A microtiter-based assay for protein kinase activity suitable for the analysis of large numbers of samples, and its application to the study of Drosophila learning mutants

We have developed a microtiter-based assay for protein kinase activity which depends on the immobilization of substrate proteins to nitrocellulose. The technique makes use of a filtration manifold, allowing as much as a 10-fold increase in efficiency as compared to other protein kinase assays. We have used this assay to measure cAMP-dependent protein kinase (PKA) in Drosophila learning and memory mutants, with exogenous and endogenous substrates. An alteration was found in the affinity of PKA in the mutant turnip. The procedure should be useful for rapid screening of mutants and drugs and could be adapted to additional types of protein kinases as well as protein phosphatases.     

Generation of a polyclonal antibody that simultaneously detects multiple Ser/Thr protein kinases

In order to obtain a polyclonal antibody that recognizes various protein kinases, a peptide corresponding to an amino acid sequence of a highly conserved subdomain (subdomain VIB) of the protein kinase family was synthesized and used for immunization. When the synthetic peptide, CVVHRDLKPENLLLAS, was coupled to keyhole limpet hemocyanin (KLH) and used to immunize rabbits, polyclonal antibodies that detected multiple protein kinases on a Western blot were generated. One of the antibodies obtained, KI98, detected a variety of purified Ser/Thr protein kinases, such as calmodulin-dependent protein kinase II (CaM-kinase II), calmodulin-dependent protein kinase IV (CaM-kinase IV), cAMP-dependent protein kinase, protein kinase C, and Erk2. The antibody detected as low as 0.2 ng of protein kinases blotted onto a nitrocellulose membrane by dot-immunobinding assay. When a rat brain extract was analyzed with this antibody, various protein kinases were simultaneously detected. The present anti-peptide antibody with a broad spectrum of cross-reactivity to multiple protein kinases may be a powerful tool for comprehensive analysis focused on protein kinases.     

Identification and structure-activity relationship of 2-morpholino 6-(3-hydroxyphenyl) pyrimidines, a class of potent and selective PI3 kinase inhibitors

PI3 Kinases are a family of lipid kinases mediating numerous cell processes such as proliferation, migration, and differentiation. The PI3 kinase pathway is often de-regulated in cancer through PI3Kα overexpression, gene amplification, mutations, and PTEN phosphatase deletion. PI3K inhibitors represent therefore an attractive therapeutic modality for cancer treatment. Herein we describe a novel series of PI3K inhibitors sharing a pyrimidine core and showing significant potency against class I PI3 kinases in the biochemical assay and in cells. The discovery, synthesis and SAR of this chemotype are described.     

Bioluminescent kinase strips: A novel approach to targeted and flexible kinase inhibitor profiling

In addition to target efficacy, drug safety is a major requirement during the drug discovery process and is influenced by target specificity. Therefore, it is imperative that every new drug candidate be profiled against various liability panels that include protein kinases. Here, an effective methodology to streamline kinase inhibitor profiling is described. An accessible standardized profiling system for 112 protein kinases covering all branches of the kinome was developed. This approach consists of creating different sets of kinases and their corresponding substrates in multi-tube strips. The kinase stocks are pre-standardized for optimal kinase activity and used for inhibitor profiling using a bioluminescent ADP detection assay. We show that these strips can routinely generate inhibitor selectivity profiles for small or broad kinase family panels. Lipid kinases were also assembled in strip format and profiled together with protein kinases. We identified two specific PI3K inhibitors that have off-target effects on CK2 that were not reported before and would have been missed if compounds were not profiled against lipid and protein kinases simultaneously. To validate the accuracy of the data generated by this method, we confirmed that the inhibition potencies observed are consistent with published values produced by more complex technologies such as radioactivity assays.     

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.     

Advances in chemical proteomic evaluation of lipid kinases—DAG kinases as a case study

Advancements in chemical proteomics and mass spectrometry lipidomics are providing new opportunities to understand lipid kinase activity, specificity, and regulation on a global cellular scale. Here, we describe recent developments in chemical biology of lipid kinases with a focus on those members that phosphorylate diacylglycerols. We further discuss future implications of how these mass spectrometry–based approaches can be adapted for studies of additional lipid kinase members with the aim of bridging the gap between protein and lipid kinase–focused investigations.     

