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.

Chromatin-associated protein kinases in human normal and benign hyperplastic prostate.

Nuclear phosphoproteins and protein kinases of human normal and benign hyperplastic prostate (BPH) were studied in an effort to delineate their properties and to identify any underlying differences therein. Chromatin-associated protein kinases active towards phosvitin, lysine-rich histone, and endogenous nonhistone proteins were characterized in human prostatic nuclei. The general properties of the human chromatin-associated prostatic protein kinases were similar to those of rat ventral prostate chromatin. Polyamines stimulated the phosphorylation of endogenous nonhistone proteins and phosvitin. Protein kinases active towards phosvitin and lysine-rich histones were unaltered in chromatin from BPH tissue as compared with the normal prostate. However, phosphorylation of chromatin-associated nonhistone proteins was markedly enhanced (average, 123%) in BPH tissue as compared with the normal tissue. The results indicate a change in the protein kinase reaction specifically involving chromatin-associated nonhistone proteins of BPH tissue as compared with normal human prostate.

Polyamine-like effects of aminoglycosides on various messenger-independent protein kinase reactions.

Gentamicin and several other aminoglycoside antibiotics in millimolar concentrations directly stimulate the phosphorylation of casein by purified preparations of cAMP- and Ca2+-independent protein kinases PK-C2 (equivalent to cytosolic casein kinase II) and its nuclear counterpart PK-N2 from rat liver and ventral prostate. These stimulatory effects of aminoglycoside antibiotics were similar to those exerted by the aliphatic polyamine spermine. Phosphorylation of casein by purified preparations of messenger-independent protein kinases PK-C1 (equivalent to cytosolic casein kinase I) and its nuclear counterpart PK-N1 was much less enhanced by spermine and the aminoglycoside antibiotics tested. Stimulations of PK-N2 reactions evoked by gentamicin or spermine (at 0.5 and 1.0 mM) were not additive. Several amino sugars tested were without effect on these protein kinases. Methylglyoxal bis(guanylhydrazone) which is known to block the stimulatory effects of polyamines on certain other enzymes did not alter spermine-stimulated phosphorylation of casein catalyzed by PK-N2 preparations.

Differential regulation of prostatic protein kinase C isozymes by androgens.

Multiple isozymes of Ca2+/phospholipid-dependent protein kinase (PKC) were isolated from the rat ventral prostate. The enzyme exists mainly as type II (beta), and type III (alpha) forms, and it is possible that type II isozyme may comprise the subspecies beta 1 and beta 2. The total and specific activities of prostatic PKC isoforms were reduced in castrated animals; this decrease was specific since administration of androgens to castrated animals reversed such a decline. Also, there was a differential response to androgen deprivation so that type III isozyme declined at a faster rate than that of type II. Thus, our studies show for the first time that PKC of the rat ventral prostate comprises multiple isozymes, and that the activity of these various forms are differentially regulated by androgens.

Modulation of cofactor requirement for the activation of protein kinase C by heparin. Possible effect at the regulatory domain.

Heparin was found to stimulate the phosphorylation of histone H1 but not protamine sulfate catalyzed by Ca2+/phospholipid-dependent protein kinase (protein kinase C or PKC). The effect of heparin on histone H1 phosphorylation appeared to be due to an increase in phosphatidylserine affinity for PKC activation in the presence of heparin. This effect of heparin was abolished when trypsinized, cofactor-independent, PKC was employed to phosphorylate histone H1. These studies suggest that heparin acts at the regulatory domain of PKC, and emphasize the importance of the negative charge in influencing the accessibility of the substrate to PKC action.

Antiserum to carcinoembryonic antigen recognizes a phosphotyrosine-containing protein in human colon cancer cell lines.

