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PubMedDate not supplied

Protein kinases in HeLa cells and in human cervix carcinoma.

Extracts of HeLa cell fractions were analyzed by DEAE- and phospho-cellulose chromatography for their range of cyclic AMP-dependent and -independent protein kinase activities phosphorylating histone and/or phosvitin; extractions were by phosphate buffered saline (soluble protein kinases) and the non-ionic detergent NP-40 (membrane-bound protein kinases). The soluble fraction contained (i) cyclic AMP-dependent histone kinases type I and II as evidenced by their behaviour on DEAE-cellulose and inhibition by specific heat- and acid-stable protein kinase inhibitor (PKI) in a dose-related manner; both types I and II as well as their purified catalytic subunit also phosphorylated protamine and - with very low efficiency - casein but not phosvitin; (ii) a histone kinase (H), insensitive to cyclic AMP and PKI, also accepting protamine as substrate but not either casein or phosvitin; (iii) a phosvitin kinase (P), insensitive to cyclic AMP and PKI, which also phosphorylates casein but not histone or protamine. These four enzyme species were also found in NP-40 extracts of 27000 x g residues which, however, contained further histone and phosvitin kinase activities as yet unspecified. NP-40 extracts of the microsomal fraction possessed, besides unspecified histone and phosvitin kinase activity, only the phosvitin kinase P and appeared to be devoid of histone kinases I, II, and H. The occurrence and ratios of the protein kinases classified suggest an ordered distribution over the diverse subcellular fractions of HeLa cells. The overall pattern of soluble and membrane-bound histone and phosvitin kinases in extracts of cervix carcinoma tissue, the in vivo correlate of HeLa cells, closely resembled that of similar extracts of HeLa cells. HeLa cells hence appear, despite their long in vitro history, to express protein kinase activities similar to those of their in vivo ancestors, recommending them as a subject for the study of (certain) human protein kinase systems.

Chromatography, DEAE-Cellulose↗
PubMed1981

Studies on the heterogeneity of the 5' ends of the protamine mRNAs from rainbow trout testis.

The structures of the 5' termini of the protamine mRNAs (PmRNAs) have been investigated by inhibiting their translation in wheat-germ extracts in the presence of 7-methyl guanosine 5'-phosphate (m7-GMP), an analogue of 'cap' structure in mRNAs. Second, the cap structures on PmRNAs were examined by labelling the RNA at the 5' end with T4 polynucleotide kinase and [gamma-32P]ATP before and after removal of these structures with tobacco acid pyrophosphatase and alkaline phosphatase. The results indicate that cap structures of the PmRNAs are heterogeneous. It appears that the mRNAs coding for protamine components CI and CIII have at least a cap 1 structure while the mRNAs coding for CII do not appear to be capped or methylated.

Animals↗
PubMed1977

[Some properties of cytoplasmic thymidine kinase and nucleoside phosphotransferase from rat liver].

The activities of two deoxythymidine-phosphorylating enzymes--thymidine kinase and nucleoside phosphotransferase--were found in the cytoplasmic fraction of normal and regenerating rat liver. The specific activity of nucleoside phosphotransferase appeared to be by 50% higher than that of thymidine kinase. Nucleoside phosphotransferase has a broad specificity for the phosphate donor. This enzyme is more stable to heating and prolonged dialysis as compared to thymidine kinase. The enzymes respond differently to the addition of d-TTP, d-CTP and sturins A and B: thymidine kinase is strongly inhibited by these agents whereas nucleoside phosphotransferase is insensitive to d-TTP and d-CTP and is only slightly inhibited by sturins. On the other hand the activity of nucleoside phosphotransferase is considerably decreased after addition of ATP. Changes in the activities of both enzymes during 50 hrs following partial hepatectomy were studied. Two activity maxima were observed at 20-22 and 40-46 hrs of regeneration. Using polyacrylamide gel electrophoresis, three isoforms of both enzymes were found. The ratio between the isoenzyme content of the two enzymes from the cytoplasmic fraction of regenerating liver varied as compared to normal.

