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1.
Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria 总被引:2,自引:0,他引:2
Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80-90% was found in the intermembrane fraction, while the rest was associated with mitoplasts. The intermembrane protein kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplasts. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinases, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight. 相似文献
2.
Stefano Ferrari Vittorio Moret Noris Siliprandi 《Molecular and cellular biochemistry》1990,97(1):9-16
Summary Incubation of rat liver mitochondria in the presence of either [32P] Pi or
32
y
-P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [32P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca2+, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [32P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed. 相似文献
3.
Giuseppe Piedimonte Lucia Silvotti Angelo F. Borghetti Solange Chamaret Luc Montagnier 《Journal of cellular biochemistry》1986,32(2):113-123
A tyrosine protein kinase activity has been detected in the mitochondrial fraction purified from human fibroblasts. By enzymatic and sedimentation analysis this activity appeared to be localized in the mitochondrial outer membrane. Mitochondrial tyrosine phosphorylation was strictly dependent on the presence of Mn2+ ions. An inverse relationship between cell proliferation and mitochondrial protein phosphorylation on tyrosine residues has been found: a marked increase in the mitochondrial tyrosine kinase activity occurred when a significant reduction in the growth rate followed serum step-down. In mitochondria purified from resting cells, a protein band with apparent molecular weight of 50 kd appeared to be phosphorylated on tyrosine. 相似文献
4.
Zuzana Technikova-Dobrova Anna Maria Sardanelli Maurizio Rosario Stanca Sergio Papa 《FEBS letters》1994,350(2-3):187-191
A study is presented of the cAMP-dependent phosphorylation in bovine heart mitochondria of three proteins of 42, 16 and 6.5 kDa associated to the inner membrane. These proteins are also phosphorylated by the cytosolic cAMP-dependent protein kinase and by the purified catalytic subunit of this enzyme. In the cytosol, proteins of 16 and 6.5 kDa are phosphorylated by the cAMP-dependent kinase. It is possible that cytosolic and mitochondrial cAMP-dependent kinases phosphorylate the same proteins in the two compartments. 相似文献
5.
《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,714(2):257-264
A cyclic nucleotide-independent protein kinase of human platelets, which phosphorylated histones, myelin basic protein and protamine and did not catalyze the phosphorylation of acidic proteins such as casein, phosvitin and myosin light chain, has been purified approx. 1,500-fold from the crude extract by steps of DEAE-cellulose, Sephadex G-200, hydroxylapatite and phosphoryl cellulose column chromatography. The substrate phosphorylation by this kinase was markedly enhanced by calmodulin even in the absence of Ca2+, when mixed histone was used as a substrate. The interaction of the kinase with mixed histone resulted in an irreversible inactivation of the enzyme. Calmodulin prevented this inactivation, and this compound produced an apparent increase in histone phosphorylation by the kinase. It should be noted that acidic polypeptides such as troponin-C, phospholipids and nucleic acids have a similar ability. The addition of Ca2+ reduced the effect of calmodulin more than the effects of other acidic compounds. 相似文献
6.
Treatment of cultured human hepatoma HepG2 cells with the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), results in an increase in tyrosine phosphorylation of several proteins, including the focal adhesion kinase (FAK) and paxillin using anti-phosphotyrosine Western blotting and immunoprecipitation. However, when cells are in suspension or in the presence of cytochalasin D which disrupts the intracellular network of actin microfilaments, TPA loses its ability to stimulate tyrosine phosphorylation of FAK and paxillin but it still activates mitogen-activated protein kinase (MAPK) and induces PKC translocation from cytosol to the membrane in HepG2 cells. On the other hand, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, blocks TPA-induced MAPK activation but has no effect on TPA-induced tyrosine phosphorylation. Our findings suggest that TPA-induced tyrosine phosphorylation of FAK and paxillin in human hepatoma cells is PKC dependent and requires the integrity of the cell cytoskeleton but is uncoupled to the signal transduction pathway of PKC leading to the translocation of PKC and MAPK activation. 相似文献
7.
