Assay Of Protein Kinase Activity Research Articles

A novel cyclic GMP-dependent protein kinase is expressed in the ring stage of the Plasmodium falciparum life cycle.

Cyclic GMP-dependent protein kinases (PKGs) are the major mediators of the cGMP signal transduction pathway and regulate a variety of physiological effects. We report here the characterization of an unusual PKG from the human malaria parasite Plasmodium falciparum (designated PfPKG). The 97.5 kDa protein contains some of the structural features of mammalian PKGs but, uniquely, contains a third predicted cGMP binding site and a degenerate fourth. Using both protein kinase activity assays and Western blotting with native P. falciparum proteins, we demonstrate here that PfPKG is expressed predominantly in the ring stage of the life cycle, suggesting a role in the development of asexual blood stage parasites. An Escherichia coli-derived recombinant protein (PfPKG2, Met115-Phe853) was purified and shown to have phosphotransferase activity in terms of both substrate phosphorylation and auto-phosphorylation. This activity was stimulated at least fivefold by 1.0 microM cyclic GMP, but was not stimulated by cAMP or by 8-pCPT-cGMP, which is a potent activator of mammalian PKGs. Several protein kinase inhibitors exhibited a range of inhibitory effects on PfPKG activity. Biochemical analysis therefore shows that PfPKG is distinct from mammalian PKGs with respect to both cyclic nucleotide analogue activation and inhibition profiles.

Rapid Activation of ERK1/2 Mitogen-Activated Protein Kinase by Corticosterone in PC12 Cells

Although the nongenomic effects of glucocorticoids have been well acknowledged, its precise intracellular signal transduction pathway remains to be elucidated. The present study using Western immunoblot and protein kinase activity assay, for the first time, showed that corticosterone (B) can induce a rapid activation of Erk1/2 mitogen-activated protein kinase (MAPK) in PC12 cells. The dose–response curve was bell shaped, with the maximal activation at 10−9 M in 15 min. The results from immunofluorescence staining also revealed that the activated Erk1/2 MAPK was translocated from cytoplasm to nucleus of PC12 cells in 15 min. Activation of Erk1/2 MAPK by B was apparently not mediated by the classical cytosolic steroid receptors, for B-BSA can induce the phosphorylation of Erk1/2 MAPK, but the antagonist (RU38486) cannot block the phosphorylation of Erk1/2 MAPK induced by B. Phosphorylation of Erk1/2 MAPK induced by B was not affected by a tyrosine kinase inhibitor (genistein), suggesting that the pathway did not involve the tyrosine kinase activity. On the other hand, protein kinase C activator (PMA) can activate and protein kinase C inhibitor (Gö6976) can block the activation of Erk1/2 MAPK induced by B. Taken together, these data clearly demonstrated that B might act via putative membrane receptor and rapidly activate Erk1/2 MAPK through protein kinase C α in PC12 cells.

A non-radioactive assay for the cAMP-dependent protein kinase activity in rat brain homogenates and age-related changes in hippocampus and cortex

Cyclic AMP-dependent protein kinase (PKA) activity was involved in a number of brain functions such as cognitive process or aging. The measurement of PKA activity is traditionally based on the use of [ 32P]ATP in phosphorylation of specific protein. Recently non-isotopic PKA assays have been developed, but none has been tested on brain homogenates. This work aimed to adapt a fluorimetric method of PKA activity into a novel assay never applied before in brain homogenate, and to characterize the enzyme activity and ratio in hippocampus and cortex from rats of different ages. Optimal conditions of homogenization and enzyme protection were determined. The method was sensitive and reproducible (intra-assay and interassay variation was 5.0% and 9.0%, respectively). In hippocampal cytosol, PKA activity was 27±8 and 80±9 nmol/min per mg protein in basal and cAMP-stimulated activity, repectively, and accounted for 80% of total cell PKA activity. The non-PKA activity, assessed by the use of the PKA specific inhibitor (PKI) accounted for 49.0% and 65.0% of endogenous levels in cytosol and membrane, respectively. cAMP-augmenting drugs effects were measured and increase of 53%, 273% and 118% over basal by 10 μM isoproterenol, 100 μM forskolin, 1 μM Sp-AMP, respectively, was observed. With respect to the changes in animal age, PKA activity increased from newborn to the mature rats but decreased in older rats. The PKA ratio was higher in cytosol than in particulate fraction, and was decreased in hippocampal sample from old rats ( P<0.05). This last result was interpreted as related to the loss of cognitive capacities in old animals.