Animals↗
PubMed1983

Regulation of yeast trehalase by a monocyclic, cyclic AMP-dependent phosphorylation-dephosphorylation cascade system.

Mutation at the GLC1 locus in Saccharomyces cerevisiae resulted in simultaneous deficiencies in glycogen and trehalose accumulation. Extracts of yeast cells containing the glc1 mutation exhibited an abnormally high trehalase activity. This elevated activity was associated with a defective cyclic AMP (cAMP)-dependent monocyclic cascade which, in normal cells, regulates trehalase activity by means of protein phosphorylation and dephosphorylation. Trehalase in extracts of normal cells was largely in a cryptic form which could be activated in vitro by ATP . Mg in the presence of cAMP. Normal extracts also exhibited a correlated cAMP-dependent protein kinase which catalyzed incorporation of label from [gamma-32P]ATP into protamine. In contrast, cAMP had little or no additional activating effect on trehalase or on protamine phosphorylation in extracts of glc1 cells. Similar, unregulated activation of cryptic trehalase was also found in glycogen-deficient strains bearing a second, independently isolated mutant allele, glc1-2. Since trehalase activity was not directly affected by cAMP, the results indicate that the glc1 mutation results in an abnormally active protein kinase which has lost its normal dependence on cAMP. Trehalase in extracts of either normal or mutant cells underwent conversion to a cryptic form in an Mg2+-dependent, fluoride-sensitive reaction. Rates of this reversible reduction of activity were similar in extracts of mutant and normal cells. This same, unregulated protein kinase would act on glycogen synthase, maintaining it in the phosphorylated low-activity D-form. The glc1 mutants provide a novel model system for investigating the in vivo metabolic functions of a specific, cAMP-dependent protein kinase.

Adenosine Triphosphate↗
PubMed1986

Triphenylethylenes: a new class of protein kinase C inhibitors.

The Ca2+- and phospholipid-dependent phosphotransferase activity of protein kinase C was inhibited by the triphenylethylene compounds clomiphene [drug concentration causing 50% inhibition (IC50) = 25 microM], 4-hydroxytamoxifen (IC50 = 25 microM), and N-desmethyltamoxifen (IC50 = 8 microM). The Ca2+- and phospholipid-independent phosphorylation of protamine sulfate, which is catalyzed by protein kinase C, was not inhibited by the triphenylethylenes, suggesting that they do not interact directly with the active site of protein kinase C. The inhibitory potency of each triphenylethylene was reduced when the phospholipid concentration was increased, providing evidence that these drugs inhibited protein kinase C by interacting with phospholipids. The potencies of the effects of the triphenylethylenes on protein kinase C in the lipid environment of intact cells were evaluated by determining their efficacies in the inhibition of [3H]phorbol 12,13-dibutyrate (PDBu) binding to mouse embryo C3H/10T1/2 cells. Micromolar concentrations of each drug inhibited [3H]PDBu binding in these cells. N-Desmethyltamoxifen, 4-hydroxytamoxifen, and tamoxifen inhibited protein kinase C with the same order of potency as that which has been reported for their inhibition of MCF-7 cell growth by Reddel et al. (1983). N-Desmethyltamoxifen and 4-hydroxytamoxifen were also more potent than tamoxifen in the inhibition of the growth of mouse embryo fibroblast C3H/10T1/2 cells. These correlations suggest that the mechanism of growth inhibition by tamoxifen and its metabolites includes interactions with protein kinase C.

Animals↗
PubMed1975

Characterization of a protein kinase and two phosphate acceptor proteins from vaccinia virions.

