A homogeneous fluorescence polarization assay adaptable for a range of protein serine/threonine and tyrosine kinases

Recently, a new technology for high-throughput screening has been developed, called IMAP(patent pending). IMAP technology has previously been implemented in an assay for cyclic nucleotide phosphodiesterases (PDE). The authors describe the development of a homogeneous, non-antibody-based fluorescence polarization (FP) assay for a variety of protein kinases. In this assay, fluorescently labeled peptide substrate phosphorylated by the kinase is captured on modified nanoparticles through interactions with immobilized metal (M(III)) coordination complexes, resulting in a change from low to high polarization values. This assay is applicable to protein kinases that phosphorylate serine, threonine, or tyrosine residues. The IMAP platform is very compatible with high-throughput robotics and can be applied to the 1536-well format. As there are hundreds of different kinases coded for in the human genome, the assay platform described in this report is a valuable new tool in drug discovery.     

An improved zinc cocktail-mediated fluorescence polarization-based kinase assay for high-throughput screening of kinase inhibitors

During the past few years, high-throughput screening (HTS) has provided a useful resource to researchers involved in the development of kinase inhibitors as a novel therapeutic modality. However, with all the choices among kinase assays, there is not yet a one-size-fits-all assay. Therefore, selection of a specific kinase assay is a daunting task. HTS assays should be homogeneous, cost effective, use nonradioactive reagents, generic and not time consuming. Here, we report an improved method of assaying protein kinase activity using a zinc cocktail in a fluorescence polarization-(FP) based format. Assay conditions were standardized manually and validated in a HTS format using a liquid handler. We validated this assay for both serine/threonine and tyrosine (receptor/nonreceptor) kinases. The results obtained in the HTS assay system were comparable to the commercially available fluorescence-based assay. We suggest that the reported assay is a cost-effective alternative to the IMAP-based generic kinase assay.     

Screening serine/threonine and tyrosine kinase inhibitors for histidine kinase inhibition

Histidine kinases of bacterial two-component systems are promising antibacterial targets. Despite their varied, numerous roles, enzymes in the histidine kinase superfamily share a catalytic core that may be exploited to inhibit multiple histidine kinases simultaneously. Characterized by the Bergerat fold, the features of the histidine kinase ATP-binding domain are not found in serine/threonine and tyrosine kinases. However, because each kinase family binds the same ATP substrate, we sought to determine if published serine/threonine and tyrosine kinase inhibitors contained scaffolds that would also inhibit histidine kinases. Using select assays, 222 inhibitors from the Roche Published Kinase Set were screened for binding, deactivation, and aggregation of histidine kinases. Not only do the results of our screen support the distinctions between ATP-binding domains of different kinase families, but the lead molecule identified also presents inspiration for further histidine kinase inhibitor development.     

Crystal structure of aurora-2, an oncogenic serine/threonine kinase

Aurora-2 is a key member of a closely related subgroup of serine/threonine kinases that plays important roles in the completion of essential mitotic events. Aurora-2 is oncogenic and amplified in various human cancers and could be an important therapeutic target for inhibitory molecules that would disrupt the cell cycle and block proliferation. We report the first crystal structure of Aurora-2 kinase in complex with adenosine. Analysis of residues in the active site suggests differences with structurally and biologically related protein kinases. The activation loop, which contains residues specific to the Aurora family of kinases, has a unique conformation. These results provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of the Aurora kinases.     

Molecular imaging of protein kinases

Protein kinases are important regulators of signal-transduction pathways. Dysregulated kinase activity is observed in a variety of human diseases such as cancer, making them targets for the development of molecular therapies. Identification of new drugs is greatly aided by molecular imaging tools which enable real time, non-invasive, dynamic and quantitative imaging of kinase activity in vivo. We have recently described a new reporter platform based on conformation dependent complementation of firefly luciferase to monitor serine/threonine kinase (Akt) activity. The reporter system provides unique insights into the pharmacokinetics and pharmacodynamics of drugs that modulate kinase activity in living subjects and also provide a platform for cell based high-throughput drug screening for modulators of kinase activity.     

Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine.

A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine. The gene was designated SPK1 for serine-protein kinase. Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.     

Serine/threonine kinases in the nervous system.

Three principal serine/threonine kinases that catalyze protein phosphorylation in response to second messengers are: cAMP-dependent protein kinase, multifunctional Ca2+/calmodulin-dependent protein kinase, and protein kinase C. Studies are now focusing on the distinct isoforms of these kinases that may subserve specific functions in some systems, and on providing a more molecular understanding of kinase functions. Combined genetic and biochemical approaches are beginning to be used to define unique roles for these kinases.     

Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues.

MEK is a family of dual specific protein kinases which activate the extracellular signal-regulated kinases by phosphorylation of threonine and tyrosine residues. MEK itself is activated via serine phosphorylation by upstream activator kinases, including c-raf, mos and MEK kinase. Here, we report the activation phosphorylation sites of human MEK1 and yeast STE7 kinase as determined by a combination of biochemical and genetic approaches. In human MEK1, substitution of either serine residue 218 or 222 with alanine completely abolished its activation by epidermal growth factor-stimulated Swiss 3T3 cell lysates or immunoprecipitated c-raf, suggesting that both serine residues are required for MEK1 activation. Phosphopeptide analysis demonstrated that serine residues 218 and 222 of human MEK1 are the primary sites for phosphorylation by c-raf. These two serine residues are highly conserved in all members of the MEK family, including the yeast STE7 gene product, a MEK homolog in the yeast mating pheromone response pathway. Mutation of the corresponding residues in STE7 completely abolished the biological functions of this gene. These data demonstrate that MEK is activated by phosphorylation of two adjacent serine/threonine residues and this activation mechanism is conserved in the MEK family kinases.     

Mycobacterium tuberculosis serine/threonine kinases PknB, PknD, PknE, and PknF phosphorylate multiple FHA domains

The physiologic roles and the substrates of the Mycobacterium tuberculosis (Mtb) serine/threonine kinases are largely unknown. Here, we report six novel interactions of PknB, PknD, PknE, and PknF with the Forkhead-Associated (FHA) domains of Rv0020c and the putative ABC transporter Rv1747. Purified PknB and PknF kinase domains phosphorylated multiple FHA-domain proteins in vitro. Although they remain to be verified in vivo, these reactions suggest a web of interactions between STPKs and FHA domains.     

WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II

We have cloned and characterized a novel mammalian serine/threonine protein kinase WNK1 (with no lysine (K)) from a rat brain cDNA library. WNK1 has 2126 amino acids and can be detected as a protein of approximately 230 kDa in various cell lines and rat tissues. WNK1 contains a small N-terminal domain followed by the kinase domain and a long C-terminal tail. The WNK1 kinase domain has the greatest similarity to the MEKK protein kinase family. However, overexpression of WNK1 in HEK293 cells exerts no detectable effect on the activity of known, co-transfected mitogen-activated protein kinases, suggesting that it belongs to a distinct pathway. WNK1 phosphorylates the exogenous substrate myelin basic protein as well as itself mostly on serine residues, confirming that it is a serine/threonine protein kinase. The demonstration of activity was striking because WNK1, and its homologs in other organisms lack the invariant catalytic lysine in subdomain II of protein kinases that is crucial for binding to ATP. A model of WNK1 using the structure of cAMP-dependent protein kinase suggests that lysine 233 in kinase subdomain I may provide this function. Mutation of this lysine residue to methionine eliminates WNK1 activity, consistent with the conclusion that it is required for catalysis. This distinct organization of catalytic residues indicates that WNK1 belongs to a novel family of serine/threonine protein kinases.     

A fluorescence lifetime-based assay for serine and threonine kinases that is suitable for high-throughput screening

We describe the development of a novel method for the assay of serine/threonine protein kinases based on fluorescence lifetime. The assay consists of three generic peptides (which have been used by others in the assay of >140 protein kinases in various assay formats) labeled with a long lifetime fluorescent dye (14 or 17 ns) that act as substrates for protein kinases and an iron(III) chelate that modulates the fluorescence lifetime of the peptide only when it is phosphorylated. The decrease in average fluorescence lifetime as measured in a recently developed fluorescence lifetime plate reader (Edinburgh Instruments) is a measure of the degree of phosphorylation of the peptide. We present data showing that the assay performs as well as, and in some cases better than, the “gold standard” radiometric kinase assays with respect to Z′ values, demonstrating its utility in high-throughput screening applications. We also show that the assay gives nearly identical results in trial screening to those obtained by radiometric assays and that it is less prone to interference than simple fluorescence intensity measurements.     

