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PubMed1994

Expression and characterization of a rac family protein kinase of Entamoeba histolytica.

We previously reported the isolation from Entamoeba histolytica of a novel rac family protein kinase gene, termed Ehrac1, for "related to cAMP-dependent protein kinases and protein kinase Cs". To study the function and properties of this kinase gene further, we fused the full-length coding region and the truncated catalytic domain of the Ehrac1 gene in frame with the gene encoding glutathione S-transferase in the pGEX-KG vector and expressed the fusion in Escherichia coli. The thrombin-cleaved and uncleaved fusion proteins, GST-Ehrac1 and GST-Ehrac1-c (catalytic domain), were purified and found to exhibit similar protein kinase activities. The Ehrac1 fusion kinase was found to phosphorylate serine/threonine residues exclusively in vitro. The preferred substrate for the enzyme was histone H1 with a Km of approx. 14 microM. Histone H3 and kemptide were phosphorylated at about half the rate of histone H1. Protamine, enolase, bovine serum albumin, and poly (Glu:Tyr) were not substrates for the enzyme. The protein kinase activity was higher in the presence of Mn2+ than Mg2+. Neither cAMP, Ca2+, nor Ca2+/calmodulin stimulated enzyme activity. The pH optimum of the enzyme was 7.5. The Ehrac1 kinase can utilize GTP as well as ATP as a phosphate donor with an apparent Km of 80 microM. Enzyme activity was inhibited 30-40% by a crude cAMP-dependent protein kinase inhibitor from rabbit and by thiol reagents. The expression and purification of enzymatically active Ehrac1 protein kinase should allow further analysis of the regulation and signal transduction pathways of E. histolytica.

Animals↗
PubMed1987

Purification of the catalytic subunit of protein phosphatase-1 from Drosophila melanogaster.

The catalytic subunit of phosphatase-1 has been purified from Drosophila melanogaster by precipitation with (NH4)2SO4 and ethanol, by affinity chromatography on heparin-Sepharose and by fast protein liquid chromatography on Mono Q beads. The preparation is homogeneous as tested by SDS gel electrophoresis and has a molecular mass of 33,000. The phosphatase specifically dephosphorylates the beta subunit of phosphorylase kinase. Its phosphorylase phosphatase activity is inhibited by inhibitor-1, inhibitor-2, protamine and histone H2B while is stimulated by histone H1.

Animals↗
PubMed1982

Isolation and purification of a new 105 kDa protein kinase from rat liver nuclei.

A protein kinase was purified from rat liver nuclei by affinity chromatography on poly(L-lysine)-agarose and protein kinase inhibitor-Sepharose 4B columns. The relative molecular weight of this protein kinase is 105000 (determined by 10% SDS-polyacrylamide slab gel electrophoresis, gel filtration on Sephadex G-200 and sedimentation velocity ultracentrifugation). Its pH optimum is between 8.0 and 9.0. It is active over a wide range of mg2+ concentrations, and its activity is stimulated by several small acidic proteins (calmodulin, lactalbumin, parvalbumin, protein kinase inhibitor and troponin C). The enzyme phosphorylates a variety of substrates including casein, histones, protamine and the synthetic basic polypeptides, poly(L-arginine) and poly(L-lysine).

Adenosine Triphosphate↗
PubMed2000

Purification and characterization of a feline hepatic insulin receptor.

