Endogenous Protein Kinase Activity Research Articles

A permeability change of myelin membrane vesicles towards cations is induced by MgATP but not by phosphorylation of myelin basic protein

The existence of an endogenous protein kinase activity and protein phosphatase activity in myelin membrane from mammalian brain has now been well established. We found that under all conditions tested the myelin basic protein is almost the only substrate of the endogenous protein kinase in myelin of bovine brain. The protein kinase activity is stimulated by Ca 2+ in the micromolar range. Optimal activity is reached at a free Ca 2+ concentration of about 2 μM. Myelin membrane vesicles were prepared and then shown to be sealed by a light-scattering technique. After preloading with 45Ca 2+, 86Rb +, or 22Na +, the self-diffusion (passive outflux) of these ions from myelin membrane vesicles was measured. Ionophores induced a rapid, concentration-dependent outflux of 80–90% of the cations, indicating that only a small fraction of the trapped ions was membrane bound. There was no difference in the diffusion rates of the three cations whether phosphorylated (about 1 mol phosphate per myelin basic protein) or non-phosphorylated vesicles were tested. In contrast, a small but significant decrease in permeability for Rb + and Na + was measured, when the vesicles were pretreated with ATP and Mg 2+.

The phosphorylation of the proteins of rat liver heterogeneous ribonucleoprotein particles by an endogenous kinase activity

An endogenous protein kinase activity of liver heterogeneous nuclear ribonucleoprotein particles has been characterized and the particle proteins which it phosphorylates in vitro have been fractionated on two-dimensional polyacrylamide gels. The activity is dependent on Mg 2+ and is further stimulated by cyclic AMP, polyamines and Mn 2+.

Phosphorylation of membranes from the rat gastric mucosa

Gastric mucosal membranes derived primarily from parietal cells were found to contain endogenous protein kinase systems as well as several phosphate-accepting substrates. One specific membrane protein with a molecular weight of 88 000 was phosphorylated only in the presence of calcium, while the degree of phosphorylation of three other membrane proteins was similarly increased. The activity of the calcium-dependent protein kinase was found to be totally inhibited in the presence of trifluoperazine, a phenothiazine known to specifically inactivate calmodulin. These results suggest that a calmodulin- and calcium-dependent phosphorylation system may be a component of the parietal cell membrane. Phosphorylation of the membrane proteins was not affected by either cyclic AMP or cyclic GMP. The heat-stable inhibitor protein of cyclic AMP-dependent protein kinase did not inhibit the endogenous protein kinase activity suggesting that the membrane enzyme is not similar to the cytosolic protein kinase. However, the catalytic subunit of the soluble enzyme was capable of phosphorylating a number of membrane proteins indicating that after maximal autophosphorylation of the gastric membranes, phosphate-acceptor sites are still available to the cytosolic cyclic AMP-dependent protein kinase.

Phosphorylation of heavy sarcoplasmic reticulum vesicles: identification and characterization of three phosphorylated proteins.

Heavy sarcoplasmic reticulum vesicles derived from the terminal cisternae of the sarcoplasmic reticulum have been shown to contain endogenous protein kinase activity and associated substrate proteins. Heavy vesicles were phosphorylated at room temperature in 5mm MgCl2, 1mm EGTA, 10mm HEPES (pH 7.4) and 10 μm γ-32P-ATP.32P-phosphoproteins were determined by sodium dodecyl sulphate gel electrophoresis and autoradiography. In the absence of ethylene glycol bis (β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), there was little phosphorylation due to the high level of ATPase activity. Phosphorylation of three proteins of 64,000 daltons (E1), 42,000 daltons (E2), and 20,000 daltons (E3) was observed in the presence of 1mm EGTA. Phosphorylation of these proteins wascAMP-independent, hydroxylamine-resistant, and was seen without the addition of protein kinase. In the presence of HgCl2 (2.5mm) or sodium deoxycholate (1%) no protein phosphorylation was observed. ProteinE1 was heavily phosphorylated in the presence of 200mm KCl, while its phosphorylation was inhibited by 20 μm sodium dantrolene, an inhibitor of Ca2+ release. PhosphoproteinE3 was found in light and heavy sarcoplasmic reticulum vesicles whileE1 andE2 were found only in heavy vesicles. The phosphoproteinE2 had the properties of an intrinsic membrane protein while the proteinE1 bejaved as an extrinsic membrane protein. ProteinsE2 andE3 corresponded in mobility to minor sarcoplasmic reticulum proteins whileE1 had the same mobility as calsequestrin. The presence of high calcium (5mm) during electrophoresis caused calsequestrin to run at a lower molecular weight (≈56,000 instead of 64,000 daltons), and correspondingly the phosphoproteinE1 ran at a lower molecular weight. Finally, calsequestrin purified by a double gel electrophoresis method has been shown to be phosphorylated.