Regulation of cyclic guanosine monophosphate-dependent protein kinase in human uterine tissues during the menstrual cycle.

Contractility of uterine smooth muscle is essential for the cyclic shedding of the endometrial lining and also for expulsion of the fetus during parturition. The nitric oxide (NO)-cGMP signaling pathway is involved in smooth muscle relaxation. The downstream target of this pathway essential for decreasing cytoplasmic calcium and muscle tone is the cGMP-dependent protein kinase (PKG). The present study was undertaken to localize expression of PKG in tissues of the female reproductive tract and to test the hypothesis that uterine smooth muscle PKG levels vary with the human menstrual cycle. Immunohistochemistry was used to localize PKG in myometrium, cervix, and endometrium obtained during proliferative and secretory phases. The PKG was localized to uterine and vascular smooth muscle cells in myometrium, stromal cells in endometrium, and a small percentage of cervical stromal cells. Using Western blot analysis and protein kinase activity assays, the expression of PKG was reduced significantly in progesterone-dominated uteri compared with myometrium from postmenopausal women or women in the proliferative phase. These findings support a role for PKG in the control of uterine and vascular smooth muscle contractility during the menstrual cycle.

Intracellular Ca2+ Mobilization and Kinase Activity during Acylated Homoserine Lactone-dependent Quorum Sensing in Serratia liquefaciens

Quorum sensing in Gram-negative bacteria involves acylated homoserine lactones (AHLs) and a transcription factor, activated by the AHLs. In this study, a possible involvement of intracellular Ca(2+) as second messenger and/or protein kinase activity during signal transduction is analyzed. When N-hexanoyl-l-homoserine lactone was added to a suspension of Fura-2-loaded Serratia liquefaciens, there was a decline in [Ca(2+)](i), measured as a decrease in the Fura-2 fluorescence ratio. As controls, the addition of the signal molecule N-3-oxohexanoyl-l-homoserine lactone, which is not produced by S. liquefaciens, did not induce changes in [Ca(2+)](i). Using a protein kinase activity assay on AHL-stimulated cells, an increase in kinase activity after N-butanoyl-l-homoserine lactone stimulation of S. liquefaciens cells was detected, whereas the kinase activity induced by N-3-oxohexanoyl-l-homoserine lactone was not statistically significant. The conclusion from this study is that changes in [Ca(2+)](i) are involved in quorum sensing signal transduction in the Gram-negative bacteria S. liquefaciens. We also conclude that kinase activity is induced in S. liquefaciens upon AHL stimulation. We suggest that the transient intracellular [Ca(2+)] changes and kinase activity, activated by the AHL signal, are critical for the quorum-sensing signal transduction.

HAESA, an Arabidopsis leucine-rich repeat receptor kinase, controls floral organ abscission

Abcission, the natural shedding of leaves, flowers and fruits, is a fundamental component of plant development. Abscission is a highly regulated process that occurs at distinct zones of cells that undergo enlargement and subsequent separation. Although some components of abscission, including accumulation of the hormone ethylene and cell wall-degrading enzymes, have been described, the regulatory pathways remain largely unknown. In this paper we describe a critical component required for floral organ abscission in Arabidopsis thaliana, the receptor-like protein kinase HAESA. Histochemical analysis of transgenic plants harboring a HAESA promoter:: beta-glucuronidase reporter gene and in situ RNA hybridization experiments show HAESA expression in the abscission zones where the sepals, petals, and stamens attach to the receptacle, at the base of pedicels, and at the base of petioles where leaves attach to the stem. Immunodetection, immunoprecipitation, and protein kinase activity assays reveal HAESA is a plasma membrane serine/threonine protein kinase. The reduction of function of HAESA in transgenic plants harboring an antisense construct results in delayed abscission of floral organs, and the severity of the phenotype is directly correlated with the level of HAESA protein. These results demonstrate that HAESA functions in developmentally regulated floral organ abscission.