The phosphorylation of two purified vaccinia virus proteins (Acceptors I and II) by a protein kinase isolated from vaccinia virus cores has been studied. Phosphorylation of viral acceptor proteins by the purified enzyme was dependent on the presence of ATP, Mg2+, and protamine or other basic proteins, and was maximal at alkaline pH values. Cyclic mononucleotides did not stimulate the vaccinia protein kinase under a variety of conditions. Protamine, however, was shown to function as an enzyme activator. In its presence, the purified vaccinia protein kinase phosphorylated mainly serine residues in Acceptor I, and predominantly threonine residues in Acceptor II. Phosphorylation of protamine accounted for less than 1% of the total 23P incorporation. Tryptic peptide maps prepared from 32P-labeled Acceptors I and II demonstrated that they contained different labeled peptide sequences and were, therefore, distinct protein species. From additional studies on both purified and virus-associated protein kinase it was concluded that various proteins affected the protein kinase reaction in one of three ways. One class of proteins served as phosphate acceptors, but only when another activator protein was present. A second class consisted of proteins that were strong activators but poor phosphate acceptors. The third class contained proteins that were fair phosphate acceptors, but which also activated the phosphorylation of other acceptor proteins.

Adenosine Triphosphate↗
PubMed1996

Ergocalciferol and cycloheximide in vivo stimulate protein kinase C of intestinal crypt cells.

Previous reports have suggested that 1,25-dihydroxychole-calciferol regulated cellular differentiation via its effects on protein kinase C activity. This study examined the in vivo effects of ergocalciferol on the activity of protein kinase C, and whether the differentiation of crypt intestinal cells is dependent on the activation of this enzyme. Ergocalciferol in saline was injected intramuscularly into rats and the animals sacrificed 24 hr after fasting. Protein kinase C specific activity was determined from the rate of incorporation of 32p-ATP into protamine. Injections of 60 micrograms ergocalciferol/200 g of body wt, raised protein kinase C specific activity to 59818 +/- 4010 (SEM, n = 5) cpm 32p-protamine/min/mg cell protein, compared with a control of 46173 +/- 4612 (P < 0.0005). Optimal specific activities were seen within 72 hr of injection. Administration of 120 micrograms ergocalciferol/200 g of body wt, raised the concentrations of serum calcium to 9.8 and 10.4 mg/dl following the intramuscular injection by 24 and 72 hr, respectively, compared with a control of 7.7 mg/dl. Actinomycin D (intramuscular, 100 micrograms/200 g of body wt) together with ergocalciferol (120 micrograms/200 g of body wt) reduced protein kinase C activity by 51% 24 hr after injection. Cycloheximide blocked the activation, but when injected alone stimulated endogenous protein kinase C activity by 34% 24 hr injection. The study shows activation of crypt protein kinase C by ergocalciferol. The inhibition of activation by actinomycin D and cycloheximide suggests the involvement of both transcriptional and translational processes in this activation.

Animals↗
PubMed1989

In vitro tyrosine phosphorylation studies on RAS proteins and calmodulin suggest that polylysine-like basic peptides or domains may be involved in interactions between insulin receptor kinase and its substrate.

We have investigated the in vitro tyrosine phosphorylation of the HRAS and KRAS proteins by human placental insulin receptor kinase. Purified HRAS proteins are not phosphorylated by purified insulin receptor kinase. Since the tyrosine phosphorylation of calmodulin by the insulin receptor kinase in vitro requires cofactors such as protamine and poly(L-lysine), we examined the possibility that poly(L-lysine) may also potentiate the interaction between RAS proteins and the insulin receptor. We found that purified HRAS proteins are indeed phosphorylated by purified insulin receptor kinase in the presence of poly(L-lysine). In contrast, the KRAS protein, which carries an extremely basic domain (residues 172-182, Lys-Asp-Glu-Lys6-Ser-Arg), is phosphorylated by the receptor kinase without the addition of basic proteins. We then determined whether the KRAS basic domain peptide plays a role similar to that of poly(L-lysine) and found that both the HRAS protein and calmodulin are phosphorylated by the receptor kinase in the presence of the KRAS basic domain peptide. Further examination of the role of poly(L-lysine) in potentiating tyrosine phosphorylation of the HRAS protein and calmodulin by purified insulin receptor kinase indicates that poly(L-lysine) affects the conformation of these protein substrates as well as that of the receptor kinase domain. These studies suggest that polylysine-like basic proteins or domains are required to establish the interaction between insulin receptor kinase and its substrate.