Cyclodextrins enhance recombinant phosphatidylinositol phosphate kinase activity

Inositol lipid kinases have been studied extensively in both plant and animal systems. However, major limitations for in vitro studies of recombinant lipid kinases are the low specific activity and instability of the purified proteins. Our goal was to determine if cyclodextrins would provide an effective substrate delivery system and enhance the specific activity of lipid kinases. For these studies, we have used recombinant Arabidopsis thaliana phosphatidylinositol phosphate kinase 1 (At PIPK1). At PIPK1 was produced as a fusion protein with glutathione-S-transferase and purified on glutathione-Sepharose beads. A comparison of lipid kinase activity using substrate prepared in alpha-, beta-, or gamma-cyclodextrin indicated that beta-cyclodextrin was most effective and enhanced lipid kinase activity 6-fold compared with substrate prepared in Triton X-100-mixed micelles. We have optimized reaction conditions and shown that product can be recovered from the cyclodextrin-treated recombinant protein, which reveals a potential method for automating the assay for pharmacological screening.     

ProKinO: an ontology for integrative analysis of protein kinases in cancer

Background

Protein kinases are a large and diverse family of enzymes that are genomically altered in many human cancers. Targeted cancer genome sequencing efforts have unveiled the mutational profiles of protein kinase genes from many different cancer types. While mutational data on protein kinases is currently catalogued in various databases, integration of mutation data with other forms of data on protein kinases such as sequence, structure, function and pathway is necessary to identify and characterize key cancer causing mutations. Integrative analysis of protein kinase data, however, is a challenge because of the disparate nature of protein kinase data sources and data formats.

Results

Here, we describe ProKinO, a protein kinase-specific ontology, which provides a controlled vocabulary of terms, their hierarchy, and relationships unifying sequence, structure, function, mutation and pathway information on protein kinases. The conceptual representation of such diverse forms of information in one place not only allows rapid discovery of significant information related to a specific protein kinase, but also enables large-scale integrative analysis of protein kinase data in ways not possible through other kinase-specific resources. We have performed several integrative analyses of ProKinO data and, as an example, found that a large number of somatic mutations (∼288 distinct mutations) associated with the haematopoietic neoplasm cancer type map to only 8 kinases in the human kinome. This is in contrast to glioma, where the mutations are spread over 82 distinct kinases. We also provide examples of how ontology-based data analysis can be used to generate testable hypotheses regarding cancer mutations.

Conclusion

We present an integrated framework for large-scale integrative analysis of protein kinase data. Navigation and analysis of ontology data can be performed using the ontology browser available at: http://vulcan.cs.uga.edu/prokino.     

A high-throughput liposome substrate assay with automated lipid extraction process for PI 3-kinase

The signaling pathways involving lipid kinase class I phosphatidylinositol 3-kinases (PI 3-kinases) regulate cell growth, proliferation, and survival. Class I PI 3-kinases catalyze the conversion of PI (4,5)P(2) to PI (3,4,5)P(3), which acts as a lipid second messenger to activate mitogenic signaling cascades. Recently, p110alpha, a class IA PI 3-kinase, was found to be mutated frequently in many human cancers. Therefore, it is increasingly studied as an anticancer drug target. Traditionally, PI 3-kinase activities have been studied using liposome substrates. This method, however, is hampered significantly by the labor-intensive manual lipid extraction followed by a low-throughput thin-layer chromatography analysis. The authors describe a high-throughput liposome substrate-based assay based on an automated lipid extraction method that allows them to study PI 3-kinase enzyme mechanism and quantitatively measure inhibitor activity using liposome substrates in a high-throughput mode. This improved assay format can easily be extended to study other classes of phosphoinositide lipid kinases.     

A pH sensitive colorometric assay for the high-throughput screening of enzyme inhibitors and substrates: a case study using kinases

We have developed an uncoupled, pH sensitive kinase assay that can be used for high-throughput screening of potential inhibitors or for determining substrate specificity. Kinases catalyze the transfer of a gamma-phosphoryl group from ATP to an appropriate hydroxyl acceptor with the release of a proton. This assay is based on the detection of this proton using an appropriately matched buffer/indicator system. The assay was used to measure the activity of four readily available kinases, including hexokinase, glucokinase, glycerokinase, and pyruvate kinase, which was run in the reverse direction. We also went on to screen a small series of mono- and diphosphonucleotides for inhibition of hexokinase as well as a modest set of potential hexokinase substrates. We determined sucrose to be a modest substrate for hexokinase with a K(m) of 1.8 +/- 0.2 mM, a k(cat) of 142 +/- 3 min(-1), and a V(max) that is 15% of that for glucose. Given the importance of kinases in a diverse array of biological functions and disease states, there is a need for a simple, rapid assay system. We feel this assay will lend itself well to meet this end. This method should be applicable to many other enzymatic reactions which involve a change in pH.     