Heat Shock Activates c-Src Tyrosine Kinases and Phosphatidylinositol 3-Kinase in NIH3T3 Fibroblasts

There is increasing evidence that cellular responses to stress are in part regulated by protein kinases, although specific mechanisms are not well defined. The purpose of these experiments was to investigate potential upstream signaling events activated during heat shock in NIH3T3 fibroblasts. Experiments were designed to ask whether heat shock activates p60 c-Src tyrosine kinase or phosphatidylinositol 3-kinase (PI 3-kinase). Using in vitro protein kinase activity assays, it was demonstrated that heat shock stimulates c-Src and PI 3-kinase activity in a time-dependent manner. Also, there was increased PI 3-kinase activity in anti-phosphotyrosine and anti-c-Src immunoprecipitated immunocomplexes from heated cells. Heat shock activated mitogen-activated protein kinase (MAPK) and p70 S6 kinase (S6K) in these cells. The role of PI 3-kinase in regulating heat shock activation of MAPK and p70 S6K was investigated using wortmannin, a specific pharmacological inhibitor of PI 3-kinase. The results demonstrated that wortmannin inhibited heat shock activation of p70 S6K but only partially inhibited heat activation of MAPK. A dominant negative Raf mutant inhibited activation of MAPK by heat shock but did not inhibit heat shock stimulation of p70 S6K. Genistein, a tyrosine kinase inhibitor, and suramin, a growth factor receptor inhibitor, both inhibited heat shock stimulation of MAPK activity and tyrosine phosphorylation of MAPK. Furthermore, a selective epidermal growth factor receptor (EGFR) inhibitor, tryphostin AG1478, and a dominant negative EGFR mutant also inhibited heat shock activation of MAPK. Heat shock induced EGFR phosphorylation. These results suggest that early upstream signaling events in response to heat stress may involve activation of PI 3-kinase and tyrosine kinases, such as c-Src, and a growth factor receptor, such as EGFR; activation of important downstream pathways, such as MAPK and p70 S6K, occur by divergent signaling mechanisms similar to growth factor stimulation.

Phosphoenolpyruvate Carboxylase Protein Kinase from Soybean Root Nodules: Partial Purification, Characterization, and Up/Down-Regulation by Photosynthate Supply from the Shoots

Phosphoenolpyruvate carboxylase (PEPC) kinase was partially purified about 3000-fold from soybean root nodules by a fast-protein liquid chromatography protocol. This protein-serine kinase has an apparent native molecular mass of about 30,000 as estimated by size-exclusion chromatography. Following electrophoresis of this partially purified PEPC-kinase preparation in a denaturing gel containing dephospho maize leaf PEPC as substrate, thein siturenaturation and assay of protein kinase activity revealed two, PEPC-dependent kinase polypeptides with molecular masses of about 32 and 37 kDa. The ∼32-kDa polypeptide was significantly more active than the ∼37-kDa catalytic subunit. The activity of this partially purified PEPC kinase, and a less purified sample, was Ca2+-insensitive. This protein kinase preparation was able to phosphorylate purified PEPCs from soybean nodules, maize leaves, and a sorghum recombinant C4PEPC. In contrast, this PEPC kinase was unable to phosphorylate a phosphorylation-site mutant form of sorghum C4PEPC (S8Y), two other soybean nodule phosphoproteins [nodulin-26 and nodulin-100 (sucrose synthase)], bovine serum albumin, and histone III-S. Followingin vitrophosphorylation of purified dephospho soybean nodule PEPC from stem-girdled plants by the partially purified nodule PEPC kinase, the former's activity and sensitivity tol-malate inhibition increased and decreased, respectively. Notably, the Ca2+-independent PEPC kinase activity in nodules from illuminated plants was markedly greater than that in nodules harvested from plants subjected to stem girdling or prolonged darkness. Furthermore, the kinase activity in nodules was controlled reversibly by illumination and extended darkness pretreatments of the parent plants, suggesting that photosynthate supply from the shoots is likely responsible for these striking changes in PEPC kinase activity observedin plantain the legume nodule.

Altered expression of protein-tyrosine phosphatase 2C in 293 cells affects protein tyrosine phosphorylation and mitogen-activated protein kinase activation.

PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is recruited to the growth factor receptors upon stimulation of cells. To investigate its role in growth factor signaling, we have overexpressed by approximately 6-fold the native PTP2C and a catalytically inactive mutant of the enzyme in 293 human embryonic kidney cells. The native PTP2C was located entirely in the cytosol, while the inactive mutant was nearly equally distributed in cytsolic and membrane fractions. Expression of the latter caused hyperphosphorylation on tyrosine of a 43-kDa protein, which was coimmunoprecipitated and co-partitioned in the plasma membrane fraction with the inactive PTP2C mutant. This protein may represent a physiological substrate of PTP2C. Overexpression of the native PTP2C enhanced epidermal growth factor (EGF)-stimulated mitogen-activated protein (MAP) kinase activity by 30%, whereas expression of the inactive mutant reduced the stimulated activity by 50%. Similar effects were observed for the activation of MAP kinase as determined by activity assay, gel mobility shift, and tyrosine phosphorylation. The data suggest that the phosphatase activity of PTP2C is partly required for MAP kinase activation by EGF and that PTP2C may function by dephosphorylating the 43-kDa membrane protein.