Calmodulin↗
PubMed1990

Isolation and characterization of a dimeric cAMP-dependent protein kinase from the fungus Saccobolus platensis.

A cAMP-dependent protein kinase from mycelia of Saccobolus platensis was characterized. The holoenzyme seems to be a dimer (i.e., regulatory subunit--catalytic subunit) of 78,000 Da, slightly activated by cAMP but susceptible to dissociation into its subunits by cAMP, or by kemptide and protamine, the best substrates for Saccobolus protein kinase. The regulatory subunit was purified to homogeneity by affinity chromatography. It is highly specific for cAMP and has two types of binding sites but failed to inhibit the phosphotransferase activity of the homologous or the heterologous (bovine heart) catalytic components. The activity of the catalytic subunit was completely abolished by the regulatory component of the bovine heart protein kinase as well as by a synthetic peptide corresponding to the active site of the mammalian protein kinase inhibitor. The data suggest that interaction between the subunits of the S. platensis protein kinase is different than that found in cAMP-dependent protein kinases from other sources. Similarities and differences between the Saccobolus protein kinase and enzymes from low eucaryotes and mammalian tissues are discussed.

Ascomycota↗
PubMed1981

Purification and characterization of a catalytic subunit of an adenosine 3':5'-monophosphate-dependent protein kinase from human erythrocyte membranes.

Protein kinase [EC 2.7.1.37] of human erythrocyte membranes was solubilized with 0.5 M NaCl in 5 mM phosphate buffer, pH 6.7 at 4 degrees C and purified on a CM-Sephadex C-50 column, followed by affinity chromatography on a histone-Sepharose 4B column. The purified protein kinase gave a single band (molecular weight; 41,000) on examination by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 8.0 and a millimolar range of concentration of Mg2+ was required for its maximum activity. Histone and protamine were well phosphorylated by the protein kinase but casein and phosvitin were poor phosphate acceptors for the enzyme. The enzymic activity was not stimulated by cyclic AMP (cAMP). A cAMP-finding protein from human erythrocyte membranes inhibited the activity of the protein kinase, but the activity was restored with cAMP. A heat stable protein inhibitor from rabbit skeletal muscle also inhibited this enzyme. From these observations, this protein kinase seemed to be a catalytic subunit of the membrane bound cAMP-dependent protein kinase. This enzyme was strongly inhibited with Ca2+ in the presence of 1 mM MgCl2. Various sulfhydryl reagents and polyamines also had inhibitory activity on the protein kinase. Natural substrates of the enzyme were investigated using heat treated membranes and 0.5 M NaCl extracted membrane residues. Band 4.1, 4.2, and 4.5 proteins were phosphorylated but band 2 (spectrin) and band 3 proteins were poor substrates for this protein kinase.

Carrier Proteins↗
PubMed1981

[Protein modulator of cyclic nucleotide-dependent protein kinases from the shrimp Palaemon adspersus].

A thermostable protein modulator of cyclic nucleotide-dependent protein kinases was isolated and purified from the tissue of the shrimp Palaemon adspersus. The molecular weight, N-terminal amino acid and amino acid composition of the protein modulator were determined and its effects on cAMP-dependent protein kinase depending on substrate and cation concentrations were investigated. The protein modulator under study is a low molecular weight (15 000) acid protein, pI-435, which exerts both strong activating and inhibitory effects on cAMP-dependent protein kinase in the reactions of phosphorylation of protamine and histones, respectively.

Amino Acids↗
PubMed1982

Purification and properties of a kinase which phosphorylates and inactivates acetyl-CoA carboxylase.