Members of the carcinoembryonic antigen (CEA) family include CEA, non-specific cross reacting antigen (NCA), and biliary glycoprotein (BGP), and appear to function as cell adhesion molecules. Immunoprecipitation and subsequent gel electrophoresis of proteins from several colon cancer cell lines labeled with [gamma-32P]ATP, under conditions designed to detect ecto-kinase-catalyzed phosphorylation of cellular proteins, revealed that polyclonal anti-CEA antiserum recognized a 175-190 kDa phosphoprotein on the surface of colon cancer cells. The ability to detect this phosphoprotein did not correlate with CEA production, and immunoprecipitation studies suggested that the phosphoprotein is BGP. Phosphoamino acid analysis of the 175-190 kDa protein showed that it contained predominantly phosphotyrosine.

Studies of prostatic cytosol protein kinases of the aging ACI rat.

Changes in prostatic cytosolic cyclic AMP (cAMP)-dependent and independent protein kinases associated with aging in ACI rats were determined. A decrease in prostatic weight and tissue protein content (but not DNA) with age was noted. The cytosolic cAMP-dependent protein kinase was present predominantly as the type II isozyme. Total content of this enzyme per prostate declined by 37% in 12-month-old rats but remained relatively stable thereafter. However, the activity ratio (+cAMP/-cAMP) increased with aging, indicating an elevation in the amount of the holoenzyme or a decrease in the amount of the free catalytic subunit. Concomitantly the specific activity (activity/unit protein in the cytosol) of this enzyme was found to be increased; this is most likely due to the loss of bulk proteins in the cytosol, and may indicate a slower loss of this enzyme protein in relation to other cytosolic proteins. By comparison, the cAMP-independent protein kinase demonstrated a marked and progressive decline in amount as well as in specific activity with aging, e.g. 80-90% decline at 12 months of age. The above changes in these enzymes did not appear to be due to low circulating androgen in the animals, since they were not reversed on administration of 3.0 mg of testosterone propionate per rat daily for four consecutive days. It is therefore suggested that the above-described changes in prostatic cytosolic protein kinases are primarily associated with cellular senescence in this organ.

A procedure for rapid isolation of cloned cDNA inserts using the polymerase chain reaction technique.

A reproducible and rapid procedure for isolation of cloned cDNA insert from a lambda gt11 cDNA library is described. The procedure relies on the polymerase chain reaction method using forward and reverse primers for lambda gt11, followed by isolation of the cloned cDNA insert by a rapid technique. The procedure should also be applicable to isolation of cDNA inserts cloned in other vectors such as lambda gt10.

Mechanisms and significance of polyamine stimulation of various protein kinase reactions.

An overview of the work on polyamine effects on certain protein kinase reactions is presented. In general, the reactions catalyzed by the messenger-independent protein kinases but not by cyclic nucleotide-, Ca2+-, Ca2+-calmodulin-, and Ca2+-anionic lipid-dependent protein kinases, are markedly enhanced by polyamines. The extent of this stimulation depends critically on the nature of the protein substrate and several other factors. A variety of other polycationic compounds including Co3+(NH3)6, polybrene, and certain aminoglycoside antibiotics exert polyamine-like effects in the same reactions. These observations suggest that the charge properties rather than any strict chemical structure play a role in the action of polyamines. Available data do not support a specific "cofactor" function of these amines for the protein kinases involved in the polyamine-stimulable reactions. It appears that the action of polyamines is mediated via their influence on the conformational status of the protein substrates thereby altering the availability of the phosphorylatable sites to the active sites on the protein kinases. Although this notion is supported by several lines of evidence, at present a role of the influence of polyamines on both the substrate and enzyme cannot be ruled out. Possible physiological relevance of the polyamine-stimulable protein kinase reactions observed in the in vitro experiments remains problematic in the absence of precise knowledge on the "effective" or free concentrations of intracellular polyamines.

A novel and simple method to assay the activity of individual protein kinases in a crude tissue extract.

Protein kinases and phosphatases play an important role in a variety of cellular functions such as cell growth, development, and gene expression (1). It is estimated that one-third of the proteins in a typical mammalian cell are phosphorylated and about 200 protein kinases and 100 protein phosphatases have been identified. In addition, perhaps 2-3% of the genes in the entire genome of an eukaryotic cell may code for protein kinases and as many as 5% of the human genes may encode protein kinases and phosphatases (2). The fact that these protein kinases and phosphatases have multiple substrates in vivo may explain their diverse physiological functions (1-3). Thus, it is of considerable interest to develop an assay system that is specific for certain protein kinases and simple enough to be used by both the novice as well as the expert in the field.