Insulin-mediated sensitization of adenylyl cyclase activation.

1. Insulin may be an important regulator of vascular function. We have previously studied lymphocyte beta-adrenoceptors as a model for the human vascular beta-adrenoceptor. To examine the effects of insulin on human beta-adrenoceptor responsiveness, adenylyl cyclase activity, cyclic AMP-dependent protein kinase activity and beta-adrenoceptor radioligand binding assays were performed on permeabilized mononuclear leukocytes. 2. With acute exposure to insulin in vitro, followed by washing and permeabilization there was a dose-dependent increase in both lymphocyte NaF-stimulated activity and beta-adrenoceptor-stimulated adenylyl cyclase activity paralleling an increase in beta-adrenoceptor-stimulated protein kinase A activity. Manganese-, forskolin- and forskolin plus guanylimidodiphosphate-stimulated adenylyl cyclase activities were not altered by insulin pretreatment. Additionally, mononuclear leukocyte beta-adrenoceptor density, proportion of externalized receptors and receptor affinity for agonist were not altered. 3. The data indicate that acute exposure to insulin sensitizes G-protein-stimulated adenylyl cyclase activity. These findings suggest a potential role for insulin in the regulation of beta-adrenoceptor responsiveness in man.

Partial Purification and Characterization of Phospho enolpyruvate Carboxylase Protein-Serine Kinase from Illuminated Maize Leaves

Following in situ renaturation and assay of protein kinase activity after denaturing electrophoresis of relatively impure samples of maize phospho enolpyruvate carboxylase (PEPC) kinase, a ∼30-kDa polypeptide was implicated as the best candidate for the PEPC kinase catalytic subunit. This kinase′s apparent native molecular weight was estimated at 28,000 by gel filtration on a calibrated Superose 12 column (HR 10/30), suggesting that the isolated PEPC kinase is monomeric. This protein-serine kinase was partially purified about 4000-fold from illuminated maize leaves by ammonium sulfate precipitation and sequential chromatography on Ultrogel AcA 54, hydroxylapatite, blue dextran-agarose, and an analytical AcA 54 column. Analysis by denaturing electrophoresis revealed that a 30-kDa polypeptide copurified with PEPC kinase activity during the final step. This highly purified kinase had an apparent K m (PEPC subunit) of 2.5 μM and a K m (total ATP) of 40 μM at pH 8.0, its pH optimum. Upon in vitro phosphorylation of dark-form (dephospho) C 4 PEPC at Ser-15 (maize PEPC) or Ser-8 (sorghum), the malate sensitivity of the target enzyme decreased significantly. The maize PEPC kinase activity was markedly inhibited by L-malate, a negative allosteric effector of its protein substrate, in a concentration- and pH-dependent manner. Comparative phosphorylation studies with the catalytic subunit of mammalian cAMP-dependent protein kinase and casein revealed that a significant part of the malate inhibition of PEPC kinase activity in vitro was due to this effector′s interaction with PEPC. The activity of both the highly purified PEPC kinase and a less pure sample prepared rapidly in the presence of various protease inhibitors wasinsensitive to Ca 2+ chelation or addition. It is concluded that the ∼30-kDa maize PEPC kinase is a low abundance, Ca 2+-independent protein-serine kinase that activates its target enzyme by the exclusive phosphorylation of the regulatory serine residue near the N terminus and the resulting decrease in feedback inhibition by L-malate.

Fast‐atom bombardment mass spectrometric analysis of cyclic nucleotide and nucleotide triphosphate analogues used in studies of cyclic nucleotide‐related enzymes

AbstractTwo isomers of adenosine 3′,5′‐cyclic monophosphate, which show agonist and antagonist activity with cyclic AMP‐dependent protein kinase, were found to yield essentially identical positive‐ion fast‐atom bombardment (FAB) mass spectra, but S1 and S2 fragments of differing relative intensities in their collision‐induced dissociations, studied using mass‐analysed ion kinetic energy (CID/MIKE) spectra. Halogen‐substituted cyclic nucleotides, used in differentiating between protein kinase cyclic nucleotide binding sites, produced FAB mass spectra and CID/MIKE spectra with fragmentations generally analogous to those of the parent cyclic nucleotides; the bromo‐derivatives showed a greater propensity for dehalogenation than the chloro‐derivatives. The adenosine triphosphate analogues, adenylyl‐(β,γ‐methylene)‐diphosphate and adenylyl‐imidodiphosphate, alternative substrates for adenylyl cyclase, showed similar fragmentations with the methylene and imido groups blocking cleavage between the β and γ phosphate groups. The fragmentations observed are discussed in the context of the use of these compounds in the assay of protein kinase and adenylyl cyclase activity by quantitative mass spectrometry.