A cell-permeable, activity-based probe for protein and lipid kinases

Protein and lipid kinases are two important classes of biomedically relevant enzymes. The expression and activity of many kinases are known to be dysregulated in a variety of diseases, and proteomic tools that can assess the presence and activity of these enzymes are likely to be useful for their evaluation. Because many of the mechanisms by which protein kinases can become unregulated involve post-translational modifications or changes in protein localization, they can only be detected by examining protein activity, sometimes within the context of the living cell. Wortmannin is a steroid-derived fungal metabolite that covalently inhibits both protein and lipid kinases. Here we describe the synthesis of three wortmannin derivatives, biotin-wortmannin, BODIPY-wortmannin, and tetramethylrhodamine-wortmannin. We demonstrate that these reagents exhibit reactivity similarly as wortmannin and react with members of the phosphatidylinositol 3-kinase and PI3-kinase related kinase families in cellular lysates. Moreover, in some cases these reagents can differentiate between the active and inactive forms of the enzyme, indicating that they are activity-based probes. The reagents also exhibit complementary properties. The biotin-wortmannin reagent is effective in the isolation of labeled proteins; all three can be used for protein labeling, and BODIPY-wortmannin is cell-permeable and can be used to label proteins within cells.     

A Homogeneous,High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel,Rapid Substrate Preparation

Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z’-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-³²P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC₅₀ of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.     

An ultrasensitive high-throughput electrochemiluminescence immunoassay for the Cdc42-associated protein tyrosine kinase ACK1

Several drugs inhibiting protein kinases have been launched successfully, demonstrating the attractiveness of protein kinases as therapeutic targets. Functional genomics research within both academia and industry has led to the identification of many more kinases as potential drug targets. Although a number of well-known formats are used for measuring protein kinase activity, some less well-characterized protein kinases identified through functional genomics present particular challenges for existing assay formats when there is limited knowledge of the endogenous substrates or activation mechanisms for these novel kinase targets. This is especially the case when a very sensitive assay is required to differentiate often highly potent inhibitors developed by late-stage medicinal chemistry programs. ACK1 is a non-receptor tyrosine kinase that has been shown to be involved in tumorigenesis and metastasis. Here we describe the development of an extremely sensitive high-throughput assay for ACK1 capable of detecting 240 fmol per well of the kinase reaction product employing a BV-tag-based electrochemiluminescence assay. This assay is universally applicable to protein tyrosine kinases using a BV-tag-labeled monoclonal antibody against phosphotyrosine. Furthermore, this assay can be extended to the evaluation of Ser/Thr kinases in those cases where an antibody recognizing the phospho-product is available.     

Evolution of Functional Diversity in the Holozoan Tyrosine Kinome

The emergence of multicellularity is strongly correlated with the expansion of tyrosine kinases, a conserved family of signaling enzymes that regulates pathways essential for cell-to-cell communication. Although tyrosine kinases have been classified from several model organisms, a molecular-level understanding of tyrosine kinase evolution across all holozoans is currently lacking. Using a hierarchical sequence constraint-based classification of diverse holozoan tyrosine kinases, we construct a new phylogenetic tree that identifies two ancient clades of cytoplasmic and receptor tyrosine kinases separated by the presence of an extended insert segment in the kinase domain connecting the D and E-helices. Present in nearly all receptor tyrosine kinases, this fast-evolving insertion imparts diverse functionalities, such as post-translational modification sites and regulatory interactions. Eph and EGFR receptor tyrosine kinases are two exceptions which lack this insert, each forming an independent lineage characterized by unique functional features. We also identify common constraints shared across multiple tyrosine kinase families which warrant the designation of three new subgroups: Src module (SrcM), insulin receptor kinase-like (IRKL), and fibroblast, platelet-derived, vascular, and growth factor receptors (FPVR). Subgroup-specific constraints reflect shared autoinhibitory interactions involved in kinase conformational regulation. Conservation analyses describe how diverse tyrosine kinase signaling functions arose through the addition of family-specific motifs upon subgroup-specific features and coevolving protein domains. We propose the oldest tyrosine kinases, IRKL, SrcM, and Csk, originated from unicellular premetazoans and were coopted for complex multicellular functions. The increased frequency of oncogenic variants in more recent tyrosine kinases suggests that lineage-specific functionalities are selectively altered in human cancers.     