Monoclonal antibodies against nuclear casein kinase NII (PK-N2).

A monoclonal antibody was produced against the nuclear casein kinase II (PK-N2) isolated from rat liver. The antibody was of the IgM class, and showed immunoreactivity towards the higher molecular weight subunit (41K Da) of the protein kinase in Western blots. The antibody was equally reactive towards the PK-N2 isolated from rat ventral prostate indicating that it can recognize the enzyme from different tissues of the rat. The antibody also detected the cytosolic casein kinase II (CK-2) suggesting significant similarity of the antigenic domains in the two forms of this protein kinase. No binding was detected with the nuclear or cytosolic casein kinase I (PK-N1 and CK-1). The antibody did not inhibit the enzyme activity or directly precipitate the enzyme, but when coupled to an affinity matrix and cross-linked with dimethylpimelimidate, it was capable of removing nearly all the PK-N2 activity from solution.

Isolation and identification of a novel cellular protein that potently activates Ca2+/phospholipid-dependent protein kinase (protein kinase C).

A novel heat-stable cellular protein that significantly stimulated Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) was identified. It is ubiquitous and has a molecular mass of over 150 kDa, and was shown to be specific for PKC. It activates PKC in the absence and presence of phospholipids; however, its maximal stimulatory potential was achieved when optimal amounts of phospholipids were also present in the reaction medium. Thus, in comparison with classical cofactors such as phospholipid, where activation of PKC mainly involves interaction with the regulatory domain of PKC, the mechanism of activation of PKC by the activator appears to involve several binding sites.

IBC's 4th Annual Conference on Signal Transduction Therapy: novel targets for therapeutic intervention.

A variety of approaches to investigate cell signalling were presented. The main goal of the speakers was to make use of signalling pathways as targets for the development of new drugs. These targets included protein kinase A anchoring, Ras-dependent transformation, growth factor receptor tyrosine kinases (trk and insulin), cytokine-mediated signalling, phospholipid lipases and immunosuppressant targeted enzymes. Each of the speakers gave a concise summary of the area involved and the rationale supporting their approaches to investigation.

Tissue-specific decline in cytosolic casein kinase II of the ventral prostate in aging rats.

An age-associated decline in the activity of cytosolic casein kinase II in the rat ventral prostate was observed. The decrease in specific and total activity of casein kinase II of prostatic cytosol obtained from 12-month old rats compared with that from 3-month old rats was 50% and 70%, respectively. This decrease was tissue specific since no such alteration in enzymic activity was found in liver, lung, or heart. The decline in activity was not due to an increase in the concentration of a casein kinase inhibitor or to altered androgenic status with aging. Rather, the reduction in the activity of the enzyme was commensurate with the decrease in the total concentration of the enzyme protein as determined by an ELISA using anti-casein kinase II antibodies. The activity of casein kinase II in the nuclear fraction of the rat prostate was not altered in aging animals. It may be concluded that the cytosolic form of casein kinase II in the prostate of older animals is regulated at the transcriptional level (but not via the androgen-receptor complex mechanism). An alternative explanation for this observation may relate to altered stability of the enzyme in the prostate of older animals.

Nature of the intrinsic protein kinases involved in phosphorylation of non-histone proteins in intact prostatic nuclei: further identification of androgen-sensitive protein kinase reactions.