Enzyme Activities Associated with an Invertebrate Iridovirus: Protein Kinase Activity Associated with Iridescent Virus Type 6 ( Chilo Iridescent Virus)

Iridescent virus type 6 was found to contain an endogenous protein kinase activity which can phosphorylate some viral proteins and exogenous basic proteins. The enzyme required a divalent metal ion but was not stimulated by cyclic nucleotides. Procedures which are known to solubilize the viral envelope indicated that the protein kinase was an internal component of the virion. Conditions for protein kinase activity are described.

Preferential phosphorylation of basic non-histone proteins by nuclear protein kinase NII from rat liver.

Rat liver nuclear protein kinase NII, which is independent of cyclic nucleotides, phosphorylated both the acidic protein casein and the basic histone preparation, soluble in 0.25 M HCl, which was extracted from the cell nuclei. When the isolated nuclei were incubated with [32P]ATP in the presence of protein kinase NII, more than 95% of the 32P-labelled proteins was recovered in the 0.25 M HCl extract. In order to analyze the substrate proteins of protein kinase NII, a histone preparation free from the endogenous protein kinase activities was used as substrate. The results demonstrated that histones were not phosphorylated, except for a faint 32P incorporation into the H3 fraction. Instead, multiple non-histone proteins were highly phosphorylated, which were present in a minor quantity in the histone preparation. The molecular weights of the main phosphorylated proteins were 72000, 68000, 56000, 47000, 46000, 43000, 38000 and 32000. Isoelectric focusing demonstrated the basic nature of the phosphorylated proteins, and as much as 72% of 32P radioactivity was distributed in the pH region higher than 7.0. Furthermore, many proteins phosphorylated in the isolated nuclei with added protein kinase NII were also found to be basic non-histone proteins. These results indicate that protein kinase NII preferentially phosphorylates in vitro a set of nuclear basic non-histone proteins.

Inhibition of microtubule assembly by phosphorylation of microtubule-associated proteins.

32P labeling of microtubular protein by endogenous protein kinase activity is shown to result from a net increase in protein-bound phosphate and is not the result of a phosphate exchange reaction between ATP and phosphoprotein. Protein phosphorylation is maximal in the presence of 0.5 mM Mg2+ and 0.25 mM ATP, resulting in approximately 2.8 nmol of phosphate/mg of protein. However, phosphorylation can be increased two-to threefold by cAMP. The protein substrates for phosphorylation either the absence or presence of cAMP are the microtubule-associated proteins which copurify with tubulin and promote microtubule assembly. Phosphorylation of microtubule-associated proteins inhibits both the rate and extent of microtubule assembly when the protein is exposed to conditions which result in dissociation of rings. These results are taken to indicate that phosphorylation modifies MAPs so that they have a reduced ability to form an assembly-competent complex with tubulin.

Tyrosine hydroxylase: a substrate of cyclic AMP-dependent protein kinase.

Data demonstrating the direct phosphorylation of tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] purified from rat pheochromocytoma by ATP, Mg2+ and cyclic AMP-dependent protein kinase catalytic subunit are presented. The incorporation of phosphate is highly correlated with the activation of the hydroxylase when either the time of preincubation or the amount of protein kinase subunit is varied. The rate of phosphorylation of tyrosine hydroylase compares favorably with that of H1 histone, a known substrate of protein kinase. Lineweaver-Burk analysis of crude or purified rat pheochromocytoma tyrosine hydroxylase activity, as a function of pterin cofactor concentration, in the absence of ATP, Mg2+, and protein kinase catalytic subunit, yields a curvilinear relationship which can be resolved into two lines, suggesting two enzyme forms with different affinities for pterin cofactor. A fraction of the hydroxylase present in the tumor exists in the activated state, presumably due to the presence of ATP and endogenous protein kinase activity. When the solubl enzyme is activated by cyclic AMP, ATP, Mg2+, and protein kinase, virtually all of the enzyme is converted to the low Km state. We conclude that tyrosine hydroxylase is a substrate of cyclic AMP-dependent protein kinase in vitro and, presumably, in vivo.