Identification of interacting proteins for calcium-dependent protein kinase 8 by a novel screening system based on bimolecular fluorescence complementation

Protein kinases are key regulators of cell function that constitute one of the largest and most functionally diverse gene families. We developed a novel assay system, based on the bimolecular fluorescence complementation (BiFC) technique in Escherichia coli, for detecting transient interactions such as those between kinases and their substrates. This system detected the interaction between OsMEK1 and its direct target OsMAP1. By contrast, BiFC fluorescence was not observed when OsMAP2 or OsMAP3, which are not substrates of OsMEK1, were used as prey proteins. We also screened for interacting proteins of calcium-dependent protein kinase 8 (OsCPK8), a regulator of plant immune responses, and identified three proteins as interacting molecules of OsCPK8. The interaction between OsCPK8 and two of these proteins (ARF-GEF and peptidyl prolyl isomerase) was confirmed in rice cells by means of BiFC technology. These results indicate that our new assay system has the potential to screen for protein kinase target molecules.     

Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding

Inositol pyrophosphates (such as IP7 and IP8) are multifunctional signaling molecules that regulate diverse cellular activities. Inositol pyrophosphates have 'high-energy' phosphoanhydride bonds, so their enzymatic synthesis requires that a substantial energy barrier to the transition state be overcome. Additionally, inositol pyrophosphate kinases can show stringent ligand specificity, despite the need to accommodate the steric bulk and intense electronegativity of nature's most concentrated three-dimensional array of phosphate groups. Here we examine how these catalytic challenges are met by describing the structure and reaction cycle of an inositol pyrophosphate kinase at the atomic level. We obtained crystal structures of the kinase domain of human PPIP5K2 complexed with nucleotide cofactors and either substrates, product or a MgF(3)(-) transition-state mimic. We describe the enzyme's conformational dynamics, its unprecedented topological presentation of nucleotide and inositol phosphate, and the charge balance that facilitates partly associative in-line phosphoryl transfer.     

In vitro protein kinase activity measurement by flow cytometry

Protein kinases are important drug targets, and a wide variety of methods have been developed for assessing their activity. A key element in developing selective kinase inhibitors is the ability to rapidly compare the effects of an inhibitor on several related or unrelated kinases. We describe a simple, nonradioactive, bead-based method for detecting kinase activity in vitro. Biotinylated peptide substrates are immobilized on beads and phosphorylation is detected with anti-phosphopeptide antibodies with no separation steps required. Phosphorylation is dependent on the amount of kinase in the assay and can be inhibited by known kinase inhibitors in a concentration-dependent manner. Using Luminex technology, we measured the activity of three kinases (PKA, PKC-μ, and Akt) on multiple substrates simultaneously. We also discuss conditions necessary to optimize measurement of the activity of several kinases in a single sample.     

Protein kinase affinity reagents based on a 5-aminoindazole scaffold

Affinity reagents that target protein kinases are powerful tools for signal transduction research. Here, we describe a general set of kinase ligands based on a 5-aminoindazole scaffold. This scaffold can readily be derivatized with diverse binding elements and immobilized analogs allow selective enrichment of protein kinases from complex mixtures.     

Appearance of smaller lipid bodies and protein kinase activation in the lipid body fraction are induced by an increase in the nitrogen source in the Mortierella fungus

We studied the regulation of lipid body biogenesis in the oleaginous fungus Mortierella ramanniana var. angulispora by investigating culture conditions to modulate lipid body size, which we found was affected by the carbon-to-nitrogen ratio (C/N ratio) in the culture medium. Increasing the nitrogen source or decreasing the C/N ratio from 38 to 9 induced the appearance of lipid bodies with diameters less than 2-3 micro m, which are usually found at a C/N ratio of 38 in this fungus. To determine factors regulating lipid body size, we compared lipid body fractions from fungal cells cultured at different C/N ratios. We found some differences in polypeptide profiles between lipid body fractions from fungal cells cultured at different C/N ratios for 2 days when the lipid bodies were enlarged at a C/N ratio of 38. We then compared the phosphorylation of lipid body proteins, since protein phosphorylation plays a pivotal role in various aspects of signal transduction. In vitro phosphorylation in the lipid body fraction indicated that protein kinase activity toward endogenous and exogenous substrates such as histone IIIS, VIIS, and myelin basic protein increased in the lipid body fraction at a C/N ratio of 9. Further analysis by in-gel protein kinase assay indicated the presence of at least three activated protein kinases with molecular masses of 75, 72, and 42 kDa, which were also autophosphorylated. These results indicate the presence of nutrient-regulated protein kinases and increased phosphorylation in lipid bodies, which correlate with the appearance of smaller lipid bodies in this fungus. Further studies to characterize these protein kinases at the molecular level should provide new insights into the link between nutrient sensing and lipid storage.