally, using analogs of ATP, we demonstrate unique differences in the ATP-binding sites of rhIKK-i, rhTBK-1, and rhIKK-2. Thus, although these IKKs are structurally similar, their enzymatic properties may provide insights into their unique functions.     

Stimulation by phspholipid of calcium-dependent phosphorylation of endogenous proteins from mammalian tissues

The occurrence of endogenous substrate proteins for calcium-dependent protein kinase, augmented by either phospholipid or calmodulin, was examined in extracts of several tissues. Pancreas, vas deferens, adrenal and liver were found to contain substrate proteins for phospholipid-sensitive protein kinase. Phosphorylation of pancreatic substrate protein for phospholipid-senstivie protein kinase was rapid and highly sensitive to Ca²⁺, being detectable within 15 s a following exposure to Ca²⁺ and phosphatidylserine and at concentrations of Ca²⁺ as low as 0.5 μM. These findings suggest that the phospholipid-sensitive protien kinase system may serve to mediate some effects of Ca²⁺ in a variety of mammalian cell types.