A protein kinase which phosphorylates and inactivates acetyl-CoA carboxylase has been purified to apparent homogeneity from rat liver. The kinase was found to exist in two forms: bound to carboxylase in a complex or in a free form that is in different stages of aggregation over a wide range of molecular weights. The purification of the kinase involved first partial purification of acetyl-CoA carboxylase through polyethylene glycol precipitation and DEAE-cellulose chromatography. The kinase was then separated from acetyl-CoA carboxylase by Sepharose 2B chromatography. The molecular weight of the kinase subunit was 170,000 as determined by sodium dodecyl sulfate-gel electrophoresis. The incorporation of 1 mol of phosphate/mole of carboxylase subunit caused complete inactivation of the carboxylase. Acetyl-CoA carboxylase, inactivated by the kinase, can be dephosphorylated and reactivated when incubated with phosphorylase phosphatase. The Km values of the kinase for acetyl-CoA carboxylase and ATP are 90 nM and 20 microM, respectively. The kinase was found to be cyclic AMP-independent, but activated by CoA. The protein kinase can phosphorylate acetyl-CoA carboxylase, protamine, and histones, but could not act on hydroxymethylglutaryl-CoA reductase or phosphorylase b.

Acetyl-CoA Carboxylase↗
PubMed1993

Inhibition of protein kinases by 6-dimethylaminopurine accelerates the transition to interphase in activated mouse oocytes.

Mouse oocyte activation is followed by a peculiar period during which the interphase network of microtubules does not form and the chromosomes remain condensed despite the inactivation of MPF. To evaluate the role of protein phosphorylation during this period, we studied the effects of the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) on fertilization and/or parthenogenetic activation of metaphase II-arrested mouse oocytes. 6-DMAP by itself does not induce the inactivation of histone H1 kinase in metaphase II-arrested oocytes, and does not influence the dynamics of histone H1 kinase inactivation during oocyte activation. However, 6-DMAP inhibits protein phosphorylation after oocyte activation. In addition, the phosphorylated form of some proteins disappear earlier in oocytes activated in the presence of 6-DMAP than in the activated control oocytes. This is correlated with the acceleration of some post-fertilization morphological events, such as sperm chromatin decondensation and its transient recondensation, formation of the interphase network of microtubules and pronuclear formation. In addition, numerous abnormalities could be observed: (1) the spindle rotation and polar body extrusion are inhibited; (2) the exchange of protamines into histones seems to be impaired, as judged by the morphology of DNA fibrils by electron microscopy; (3) the formation of a new nuclear envelope around the sperm chromatin proceeds prematurely, while recondensation is not yet completed. These observations suggest that the 6-DMAP-sensitive kinase(s) is (are) involved in the control of post-fertilization events such as the formation of the interphase network of microtubules, the remodelling of sperm chromatin and pronucleus formation.

Adenine↗
PubMed1991

HMG 14 and protamine enhance ligation of linear DNA to form linear multimers: phosphorylation of HMG 14 at Ser 20 specifically inhibits intermolecular DNA ligation.

HMG 14 and protamine can be used to enhance intermolecular ligation of low concentrations of linear DNA. Adding HMG 14 (50 moles per mole DNA) caused 50% of blunt-ended DNA to form predominantly dimers, and all cohesive-ended DNA to form multimers (greater than 6-mer) in response to T4 ligase. Protamine was maximally effective at 40:1, producing mostly dimers and trimers. Adding higher concentrations of HMG 14 did not affect the ligation pattern of cohesive-ended DNA, while higher concentrations of protamine inhibit the formation of multimers. Phosphorylation of HMG 14 at Ser 20 by Ca(++)-phospholipid dependent protein kinase abolished the ability of HMG 14 to stimulate intermolecular ligation, but did not substantially interfere with intramolecular ligation, or the binding of HMG 14 to linear or circular DNA as assessed by gel mobility. Thus Ser 20, which is located in the amino terminal DNA-binding domain of HMG 14, appears to modulate DNA-DNA interactions.

Animals↗
PubMed1997

Release of ecto-protein kinases by the protozoan parasite Leishmania major.