Phosphorylation of cardiac sarcolemma by endogenous and exogenous protein kinases

Sarcolemmal membranes isolated from guinea pig heart ventricles contained endogenous protein kinase activity and protein substrates for this enzyme. Phosphorylation of sarcolemma was modestly stimulated by cyclic AMP with the half-maximal stimulation at 0.5 μ m cyclic AMP. The phosphorylation of sarcolemma due to endogenous kinase was dependent on Mg 2+. The apparent affinity for Mg 2+ was found to be 1.4 and 0.53 m m in the absence and presence of 1 μ m cyclic AMP, respectively. The apparent affinity for ATP was 55 μ m. Sarcolemmal membranes were also phosphorylated by exogenous (purified) cyclic AMP-dependent protein kinase(s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of phosphorylated membranes, followed by slicing and determination of the radioactivity in the gel slices, showed that endogenous protein kinase activity promoted the phosphorylation of specific protein peaks, arbitrarily designated a–g in order of increasing relative mobility (relative molecular weights 125,000, 110,000, 86,000, 58,000, 48,000, 22,000, and 16,000, respectively); peak e (48,000) was the major phosphorylated band. Exogenous protein kinase stimulated the phosphorylation of all peaks. However, the degree of stimulation of the low molecular weight peaks f and g was more marked. Results obtained after treatment of phosphorylated membranes with hydroxylamine at acid pH indicated the absence of any significant amount of acyl phosphate-type incorporation of phosphate. Purified phosphoprotein phosphatase from rabbit liver effected dephosphorylation of previously phosphorylated sarcolemma; this treatment resulted in dephosphorylation of all peaks (a–g). Pretreatment of sarcolemma with trypsin (membrane to trypsin ratio of 100) was found to markedly reduce both the total membrane phosphorylation as well as relative phosphorylation of peaks c, f, and g. On the other hand, pretreatment of sarcolemma with phospholipase c slightly stimulated total membrane phosphorylation with nondiscriminatory enhancement of the phosphorylation of all peaks. Microsomal membrane vesicles (enriched in sarcoplasmic reticulum fragments) isolated from guinea pig heart ventricle also contained endogenous protein kinase activity. Cyclic AMP modestly increased the kinase. Polypeptides of molecular weights 56,000, 22,000, and 16,000 were found to be phosphorylated. Exogenous (purified) cyclic AMP-dependent protein kinase increased the phosphorylation of microsomes and of 22,000 and 16,000 molecular weight polypeptides.

Studies on Mitochondrial Protein Kinase Activity of Porcine Corpora Lutea*

Activation of mitochondrial protein kinase and its role in regulation of [4-14MC]cholesterol conversion to [4- 14C]pregnenolone and [4-14C]progesterone were studied using mitochondria prepared from porcine corpora lutea. The presence of a cAMP-dependent protein kinase in luteal mitochondria was confirmed by using highly purified samples prepared by large scale zonal centrifugation. Slices of corpora lutea secreted almost 3 times more progesterone when incubated for 30 min with 10 μg LH/ml. The LH treatment of luteal tissue also resulted in an increase in endogenous protein kinase activity (measured without cAMP) of mitochondria from 45.0 to 60.7 pmol 32P/mg protein min. However, the LH treatment of corpora lutea did not significantly change [4-14C]cholesterol conversion activity. When isolated intact luteal mitochondria were incubated with cAMP, almost all of themitochondrial protein kinase was activated, but there was no change in the [4-14C]cholesterol conversion activity of these mitochondria. The addi...

Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg 2+ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.

Membrane-bound protein kinase activity in Jerusalem artichoke rhizome tissues

A membrane-bound protein kinase, which catalyses the phosphorylation of endogenous substrates, has been isolated from homogenates of Jerusalem artichoke rhizome tissues by differential and discontinuous sucrose-gradient centrifugation. Electron microscopic analysis revealed that the membrane fractions showing endogenous protein kinase activity contained vesicles of various sizes and that most of these structures were stained by phosphotungstic acid/chromic acid, a stain that is known to be almost specific for plant plasma membranes. The enzyme has an almost absolute requirement for Mg 2+. It is unaffected by cyclic AMP, indoleacetic acid, gibberellic acid and fusicoccin. Adenosine and its cytokinin derivative isopentenyladenosine were inhibitory. When acidic (casein and phosvitin) or basic (histone and protamine) proteins were used as exogenous substrates only acidic proteins are phosphorylated by the enzyme. The endogenous phosphorylated substrates have been characterized by SDS-polyacrylamide gel electrophoresis.