Characterization of cAMP-dependent protein kinase activation by CCK in rat pancreas

This study reports on the use of a new sensitive assay of cAMP-dependent protein kinase activity to examine the effect of cholecystokinin (CCK) on the cAMP second messenger cascade in rat pancreatic acini. Treatment of acini with both low (pM) and high (nM) concentrations of CCK was associated with an increase in cAMP-dependent protein kinase activity. The increases in kinase activity were detected in the absence of phosphodiesterase inhibition, a condition required to detect a measurable increase in cellular cAMP in these cells. Furthermore, the cAMP cascade was dissociated from the secretory effects of CCK, since the CCK analogue, OPE, mediates enzyme secretion but does not increase cellular cAMP levels or kinase activity.

Assay of tyrosine protein kinase activity from HL-60 by high-performance liquid chromatography for specificity studies

Using a partially purified HL-60 tyrosine protein kinase, we designed a new HPLC method for the measurement of tyrosylphosphorylation of angiotensin II. The present method uses reversed-phase chromatography and elution involving an acetonitrile gradient containing the counterion tetrabutylammonium phosphate. The peptide substrate, [γ- 32P]ATP, the cosubstrate, and 32P-labeled phosphorylated peptides were quantified online by measuring the Cerenkov effect. Injections, separation, and analysis were performed automatically. Furthermore, the method permits a direct visualization of peptide substrate phosphorylation and has a potentially universal application; i.e., it is usable with any kind of peptide in a given range of hydrophobicity. This assay was designed for specificity studies, which are of major importance at the molecular level, in order to understand active site topology and the biophysical requirements of tyrosine protein kinases. As examples, data on chromatography separations of angiotensin II analogs (five to ten amino acids in length) are presented, as well as for other peptide substrates such as RR-src, the pp60src autophosphorylation site-derived peptide, and minigastrin. We adapted our experimental conditions to accommodate crude extracts from HL-60 cells. Preliminary experiments clearly indicated that other biological sources can be used. Despite the existence of numerous methods published in the literature for the measurement of kinase activities, the method presented herein is the only one to the author's knowledge that can be used in and has been assessed for specificity studies. Peptides do not require particular features such as charged residues (i.e., arginine) to be analyzed.

Insulin, oxytocin, and vasopressin stimulate protein kinase C activity in adipocyte plasma membranes.

Incubation of isolated rat adipocytes with insulin, vasopressin, or oxytocin increased plasma membrane-bound protein kinase C (PKC) activity by 100-400%. PKC activity was assayed by a procedure that is virtually background-free, thus permitting assay of protein kinase activity in highly diluted samples of solubilized membranes. Hormone-dependent increases in PKC activity were limited to plasma membranes. Stimulation of the kinase was half-maximal with 70 pM insulin, and the hormone effect was rapid. Oxytocin and vasopressin produced effects on PKC similar to insulin, but the magnitude of the vasopressin stimulation exhibited seasonal variations. Treatment of cells with phorbol 12-myristate 13-acetate (PMA) resulted in a loss of PKC activity from the cytosol and a gain in plasma membrane activity, indicative of translocation of the enzyme. With activity measurements it was not possible to determine if insulin stimulated a translocation of the kinase. However, Western blot analysis of plasma membranes with polyclonal antibodies directed against PKC suggest that at least some of the insulin-stimulated PKC activity resulted from enzyme translocation.

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.

Enzyme activity dot blots: A rapid and convenient assay for acetyltransferase or protein kinase activity immobilized on nitrocellulose

Methods are described for assaying ( Tetrahymena) histone acetyltransferase activity and ( Drosophila) casein kinase II activity by spotting extracts on nitrocellulose filters. The methods are quantitative over a wide range of enzyme concentrations and are almost as sensitive as liquid assays. Examples are presented for illustrating the use of these methods for enzyme purification, concentration, and desalting, as well as for electrophoretic blotting from agarose gels. A simple method for autoradiographic enhancement of nitrocellulose filters is also described.