OBJECTIVE: To elucidate the functional characteristics of a highly purified soluble liver insulin receptor in cats. SAMPLE POPULATION: Frozen livers from domestic cats were obtained commercially. PROCEDURES: The feline hepatic insulin receptor was purified from Triton X-100 solubilized plasma membranes by the use of several chromatography matrices, including affinity chromatography on an insulin-Sepharose matrix. RESULTS: The receptor, although not homogeneous, was purified 3,000-fold. Two silver-stained protein bands were identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weight of 134,000 and 97,000, which are similar to insulin receptors isolated from other animals. This isolated receptor had steady-state insulin binding by 40 minutes at 24 C. Optimal insulin binding occurred at pH 7.8 and with 150 mM NaCl. Under these conditions, a curvilinear Scatchard plot was obtained with the isolated receptor. Using a 2 binding-site model, the feline insulin receptor had a high-affinity low-capacity site with a dissociation constant (KD; nM) of 3 and a low-affinity high-capacity site with a K(D) of 1,180. The receptor also had tyrosine kinase activity toward an exogenous substrate that was stimulated by insulin and protamine. CONCLUSIONS AND CLINICAL RELEVANCE: Many of the reported characteristics of the liver insulin receptor in cats are similar to those for the receptor isolated from other animals and tissues, although some differences exist. These similarities suggest that characterization of the feline insulin receptor is important to understanding insulin resistance in cats with diabetes as well as in humans with diabetes.

Animals↗
PubMed1986

Probable occurrence of brain basic protein factor I, an activator of phosphoprotein phosphatases.

Basic protein factor I (BPFI was purified to homogeneity from bovine brain by boiling and trichloroacetic acid-precipitation of tissue homogenate, followed by DEAE-cellulose, Sephadex G-150, Affi-Gel-phenothiazine, and Bio-Gel P-6DG chromatographic procedures. The preparation appeared as a single protein band in the SDS-polyacrylamide gel electrophoresis with a minimal Mr of 13,200. The factor was a basic protein as indicated by an estimated isoelectric point of pH 8.3 and a high content of amino acids including arginine, histidine, lysine and others. In the absence of Mn2+, the factor stimulated the phosphoprotein phosphatases (PPase) from rabbit brains. Unlike histones or protamine, the factor was a poor substrate for megamodulin-dependent protein kinase. In addition, the factor did not interact significantly with E. coli megamodulin.

Amino Acids↗
PubMed1977

Purification and properties of a nuclear protein kinase from rat mammary gland.

A nuclear protein kinase that shows a high degree of substrate specificity for the phosphorylation of the acidic proteins casein, phosvitin and non-histone chromatin proteins, rather than the basic proteins histones and protamine, was partially purified from lactatingrat mammary gland. The enzyme is associated with the acidic protein fraction of chromatin. Nuclear kinase requires Co(2+) for activity, and other bivalent cations such as Mg(2+) and Mn(2+) can substitute partially for Co(2+). The kinase is further activates (2-3-fold) by various salts, their concentration for maximum stimulation being: NaCl, 150mm; KCl, 200mm; sodium acetate, 300mm. The sedimentation coefficient of the nuclear kinase is 8.9S and its mol.wt. is approx. 300000 by gel-exclusion chromatography. The enzyme is not activated by cyclic AMP or cyclic GMP and is inhibited neither by the regulatory subunit of mammary cyclic AMP-dependent protein kinase nor by the heat-stable protein kinase inhibitor from ox heart. Analysis of (32)P-labelled protein products reveals that the kinase transfers the terminal phosphate of ATP to serine and threonine residues of proteins. The enzyme, however, has specificity for the phosphorylation of threonine in casein and serine in phosvitin. Molecular size and enzymic characteristics of the nuclear protein kinase are clearly different from those of the cytosol enzyme previously characterized.

Animals↗
PubMed1981

Purification and characterization of two cyclic AMP-independent casein/glycogen synthase kinases from rat liver cytosol.