The 27 kDa protein, a major component of rat liver gap junctions, was shown to be phosphorylated in vitro by protein kinase C. The stoichiometry of the phosphorylation indicated that approx. 0.33 mol phosphate was incorporated per mol 27 kDa protein. Phosphorylation was entirely dependent on the presence of calcium and was virtually specific for serine residues. For comparison, the gap junction protein was also examined for its phosphorylation by cAMP-dependent protein kinase, the extent of phosphorylation being one-tenth that exerted by protein kinase C. 相似文献
8.
Pusapati GV Eiseler T Rykx A Vandoninck S Derua R Waelkens E Van Lint J von Wichert G Seufferlein T 《The Journal of biological chemistry》2012,287(12):9473-9483
The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin. 相似文献
9.
G Piedimonte S Chamaret C Dauguet A F Borghetti L Montagnier 《Journal of cellular biochemistry》1988,36(1):91-102
Tyrosine protein kinase activity has been detected in the mitochondrial fraction purified from sarcoma 180 tumor cells. Following hypotonic disruption of mitochondria, tyrosine kinase activity appeared to cosediment with monamine oxidase, marker enzyme of mitochondrial outer membrane; meanwhile, serine and threonine kinases were found to be associated with the inner membrane and matrix of mitochondria. Mitochondrial tyrosine kinase(s) showed thermosensitivity and Mn2+ dependence, useful properties for its characterization and separation from tyrosine kinases associated with other particulate fraction and from serine and threonine kinases associated with mitochondria. Following in vitro incubation of mitochondria with labelled ATP as substrate and analysis by PAGE, a complex pattern of phosphotyrosine containing proteins with a major band of 50-55 kilodaltons resulted. 相似文献
10.
Site-specific phosphorylation of L-form starch phosphorylase by the protein kinase activity from sweet potato roots 总被引:1,自引:0,他引:1
A 78-amino acid insertion (L78) is found in the low-affinity type (L-form) of starch phosphorylase (L-SP, EC 2.4.1.1). This
insertion blocks the starch-binding site on the L-SP molecule, and it decreases the binding affinity of L-SP toward starch.
The computational analysis of the amino acid sequence on L78 predicts several phosphorylation sites at its Ser residues. Indeed,
from the immunoblotting results using antibodies against phosphoamino acids, we observed that the purified L-SP from mature
sweet potato (Ipomoea batatas) roots is phosphorylated. This observation led us to the detection of a protein kinase activity in the protein fraction of
the crude extract from the sweet potato roots. The kinase was partially purified by liquid chromatography, and its native
molecular mass was estimated as 338 kDa. An expressed peptide (L78P) containing the essential part of L78 was intensively
phosphorylated by the kinase. However, H-SP (the high-affinity isomer of SP lacking the L78 insertion) and the proteolytic
modified L-SP, which lost its L78 fragment, could not be phosphorylated. Furthermore, using L78P mutants by site-directed
mutagenesis at Ser residues on L78, we demonstrate that only one Ser residue on L78 is phosphorylated by the kinase. These
results imply that this kinase is specific to L-SP, or more precisely, to the L78 insertion. The in vitro phosphorylated L-SP
shows higher sensitivity to proteolytic modification, but has no change in its kinetic parameters.
H.M. Chen and C.T. Lin contributed equally to this work. 相似文献
11.
Jorma Palvimo Anitta Mahonen Pekka H. Menp 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1987,931(3)
Chromosomal high-mobility-group (HMG) proteins have been examined as substrates for calcium/phospholipid-dependent protein kinase C. Protein kinase C from rat brain phosphorylated efficiently both HMG 14 and HMG 17 derived from calf thymus and the reactions were calcium/phospholipid-dependent. About 1 mol of 32P was incorporated per mol of HMG 14 and HMG 17. Phosphopeptide mapping suggested that the same major site was phosphorylated in both proteins at serine. The apparent Km values for HMG 14 and HMG 17 were about 5 μM. HMG 14, HMG 17 and the five histone H1 subtypes prepared from rat thymus, liver and spleen were phosphorylated by the kinase. HMG 14 and HMG 17 from transformed human lymphoblasts (Wi-L2) were also phosphorylated in a calcium/phospholipid-dependent manner. HMG 1 and HMG 2 from the tissues examined were found to be poor substrates for the kinase. 相似文献
12.