Monoclonal antibodies to an interferon-induced Mr 68,000 protein and their use for the detection of double-stranded RNA-dependent protein kinase in human cells.

Extracts from interferon-treated human cells show an enhanced level of a double-stranded RNA-dependent protein kinase activity that is manifested by the phosphorylation of an endogenous Mr 69,000-72,000 protein in its phosphate-saturated state. By using a highly purified protein kinase fraction from interferon-treated human Daudi cells, we can now describe the preparation of murine monoclonal antibodies directed against this phosphoprotein, the Mr of which in its native state is found to be 68,000. These monoclonal antibodies (class IgG1) can identify the electrophoresed protein (p68) in polyacrylamide gels by the electrophoretic transfer blotting technique. Immunoprecipitates formed after incubation of extracts from interferon-treated human cells with the monoclonal antibodies can be conveniently recovered by protein A-Sepharose. Such immune complex preparations have associated protein kinase activity--i.e., addition of [gamma-32P]ATP results in the phosphorylation of p68 and added substrates, calf thymus histone, and eukaryotic initiation factor 2. Immune complex preparations from [35S]methionine-labeled extracts show the specific immunoprecipitation of p68. In addition, several other [35S]methionine-labeled proteins are bound unspecifically in these immune complexes prepared under similar experimental conditions as for the assay of protein kinase activity. These unspecifically bound proteins can be washed out by using a buffer containing detergents or high concentrations of KCl and magnesium acetate. Immune complex preparations washed similarly with these buffers still retain p68 but lose their capacity to phosphorylate p68 or exogenous substrates. These results indicate that p68 by itself has no protein kinase activity. The induction of [35S]methionine-labeled p68 in Daudi cells occurs with as little as 1 unit of human alpha interferon, with maximal synthesis between 6 to 9 hr after the addition of interferon. Actinomycin D blocks this induction.

LH-dependent steroid production and protein phosphorylation in culture of rat tumour leydig cells

The possible cause of the decreased responsiveness of cultured Leydig cells to stimulation with LH was investigated with rat tumour Leydig cells cultured at 32°C for 2 days. Pregnenolone production and phosphorylation of proteins were studied in combination with the activity of cyclic AMP-dependent protein kinase. Pregnenolone production in cultured tumour Leydig cells decreased from 45 ± 16 on day 0 to 14 ± 7 on day 2 (ng/60 min/mg protein) under basal conditions and from 226 ± 108 on day 0 to 30 ± 24 on day 2 (ng/60 min/ng protein) under LH-stimulated conditions (means ±SD, n = 4).This decrease may be accounted for by decreased capacity of cholesterol side-chain cleavage which decreased (in the presence of 25-hydroxycholesterol) from 1.68±0.26 on day 0 to 0.74 ± 0.48 on day 2 (μg/60 min/mg protein; means ±SD, n = 4) and decreased activity of cyclic AMP-dependent protein kinase (as apparent from a direct assay of protein kinase activity and the extent of phosphorylation of LH-dependent phosphoproteins).

Thyroid hormone increases type I adenosine 3', 5'-monophosphate-dependent protein kinase and casein kinase activities in rat liver cytosol: analysis of protein kinases by polyacrylamide disc gel electrophoresis.

We investigated the action of thyroid hormone on each protein kinase in rat liver cytosol. Kinases were analyzed by polyacrylamide disc gel electrophoresis and isoelectric focusing in polyacrylamide gel. Polyacrylamide disc gel electrophoresis separated cAMP-dependent protein kinase type I (Rf = 0.35), type II (Rf = 0.44), their catalytic subunit (Rf = 0.26), and cAMP-independent protein kinase (Rf = 0.50). Casein kinase was detected at Rf = 0.37. In addition to the catalytic subunit with Rf = 0.26, another catalytic subunit was found at Rf = 0.44 when the cytosol was preincubated with cAMP. The administration of T3 (20 micrograms/100 g BW for 3 days) to hypothyroid rats increased enzyme activities of type I holoenzyme and casein kinase by 48%. Free catalytic subunit, separated from holoenzyme, had the same level of enzyme activity in both groups, suggesting greater endogenous dissociation of type I holoenzyme in hypothyroid rats. When heat-inactivated rat liver cytosol was used as substrate in the assay of protein kinase activity, the peak enzyme active in phosphorylating the cytosol corresponded to the casein kinase peak. Our data indicate that casein kinase is the main enzyme that mediates phosphorylation of endogenous proteins in rat liver cytosol, and that T3 treatment increases the activity of casein kinase and of type I cAMP-dependent protein kinase.