A fluorescence lifetime based binding assay to characterize kinase inhibitors

The authors present a fluorescence lifetime-based kinase binding assay that identifies and characterizes compounds that bind to the adenosine triphosphate (ATP)-binding pocket of a range of tyrosine and serine/threonine kinases. The assay is based on displacement of an Alexa Fluor 647 conjugate of staurosporine from the ATP-binding site of a kinase, which is detected by a change in the fluorescence lifetime of the probe between the free (displaced) and kinase-bound states. The authors screened 257 kinases for specific binding and displacement of the Alexa Fluor 647-staurosporine probe and found that approximately half of the kinases tested could potentially be assayed with this method. They present inhibitor binding data against 4 selected serine/threonine kinases and 4 selected tyrosine kinases, using 6 commonly used kinase inhibitors. Two of these kinases were chosen for further studies, in which inhibitor binding data were compared to inhibition of kinase activity using 2 separate activity assay formats. Rank-order potencies of compounds were similar, but not identical, between the binding and activity assays. It was postulated that these differences could be caused by the fact that the assays are measuring distinct phenomena, namely, activity versus binding, and in a purified recombinant kinase preparation, there can exist a mixture of active and nonactivated kinases. To explore this possibility, the authors compared binding affinity for the probe using 2 kinases in their respective nonactivated and activated (phosphorylated) forms and found a kinase-dependent difference between the 2 forms. This assay format therefore represents a simple method for the identification and characterization of small-molecule kinase inhibitors that may be useful in screening a wide range of kinases and may be useful in identifying small molecules that bind to kinases in their active or nonactivated states.     

Molecular identification of a soybean protein kinase gene family by using PCR

In this study we report identification of six members of a protein kinase gene family from soybean (Glycine max L.). Two fully degenerate oligonucleotide primers corresponding to two conserved motifs (DLK-PENV and GTHEYLAPE) in the catalytic domains of eukaryotic protein serine/threonine kinases were used in a polymerase chain reaction (PCR) to amplify soybean cDNA. Sequence analysis showed that 28 of the PCR sequences represented six different putative protein serine/threonine kinases. These results not only demonstrate that catalytic domains of protein kinases are highly conserved between plants and other eukaryotes but also suggest that there are multiple genes encoding protein kinases in plants.     

Deciphering the MAP kinase pathway

MAP kinases (MAPK) are serine/threonine kinases which are activated by a dual phosphorylation on threonine and tyrosine residues. Their specific upstream activators, called MAP kinase kinases (MAPKK), constitute a new family of dual-specific threonine/tyrosine kinases, which in turn are activated by upstream MAP kinase kinase kinases (MAPKKK). These three kinase families are successively stimulated in a cascade of activation described in various species such as mammals, frog, fly, worm or yeast.In mammals, the MAP kinase module lies on the signaling pathway triggered by numerous agonists such as growth factors, hormones, lymphokines, tumor promoters, stress factors, etc. Targets of MAP kinase have been characterize tin all subcellular compartments. In yeast, genetic epistasis helped to characterize the presence of several MAP kinase modules in the same system. By complementation tests, the relationships existing between phylogenetically distant members of each kinase family have been described. The roles of the MAP kinase cascade have been analyzed by engineering various mutations in the kinases of the module. The MAP kinase cascade has thus been implicated in higher eukaryotes in cell growth, cell fate and differentiation, and in low eukaryotes, in conjugation, osmotic stress, cell wall constrct and mitosis.     

Recombinant human pim-1 protein exhibits serine/threonine kinase activity.

The protein predicted by the sequence of the human pim-1 proto-oncogene shares extensive homology with known serine/threonine protein kinases, and yet the human Pim-1 enzyme has previously been reported to exhibit protein tyrosine kinase activity both in vitro and in vivo. Recently a new class of protein kinases has been identified which exhibits both protein-serine/threonine and protein-tyrosine kinase activities. We therefore investigated the possibility that the human Pim-1 kinase likewise possesses such bifunctional enzymatic phosphorylating activities. A full-length human pim-1 cDNA was subcloned into the bacterial vector pGEX-2T and the Pim-1 protein expressed as a fusion product with bacterial glutathione S-transferase (GST). The hybrid GST-Pim-1 fusion protein was affinity purified on a glutathione-Sepharose column prior to treatment with thrombin for cleavage of the Pim-1 protein from the transferase. Pim-1 was purified and the identity of recombinant protein confirmed by amino-terminal sequence analysis. Pim-1 was tested for kinase activity with a variety of proteins and peptides known to be substrates for either mammalian protein-serine/threonine or protein-tyrosine kinases and was found to phosphorylate serine/threonine residues exclusively in vitro. Both the Pim-1-GST fusion protein and the isolated Pim-1 protein exhibited only serine/threonine phosphorylating activity under all in vitro conditions tested. Pim-1 phosphorylated purified mammalian histone H1 with a Km of approximately 51 microM. Additionally, Pim-1 exhibited low levels of serine/threonine autophosphorylating activity. These observations place the human Pim-1 in a small select group of cytoplasmic transforming oncogenic kinases, including the protein kinase C, the Raf/Mil, and the Mos subfamilies, exhibiting serine/threonine phosphorylating activity.     