Nuclei isolated from rat ventral prostate contain a number of messenger-dependent and -independent protein kinases. Studies were undertaken to determine the relative contribution of these protein kinases in phosphorylation of non-histone proteins (NHPs) in isolated nuclei. The data suggest that messenger-dependent protein kinases such as those dependent on cAMP or Ca2+/calmodulin or Ca2+/phospholipid may be present in very small amounts in intact isolated nuclei, and thus appear not to be significantly involved in phosphorylation of endogenous NHPs. Messenger-independent nuclear associated protein kinases PK-N1 and PK-N2 are known to catalyze the phosphorylation of NHPs in vitro (Goueli Sa, et al., Eur J Biochem 113: 45-51, 1980). Of these, the intrinsic heparin-sensitive PK-N2 as compared with heparin-insensitive PK-N1 appeared to be the predominant protein kinase engaged in phosphorylation of NHPs in intact nuclei. About 78-88% of NHP phosphorylation in intact nuclei was inhibited by heparin suggesting that the remaining 12-22% phosphorylation of NHPs was catalyzed via the heparin-insensitive protein kinase(s). Further, the data provide additional evidence that heparin-sensitive PK-N2 is the one that is most responsive to androgenic status in the animal.

Indomethacin and inhibition of protein kinase reactions.

Indomethacin, an inhibitor of prostaglandin biosynthesis, is useful in studies aimed at understanding the metabolism and physiological function of prostaglandins. A recent report showing that indomethacin at 10(-7) M potently inhibits the cyclic AMP-dependent protein kinases (cAMP-PrK) from ileal mucosa in the presence or absence of cyclic AMP, suggests how indomethacin may antagonize prostaglandin action on ileal mucosa. It also suggests that indomethacin might be useful in studying the properties and functions of protein kinase reactions. Inhibitors of prostaglandin biosynthesis, such as sodium salicylate and acetylsalicylate, at concentrations near 10(-2) M, have been shown to inhibit bovine diaphragm protein kinase only in the presence of cAMP, while stimulating it in the absence of cAMP. We report here that complete inhibition of cAMP-PrKs by indomethacin requires a concentration of 10(-3) M and is not tissue-specific, and that the effect of indomethacin is concentration dependent above 2 x 10(-4) M for the cAMP-dependent, and above 10(-3) M for cAMP-independent PrKs. These results contrast previous ones.

CD31 (PECAM-1), CDw32 (Fc gamma RII), and anti-HLA class I monoclonal antibodies recognize phosphotyrosine-containing proteins on the surface of human neutrophils.

Although most studies of protein phosphorylation have focused on intracellular reactions, studies have provided evidence for the existence of ectoprotein kinase activity on the surface of some cells including human neutrophils. The identification and characterization of physiologic substrates of ectoprotein kinase activity should aid the understanding of the role of this enzyme activity in cell function. Immunoprecipitation and subsequent gel electrophoresis of proteins from neutrophils labeled with [gamma-32P]ATP under conditions initially designed to detect ectoprotein kinase activity revealed that CD31, CDw32, and anti-HLA class I mAbs specifically recognize phosphoproteins on the surface of human neutrophils. Phosphorylation of these proteins was inhibited by pretreatment of cells with an impermeant sulfhydryl reagent before radiolabeling. Phosphoamino acid analysis of the proteins revealed that they contained predominantly phosphotyrosine. However, controlled proteolysis of intact cells and purified HLA class I revealed that the HLA class I heavy chain was phosphorylated on the cytoplasmic domain. These results suggest that the molecules recognized by CD31 (PECAM-1) and CDw32 (Fc gamma RII) Abs may also be phosphorylated on cytoplasmic domains under conditions originally designed to detect ectoprotein kinase activity. Phosphorylation of CD31 (PECAM-1), Fc gamma RII, and HLA class I heavy chain on tyrosine may play a role in regulating their function. These results emphasize that the demonstration that a membrane protein is an ectokinase substrate is complex and requires the definitive localization of the phosphorylated residue to the extracellular domain of the protein.

Fractionation and partial purification of rat liver nuclear protein kinases.

We have fractional and partially purified several rat liver nuclear protein kinases by utilizing endogenous (nonhistone proteins) and exogenous acidic (dephosphophosvitin) and basic (lysine-rich histone) protein substrates. Three enzymes were active towards endogenous substrates, two towards dephosphophosvitin and two towards lysine-rich histone. Of the latter only one was cAMP-dependent, however, only minimal cAMP binding activity was detected. Several features of these enzyme reactions are described revealing distinct differences in the characteristics of each of these enzymes.