Leishmania major promastigotes have externally oriented ecto-protein kinases (PK) that are capable of phosphorylating both endogenous membrane substrates and foreign proteins. Live parasites phosphorylate protamine sulfate, casein, and phosvitin but not bovine serum albumin. Addition of exogenous PK substrates, such as phosvitin or casein, induced the shedding of ecto-PK that are capable of phosphorylating protamine sulfate. No phosphorylation of protamine sulfate was seen when cell-free supernatants from promastigotes incubated with either buffer alone or bovine serum albumin were used. A second enzyme, a constitutively released PK that phosphorylates casein or phosvitin and not protamine sulfate or mixed histones, was identified and characterized. This PK is inhibited by 5 microM staurosporine, 50 microg/ml heparin, and 75 microM CKI-7, concentrations typical of the IC50 found for other eukaryotic casein kinases (CK). The constitutively shed ecto-PK specifically phosphorylated a peptide substrate for CK1 but not for CK2, suggesting that this shed PK is similar to CK1.

Amino Acid Sequence↗
PubMed1982

Purification and properties of cyclic AMP-dependent protein kinase from rat epididymis.

Two protein kinases (I and II: EC 2.7.1.37) that show a high degree of substrate specificity for protamine rather than histones, phosvitin and casein were partly purified from rat epididymal tissue. The enzymes were present in the cytosol because greater than 80% of the enzymic activity was recovered in the soluble fraction. The kinases required Mg2+ for activity although Co2+ and Mn2+ were partial substitutes. Zn2+ (1 mM) inhibited nearly completely the activity of the enzymes. Both the kinases showed high affinity for activation with cyclic AMP compared to other cyclic nucleotides. Amino acid analysis of 32P-labelled protamine product revealed that the kinases transfer the terminal phosphate of ATP to serine residues of the protein. The isoenzymes I and II showed certain differences in relation to their hydroxyapatite-chromatography profiles, pH activation profiles, heat sensitivity and Km for ATP and cyclic AMP.

Animals↗
PubMed1988

The in vitro phosphorylation of calmodulin by the insulin receptor tyrosine kinase.

Calmodulin, a ubiquitous Ca2+-binding regulatory protein, is phosphorylated exclusively on tyrosine-99 in an insulin-dependent manner by wheat germ lectin-purified preparations of insulin receptors from rat adipocyte plasma membranes. Calmodulin is phosphorylated in the presence of polylysine, histone Hf2b, and protamine sulfate, but not in the absence of these cofactors or in the presence of other basic compounds known to interact with calmodulin, such as mellitin, myelin basic protein, chlorpromazine, trifluoperazine, substance P, glucagon, polyarginine, mastoparin, beta-endorphin, spermine, spermidine, and putrescine. The incorporation of 32P into calmodulin, expressed in terms of moles of phosphate per moles of calmodulin and assayed at calmodulin concentrations of 1.2 and 0.06 microM, is 0.023 + 0.002 and 0.046 + 0.006, respectively. This low stoichiometry is likely due to the relative impurity of the receptor preparation, as similar studies not shown here, using highly purified human insulin receptors, yield a stoichiometry of 1 mol phosphate/mol calmodulin. The time course of phosphorylation is characterized by a short initial lag phase of approximately 5 min, a rapid linear rate from approximately 5 to 40 min, with a steady state of 32P incorporation being approached at approximately 60 min. The K0.5 for ATP is 104 + 18 microM. Phosphorylated calmodulin is partially purified by HPLC on a C4 column using a trifluoroacetic acid/acetonitrile gradient solvent system. Phosphoamino acid analysis and limited thrombin digestion were used to determine that the site of insulin-induced phosphorylation of calmodulin is exclusively on tyrosine-99 regardless of the basic protein cofactor used. Phosphorylated calmodulin does not exhibit the characteristic Ca2+ shift normally observed with calmodulin in electrophoretic gels, an observation that is consistent with this modification affecting the biological activity of the molecule. Thus, the tyrosine phosphorylation of calmodulin represents a potentially important post-translational modification altering calmodulin's ability to regulate a variety of enzymes involved in growth, differentiation, and metabolic regulation.

Adenosine Triphosphate↗