Two cyclic AMP-independent protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) (casein kinase 1 and 2) have been purified from rat liver cytosol by a method involving chromatography on phosphocellulose and casein-Sepharose 4B. Both kinases were essentially free of endogeneous protein substrates and capable of phosphorylating casein, phosvitin and I-form glycogen synthase, but were inactive on histone IIA, protamine and phosphorylase b. They were neither stimulated by cyclic AMP, Ca2+ and calmodulin, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. The casein and glycogen synthase kinase activities of each enzyme decreased at the same rate when incubated at 50 degrees C. Casein kinase 1 and casein kinase 2 showed differences in molecular weight, sensitivity to KCl, Km for casein and phosvitin and Ka for Mg2+, whereas their Km values for ATP and I-form glycogen synthase were similar. The phosphorylation of glycogen synthase by these kinases correlated with a decrease in the +/- glucose 6-phosphate activity ratio (independence ratio). However, casein kinase 1 catalyzed the incorporation of about 3.6 mol of 32P/85000 dalton subunit, decreasing the independence ratio from 83 to about 15, whereas the phosphorylation achieved by casein kinase 2 was only about 1.9 mol of 32P/850000 dalton subunit, decreasing the independence ratio to about 23. The independence ratio decrease was prevented by the presence of casein but was unaffected by phosphorylase b. These data indicate that casein/glycogen synthase kinases 1 and 2 are different from cyclic AMP-dependent protein kinase and phosphorylase kinase.

Animals↗
PubMed1993

The role of polybasic compounds in determining the tyrosyl phosphorylation of calmodulin by the human insulin receptor.

A highly purified human insulin receptor preparation was shown to effect receptor autophosphorylation and the phosphorylation of poly(GluTyr) but not that of calmodulin. Addition of poly-L-lysine allowed for the stoichiometric tyrosyl phosphorylation of calmodulin in a dose-dependent fashion (EC50 approximately 83 nM) with the single target residue identified at tyr99. Higher concentrations of poly-L-lysine elicited the dose-dependent inhibition of calmodulin phosphorylation (IC50 approximately 0.7 microM) by a process which did not apparently involve either stimulation of calmodulin phosphatase activity or diminished receptor kinase activity. Polybasic substances such as poly-L-arginine, histone H1 and protamine sulphate all promoted calmodulin phosphorylation by the insulin receptor in a similar biphasic dose-dependent fashion. Poly-lysine's actions proved to lack stereo-specificity in that both the D- and L-forms were equally as effective. Reduction in the chain length of poly-L-lysine species attenuated their ability to promote calmodulin phosphorylation with L-lysine proving to be ineffective. Optimal promotion of calmodulin phosphorylation was achieved at an apparently constant ratio of calmodulin to poly-L-lysine of approximately 1:4 over a 100-fold range of calmodulin concentrations. Poly-L-lysine promoted the precipitation and subsequent resolubilization of calmodulin in a fashion whose biphasic dose-dependence paralleled that seen for its action in promoting calmodulin's phosphorylation. NaCl attenuated, in apparently identical dose-dependent fashions, poly-L-lysine's ability to both elicit the precipitation of calmodulin and to promote its phosphorylation. The presence of added Ca2+ led to a small potentiation of poly-L-lysine-dependent calmodulin phosphorylation at low concentrations, with inhibition occurring at higher concentrations where Ca2+ was shown to block calmodulin precipitation by poly-L-lysine. It is suggested that calmodulin can be phosphorylated by the insulin receptor only when it is cross-linked in a multivalent fashion to a suitable polybasic substance so that it forms large multimeric aggregates. Such a requirement for the formation of an aggregate between calmodulin and a suitable polybasic species may place specific constraints on the ability of calmodulin to serve as a substrate for receptor tyrosyl kinases within the cell.

Calmodulin↗
PubMed1988

Properties of the 12-O-tetradecanoylphorbol-13-acetate-stimulated S6 kinase from rat astroglial cells.