Characterization and properties of a protein kinase associated with murine sarcoma-leukemia virus (MSV-MuLV). Endogenous phosphorylation of a unique p10 polypeptide

MSV-MuLV virions produced by the rat embryo fibroblast 78 A 1 cell line and extensively purified by treatment with trypsin (M. Rucheton, D. Blaas and Ph. Jeanteur, Biochimie, in press) were shown to contain an endogenous cyclic AMP independent protein kinase activity which is located within the viral core. Enzymatic properties were investigated and an apparent molecular weight of 40,000 daltons determined by gel filtration. It shows a marked preference for acidic exogenous substrates and especially α-casein. During endogenous reaction, only the viral protein p10 was phosphorylated.

Ectoprotein kinase activity of the isolated rat adipocyte

Intact adipocytes exhibit ectoprotein kinase activity as reflected by their ability to catalyze the transfer of the terminal phosphate of (γ- 32P) ATP to histone added to a cell suspension. This activity is substrate, time and cell number dependent. Lineweaver-Burk plots gave Km and Vmax values for ATP of 5 × 10 −5 M and 7.14 pmoles/min/1.5 × 10 5 cells. Cyclic AMP but not cyclic GMP in μM concentrations stimulates ectoprotein kinase activity. The controlled tryptic digestion of intact cells results in reduction of ectoprotein kinase activity. This activity is not due to leakage of intracellular protein kinases during the preparative procedure nor to penetration of histone into the cells. Additional phosphoproteins not accessible to endogenous protein kinase activity are also localized on the external surface of the intact fat cell.

Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic reticulum. Comparative biochemical analysis of component activities.

Sarcolemmal and sarcoplasmic reticulum membrane vesicle fractions were isolated from cardiac microsomes. Separation of sarcolemmal and sarcoplasmic reticulum membrane markers was documented by a combination of correlative assay and centrifugation techniques. To facilitate the separation, the crude microsomes were incubated in the presence of ATP, Ca2+, and oxalate to increase the density of the sarcoplasmic reticulum vesicles. After sucrose gradient centrifugation, the densest subfraction (sarcoplasmic reticulum) contained the highest (K+,Ca2+)-ATPase activity and virtually no (Na2+,K+)-ATPase activity, even when latent (Na+,K+)-ATPase activity was unmasked. In addition, the sarcoplasmic reticulum fraction contained no significant sialic acid, beta receptor binding activity, or adenylate cyclase activity. Sarcolemmal membrane fractions were of low buoyant density. Preparations most enriched in sarcolemmal vesicles contained the highest level of all the other parameters and only about 10% of the (K+,Ca2+)-ATPase activity of the sarcoplasmic reticulum fraction. The results suggest that (Na+,K+)-ATPase, sialic acid, beta-adrenergic receptors, and adenylate cyclase can be entirely accounted for by the sarcolemmal content of cardiac microsomes. Gel electrophoresis of the sarcolemmal and sarcoplasmic reticulum membrane fractions showed distinct bands. Membrane proteins exclusive to each of the fractions were also demonstrated by phosphorylation. Cyclic AMP stimulated phosphorylation by [gamma-32P]ATP of two proteins of apparent Mr = 20,000 and 7,000 that were concentrated in sarcoplasmic reticulum, but the stimulation was markedly dependent on the presence of added soluble cyclic AMP-dependent protein kinase. Cyclic AMP also stimulated phosphorylation of membrane proteins in sarcolemma, but this phosphorylation was mediated by an endogenous protein kinase activity. The apparent molecular weights of these phosphorylated proteins were 165,000, 90,000, 56,000, 24,000, and 11,000. The results suggest that sarcolemma may contain an integral enzyme complex, not present in sarcoplasmic reticulum, that contains beta-adrenergic receptors, adenylate cyclase, cyclic AMP-dependent protein kinase, and several substrates of the protein kinase.

Occurrence of a cyclic AMP-dependent protein kinase on the outer surface of rat epididymal spermatozoa

Endogenous protein kinase activity was detected on the outer surface of rat cauda epididymal spermatozoa. The kinase activity of the intact sperm cells catalyses the transfer of the terminal phosphate of exogenous [ γ 32-P] ATP to the alkali labile phosphoester bonds of exogenous calf thymus histones. There was little uptake of [ γ 32-P] ATP and phosphorylation of endogenous proteins by intact spermatozoa. The amount of histones phosphorylated by the peripherial kinase is directly proportional to the sperm numbers and the reaction is linear for approx. 5 min. Cyclic AMP (2.5 μM) activates the kinase (approx. 120%) and also causes the release of the enzyme from spermatozoa into the medium. Approx. 80% of the peripherial kinase activity is released after 30 seconds of incubation of spermatozoa.