Multiple implications of 3-phosphoinositide-dependent protein kinase 1 in human cancer

3-phosphoinositide-dependent protein kinase-1 (PDK1) is a central mediator of cellular signaling between phosphoinositide-3 kinase and various intracellular serine/threonine kinases, including protein kinase B, p70 ribosomal S6 kinase, serum and glucocorticoid-inducible kinase, and protein kinase C. PDK1 activates members of the AGC family of protein kinases by phosphorylating serine/threonine residues in the activation loop. Here, we review the regulatory mechanisms of PDK1 and its roles in cancer. PDK1 is activated by autophosphorylation in the activation loop and other serine residues, as well as by phosphorylation of Tyr-9 and Tyr-373/376. Src appears to recognize PDK1 following tyrosine phosphorylation. The role of heat shock protein 90 in regulating PDK1 stability and PDK1-Src complex formation are also discussed. Furthermore, we summarize the subcellular distribution of PDK1. Finally, an important role for PDK1 in cancer chemotherapy is proposed. In conclusion, a better understanding of its molecular regulatory mechanisms in various signaling pathways will help to explain how PDK1 acts as an oncogenic kinase in various cancers, and will contribute to the development of novel cancer chemotherapies.     

A novel transmembrane serine/threonine protein kinase gene from a rifamycin SV-producing amycolatopsis mediterranei U32.

Genomic DNA sequencing in the vicinity of methylmalonyl-CoA mutase gene (mutAB) from a rifamycin SV-producing Amycolatopsis mediterranei U32 allowed us to clone, sequence, and identify a gene encoding a novel serine/threonine protein kinase (amk). The sequence contains a complete ORF of 1821 base pairs encoding a predicted protein of 606 amino acids in length. The N-terminal domain of the protein shows significant homology to the catalytic domain of other protein kinases from both prokaryotic and eukaryotic sources. It also contains all the structural features that are highly conserved in active protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP-binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein kinase. This protein kinase gene was expressed in Escherichia coli and was shown to have the ability of autophosphorylation. The autophosphorylated site was found to be the threonine at position 164 by labeled phosphoamino acid analysis and site-directed mutagenesis. The C-terminal half of protein kinase was found to contain strong transmembrane structures by PhoA fusion protein analysis, suggesting that Amk protein kinase is a transmembrane protein. A Southern hybridization experiment showed that this type of protein kinase is distributed ubiquitously and might play significant physiological roles in the various species of streptomycetes. However, overexpression of amk gene in Streptomyces cinnamonensis showed no effect on methylmalonyl-CoA mutase activity, monensin production and the hyphae morphology. Although its biological role is still unknown, Amk protein kinase is the first transmembrane serine/threonine protein kinase described for genus Amycolatopsis.     

A generic time-resolved fluorescence assay for serine/threonine kinase activity: application to Cdc7/Dbf4

The serine/threonine protein kinase family is a large and diverse group of enzymes that are involved in the regulation of multiple cellular pathways. Elevated kinase activity has been implicated in many diseases and frequently targeted for the development of pharmacological inhibitors. Therefore, non-radioactive antibody-based kinase assays that allow high throughput screening of compound libraries have been developed. However, they require a generation of antibodies against the phosphorylated form of a specific substrate. We report here a time-resolved fluorescence assay platform that utilizes a commercially-available generic anti-phospho-threonine antibody and permits assaying kinases that are able to phosporylate threonin residues on protein substrates. Using this approach, we developed an assay for Cdc7/Dbf4 kinase activity, determined the K(m) for ATP, and identified rottlerin as a non-ATP competitive inhibitor of this enzyme.     

Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction

We report herein the development of an efficient fluorescence assay for serine/threonine kinases using a peptide array. Our approach is based on chemical reactions specific to phosphoserine and phosphothreonine residues, that is, base-mediated beta-elimination of the phosphate group and subsequent Michael addition of a thiol-containing fluorescent reagent. This procedure enables the covalent introduction of a fluorescent moiety into the phosphorylated peptide. Novel fluorescent reagents were designed for this purpose and synthesized. With these reagents, protein kinase A (PKA) and Akt-1 activities were readily detected. Our method can also be used to measure the activity of kinase inhibitors. This assay is expected to be widely applicable in kinase research.