The S6 kinase activity of astroglial cells in primary culture stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) has been studied. This activity was eluted as a single peak at 0.15 M NaCl from a DEAE-Sephacel column. The chromatography of this peak on phosphocellulose revealed an activity eluted at 0.15 M NaCl. This partially purified enzyme had a sedimentation coefficient of 3.7S; Km values were 2 X 10(-5) M for ATP and 10(-6) M for 40S ribosomal subunits. The optimal Mg2+ concentration requirement was 2-3 mM. Mn2+ and Co2+ could substitute for Mg2+ (optimum concentrations 1.5 and 0.8 mM, respectively), but these cations were strong inhibitors in the presence of Mg2+. The enzyme was inhibited by N-ethylmaleimide, indicating that it contained thiol groups. This S6 kinase used ATP, but not GTP, as a phosphate donor, and exhibited great specificity for S6 as phosphate acceptor. Whole histones and protamine were slightly phosphorylated whereas phosvitin, histone H1, and surprisingly the peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala were not phosphorylated. The TPA-stimulated S6 kinase resembles the insulin-, fibroblast growth factor- and cyclic AMP-stimulated enzymes, suggesting that several pathways might activate the same entity.

Adenosine Triphosphate↗
PubMed1981

Affinity labeling of the ATP binding site of bovine lung cyclic GMP-dependent protein kinase with 5'-p-fluorosulfonylbenzoyladenosine.

Bovine lung cyclic GMP-dependent protein kinase was covalently labeled with the ATP analog, 5-p-fluorosulfonylbenzoyladenosine. The inactivation reaction was pseudo-first order. The rate of kinase sulfonation exhibited saturation kinetics indicative of a rapid reversible binding of the reagent prior to enzyme modification. The enzyme could be protected by MgATP, MgADP, and Mg-adenylylimidodiphosphate but not by a synthetic peptide substrate. Cyclic GMP when bound to the kinase did not influence the rate of labeling. The reagent demonstrated competitive inhibition with respect to MgATP; the Ki was found to be 0.82 mM. Magnesium and cobalt ions when included in the reaction mixture accelerated the inactivation rate up to severalfold. Addition of basic polypeptides such as mixed histone, protamine sulfate, or poly-L-lysine HBr also markedly accelerated the sulfonation rate. Inactivation of the kinase with 5-'fluorosulfonyl[3H]benzoyladenosine resulted in a linear relationship between the residual phosphotransferase activity and the incorporation of up to 0.9 mol of reagent/mol of monomer.

Adenosine↗
PubMed1990

High mobility group proteins 1 and 2 function as general class II transcription factors.

High mobility group (HMG) proteins 1 and 2 are thought to be associated with chromatin enriched in active gene sequences, to stimulate endogenous transcription of class II and III genes using HMG-depleted nuclei, and to bind specific DNA sequences upstream of the coding regions of trout HMG-T and human beta-globin genes. In testing the possibility that these proteins may act as general transcription factors, the run-off transcription of trout protamine, human beta-globin, adenovirus 2 major late promoter, and herpes simplex virus (HSV) thymidine kinase genes was found to be inhibited by affinity-purified HMG-1 and -2 antibodies. The inhibition was partially relieved by exogenously added HMG-1 or -2. A complementation assay showed that the 0.15 M KCl flowthrough of HeLa nuclear extract fractionated by anion-exchange chromatography (DE-52) could be replaced by purified HMG-1 and/or -2 to complement transcription of the trout protamine gene by the 0.5 M KCl eluate fraction. Inhibition studies with heparin showed that HMG-1 and -2 were required for initiation of transcription. These results indicate an absolute requirement of HMG-1 and -2 for class II gene transcription. Western blotting and transcription reconstituted with purified factors show a copurification of HMG-1 and -2 with factor II B, described earlier by Reinberg and Roeder [(1987) J. Biol. Chem. 262, 3310-3321].

Animals↗
PubMed1984

Characterization of bovine brain calmodulin-dependent protein phosphatase.