Phosphorylation of pig brain microtubule proteins. General properties and partial characterization of endogenous substrate and cyclic AMP-dependent protein kinase

1. A simple purification procedure for microtubule proteins is described, which involves a single assembly step in vitro in the absence of glycerol, followed by centrifugation through sucrose. 2. The preparation contains 80% tubulin (mol.wt. 54000), 15-20% of a 280000-mol.wt. protein and several other minor components of intermediate molecular weight after polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and 2-mercaptoethanol. 3. In the presence of [gamma-32P]ATP, [32P]phosphate was incorporated into the 280000-mol.wt. component reaching half-maximal incorporation at 1-2 min, but no phosphorylation of tubulin was detected. Cyclic AMP (Km 0.8 micrometer) increased both the initial rate and the extent of incorporation of [32P]phosphate into this component. 4. About half of the endogenous protein kinase activity did not require cyclic AMP and was not inhibited by a heat-stable inhibitor protein from muscle. The remainder of the activity was cyclic AMP-dependent and sensitive to the inhibitor protein. A regulatory subunit was not dissociable from microtubules assembled in vitro in the presence of saturating concentrations of cyclic AMP. 5. The endogenous substrate and the endogenous protein kinase activity could be partially resolved chromatography on phosphocellulose. 6. The data show that cyclic AMP can moduate the activity of an endogenous protein kinase(s) with unusual properties and which phosphorylates a prominent microtubule-associated protein.

An effect of ATP on the permeability of transport-competent plasma membrane vesicles from mouse fibroblasts

ATP and Mg ++ produced a striking inhibition of Na +-stimulated amino acid and phosphate transport activity assayed in vesicles isolated from the plasma membrane of SV40-transformed and nontransformed mouse fibroblasts. The α-β and β-γ-methylene analogs of ATP were ineffective. Purified membrane vesicles contain an endogenous protein kinase activity which catalyzes autophosphorylation of membrane proteins in the presence of ATP. This provides an experimental approach to investigate the role of membrane protein phosphorylation in mediating permeability changes in response to changes in cellular proliferative or transformed state.

Studies on adenosine 3′,5′-phosphate-binding and adenosine 3′,5′-phosphate-dependent protein kinase activities associated with subcellular fractions of Chinese hamster ovary cells

Both cyclic AMP-binding and cyclic AMP-dependent protein kinase activities exists in Chinese hamster ovary cell extract. Competition experiments demonstrate that the binding is specific for cyclic AMP. All cellular elements including nucleus, mitochondria, plasma membrane, microsome, ribosome and cytosol contain both activities. Binding activity is highest in the cytosol and lowest in the nucleus. Each fraction contains endogenous protein kinase activity which is insensitive to cyclic AMP activation. When histone was used as a substrate, protein kinase activity in all fractions was stimulated by cyclic AMP (with the highest in cytosol and lowest in the nucleus) and inhibited by Walsh's protein kinase inhibitor.

Characterisation of an endogenous protein kinase activity in ribonucleoprotein structures containing heterogenous nuclear RNA in HeLa cell nuclei.

Ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNA) were isolated from sonically disrupted HeLa cells nuclei by sedimentation through a triple sucrose cushion. We found that these particles were able to catalyse the incorporation of radioactive phosphate from [γ32P]ATP into phosphoserine and phosphothreonine residues of endogenous proteins. Optimal conditions for enzymatic activity were defined after investigation of the influence of several parameters of the reaction. The requirement for a divalent cation was most efficiently met by 10 mM Mg2+, to a lower extent by Mn2+. An apparent Km value for ATP of 0.02 mM was determined and the enzyme was found to be sensitive to p‐hydroxymercuribenzoate. Neither cyclic AMP nor cyclic GMP stimulated the reaction over a wide range of concentrations and the same protein substrates were phosphorylated in their presence.After excluding possible contamination by other subcellular fractions, considerable evidence for the association of kinase and phosphate acceptor proteins with ribonucleoprotein structures containing hnRNA was gathered along several lines by showing that the enzymatic activity closely followed tritiated RNA pulse‐labeled in the presence of low doses of actinomycin D in the following circumstances: (a) sedimentation in sucrose gradients, (b) isopycnic banding in metrizamide density gradients in the presence or absence of Mg2+ ions and (c) adsorption to oligo(dT)‐cellulose columns under conditions where poly(A)‐containing material is adsorbed.Analysis of phosphorylated substrates by electrophoresis on polyacrylamide gels containing sodium dodecylsulphate revealed that, although they spanned a wide range of molecular weights, the most intensely labeled species were localized in bands corresponding to molecular weights of 37000 and 28000.The above pattern of polypeptides phosphorylated in vitro is compared with that of phosphoproteins labeled in vivo after exposure of cells to [32P]phosphate.