Calmodulin-dependent protein phosphatase of bovine brain exhibited a pH optimum of 7 and appeared to require sulfhydryl groups for activity. Phosphatase activity was inhibited by both NaF and ZnCl2, but was stimulated approximately 2-fold by MnCl2. The enzyme exhibited broad substrate specificity, dephosphorylating casein, troponin I, protamine, histone, and phosvitin, and was not phosphorylated by cAMP-dependent protein kinase. With 32P-labeled casein as a substrate, phosphatase was activated 15-fold by calmodulin; the dissociation constant of phosphatase for calmodulin was 30 nM. Activation of the enzyme by calmodulin as a function of Ca2+ was highly cooperative; the Hill coefficient was 4.9. At a saturating concentration of calmodulin, half-maximal activation of phosphatase was obtained at 0.3 microM Ca2+. Calmodulin increased the Vmax from 1.7 to 41 nmol mg protein-1 min-1 with no significant change in its Km. Formation of a Ca2+-dependent complex between calmodulin and the phosphatase was demonstrated by a calmodulin-Sepharose affinity column, gel-filtration chromatography, and sedimentation on a sucrose density gradient. The rate of formation and dissociation of the calmodulin X phosphatase complex was rapid and readily reversible in response to changes in Ca2+ concentration. The calmodulin X phosphatase complex consists of 1 mol of calmodulin and 1 mol of phosphatase.

Animals↗
PubMed1979

Purification and properties of troponin T kinase from rabbit skeletal muscle.

A protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) which catalyzes the phosphorylation of troponin T, phosvitin and casein has been purified over 2000 fold from rabbit skeletal muscle. The partial purification of this new enzyme, designated troponin T kinase, involves precipitation of contaminating proteins at pH 6.1, fractionation of the supernatant with (NH4)2SO4 and successive column chromatographies on DEAE-cellulose, hydroxyapatite and Sepharose 6B. The chromatographic patterns on DEAE-cellulose and hydroxyapatite columns show two peaks of troponin T kinase activity. Gel filtration experiments indicate the existence of multiple, possibly aggregated, forms of the enzyme. The purified enzyme does not catalyze the phosphorylation of phosphorylase b, troponin I, troponin C, tropomyosin, protamine, or myosin light chain 2 nor does it catalyze the interconversion of glycogen synthase I into the D form. Troponin T kinase is not affected by the addition of cyclic nucleotides or AMP to the reaction mixture. Divalent cations (other than Mg2+, required for the reaction) do not stimulate the enzyme, and several are inhibitory. Other characteristics of the reaction catalyzed by troponin T kinase, such as Km values for ATP and substrate proteins, pH optima, effect of the concentration of Mg2+, substitution of ATP for GTP have also been studied.

Animals↗
PubMed1986

Modulation of the catalytic subunit of cyclic AMP-dependent protein kinase by calmodulin, S-100 protein, parvalbumin and troponin.

The activity of the catalytic subunit of cyclic AMP-dependent protein kinase (A-PK), utilizing type II-S, III-S histone or protamine (free base) as a substrate, was augmented in the presence of regulatory protein including calmodulin, S-100 protein, parvalbumin, or troponin. However, inhibition by calmodulin or S-100 but stimulation by parvalbumin or troponin on this A-PK subunit was observed when II-S histone was replaced by V-S, VI-S, VII-S, or VIII-S histone. In addition, the stimulatory effect of calmodulin or S-100 on this A-PK subunit utilizing III-S histone was greatly diminished when half the dose of III-S was replaced by other histones in a bi-mixed form.

Animals↗
PubMed1995

Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase.

We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide reductase following expression in Escherichia coli. Autophosphorylation activity was observed when kinase assays were performed with immunoprecipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of histones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of these two proteins are similar. We further characterized the protein kinase activity of HSV-1 R1 by producing insertion and deletion mutants constructed with a plasmid expressing R1 amino acids 1 to 449. C-terminal deletion analysis identified the catalytic core of the enzyme as comprising residues 1 to 292, and this polypeptide will be useful for structural determinations by X-ray crystallography. Insertion of a 4-amino-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 completely inactivated activity, and an insertion at residue 136 reduced activity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoyladenosine, which covalently modifies conventional eukaryotic kinases at an essential lysine residue within the active site, did label HSV R1, but this labelling occurred outside the N-terminal domain. These data indicate that the HSV R1 kinase is novel and distinct from other eukaryotic protein kinases.

Adenosine↗
PubMed1989

Tyrosine-specific phosphorylation of calmodulin by the insulin receptor kinase purified from human placenta.

It has previously been demonstrated that calmodulin can be phosphorylated in vitro and in vivo by both tyrosine-specific and serine/threonine protein kinase. We demonstrate here that the insulin receptor tyrosine kinase purified from human placenta phosphorylates calmodulin. The highly purified receptors (prepared by insulin-Sepharose chromatography) were 5-10 times more effective in catalysing the phosphorylation of calmodulin than an equal number of partially purified receptors (prepared by wheat-germ agglutinin-Sepharose chromatography). Phosphorylation occurred exclusively on tyrosine residues, up to a maximum of 1 mol [0.90 +/- 0.14 (n = 5)] of phosphate incorporated/mol of calmodulin. Phosphorylation of calmodulin was dependent on the presence of certain basic proteins and divalent cations. Some of these basic proteins, i.e. polylysine, polyarginine, polyornithine, protamine sulphate and histones H1 and H2B, were also able to stimulate the phosphorylation of calmodulin via an insulin-independent activation of the receptor tyrosine kinase. Addition of insulin further increased incorporation of 32P into calmodulin. The magnitude of the effect of insulin was dependent on the concentration and type of basic protein used, ranging from 0.5- to 9.0-fold stimulation. Maximal phosphorylation of calmodulin was obtained at an insulin concentration of 10(-10) M, with half-maximal effect at 10(-11) M. Either Mg2+ or Mn2+ was necessary to obtain phosphorylation, but Mg2+ was far more effective than Mn2+. In contrast, maximal phosphorylation of calmodulin was observed in the absence of Ca2+. Inhibition of phosphorylation was observed as free Ca2+ concentration exceeded 0.1 microM, with almost complete inhibition at 30 microM free Ca2+. The Km for calmodulin was approx. 0.1 microM. To gain further insight into the effects of basic proteins in this system, we examined the binding of calmodulin to the insulin receptor and the polylysine. Calmodulin binds to the insulin receptor in a Ca2+-dependent manner, whereas it binds to polylysine seemingly by electrostatic interactions. These studies identify calmodulin as a substrate for the highly purified insulin receptor tyrosine kinase of human placenta. They also demonstrate that the basic proteins, which are required for insulin to stimulate the phosphorylation of calmodulin, do so by a direct interaction with calmodulin.

Calmodulin↗
PubMed1975

Changes in cyclic AMP-dependent protein dinase activity in Tetrahymena pyriformis during the growth cycle.

An adenosine 3':5'-monophosphate-dependent protein kinase II (ATP:protein phosphotransferase, EC 2.7.1.37) was partially purified from the cytosol fraction of an exponentially growing culture of Tetrahymena pyriformis. Protein kinase II represented approximately 90% of the cytosolic protein kinase activity. The enzyme had a high degree of substrate specificity for calf thymus and Tetrahymena histones as compared to casein, protamine and phosvitin. The enzyme incorporated the terminal phosphate of ATP into serine and threonine residues of all the histone fractions. The apparent Km of the enzyme for adenosine 3':5'-monophosphate (cyclic AMP) was 1-10-minus 8 M. Protein kinase II was also activated by other cyclic nucleotides with apparent Km values in the range 2.k-10-minus 6 M. Ther specific activity of the cyclic AMP-dependent protein kinase of Tetrahymena decreases markedly from initial high values during the transition from the lag to early log phase of growth. This is followed by a shrp increase in the activity of the enzyme as the log phase of growth progresses. The specific activity of the enzyme increases rapidly during the heat-induced synchronization of Tetrahymena cells. The capacity for rapid phosphorylation of multiple classed of organelle-specific phosphoproteins and the level of cyclic AMP were maximal in Tetrahymena during the earliest phase of growth. These results demonstrate that the cell cycle of Tetrahymena may be coordinated by marked variations in the level of cyclic AMP which in turn regulate the cyclic AMP-dependent protein kinase.

Animals↗