Intracellular phosphorylation of dCK on Ser-74 results in increased nucleoside kinase activity. We mimicked this phosphorylation by a Ser-74-Glu mutation in bacterially produced dCK and investigated kinetic parameters using various nucleoside substrates. The S74E mutation increases the kcat values 11-fold for dC, and 3-fold for the anti-cancer analogues dFdC and AraC. In contrast, the rate is decreased for the purine substrates. In HEK293 cells, we found that by comparing transiently transfected dCK(S74E)-GFP and wild-type dCK-GFP, mimicking the phosphorylation of Ser-74 has no effect on cellular localisation. We note that phosphorylation may represent a mechanism to enhance the catalytic activity of the relatively slow dCK enzyme. 相似文献
13.
It has been suggested that phosphorylation of myelin basic protein (MBP) in CNS is catalyzed by protein kinase C (PKC). In order to demonstrate that PKC in the myelin phosphorylates MBP, PKC was partially purified from rat CNS myelin by solubilization with Triton X-100 followed by a DEAE-cellulose column. MBP and histone III-S were phosphorylated in the presence of Ca2+ and phospholipid by rat myelin PKC. High voltage electrophoresis revealed that the phosphoamino acids in MBP by this kinase was serine residue, which is known to be the amino acid phosphorylated by PKC. The activity of PKC extracted from myelin was inhibited by the addition of psychosine to the incubation mixture. To confirm the presence of PKC molecule and to identify the isoform of PKC in the myelin, the solubilized myelin fraction was applied on SDS-PAGE, transferred to a nitrocellulose sheet and stained with anti-PKC monoclonal antibodies. Rat CNS myelin contained the PKC of about 80 kDa (intact PKC), and no proteolytic fragments were observed. PKC isozymes in myelin were type II and III. A developmental study from 14 to 42 postnatal days showed that PKC activity in CNS myelin seemed to parallel the deposition of myelin protein. 相似文献
14.
In Streptomyces fradiae, calcium ions induce alterations in intensity and specificity of the secondary metabolism and stimulate aerial mycelium formation and sporulation. Using in vitro labeling, we demonstrate that in S. fradiae in the late exponential growth phosphorylation of 65-kDa membrane-associated protein is also influenced by Ca(2+) added exogenously. Calcium ions at physiological concentration stimulate intensive Ca(2+)-dependent phosphorylation of 65-kDa protein at multiple sites on serine, threonine, and tyrosine residues. Assay of protein kinases in situ demonstrated in the fraction of membrane-associated proteins the presence of two autophosphorylating protein serine/threonine kinases with molecular masses of 127 kDa and 65 kDa. Autophosphorylation of both proteins is also Ca(2+)-dependent. 相似文献
15.
Yvonne Munari-Silem Bernard Rousset René Mornex 《Biochimica et Biophysica Acta (BBA)/General Subjects》1983,756(2):150-158
1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands/ Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 3000 mM, phosphorylated only phosvitin and was not retained on phophocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhbiited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent Km of 100 μg/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent Km of 25 and 28 μM, respectively. It had an absolute requirement for Mg2+ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecualr weight of 35 000 suggesting a polymeric structure of the enzyme. 相似文献
16.
Daniel L. Friedman Norman J. Kleiman Frank E. Campbell 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1985,847(2):165-176
A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [γ-32P]GTP, low levels of [γ-32P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity. 相似文献
17.
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 Mg2+ 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. 相似文献
18.
The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels. 相似文献
19.
Mathias Ziegler Wolfgang Dubiel Alexander M. Pimenov Yuri V. Tikhonov Ruslan T. Toguzov Wolfgang Henke Gerhard Gerber 《Molecular and cellular biochemistry》1990,93(1):7-12
Summary The degradation of intramitochondrial adenine nucleotides to nucleosides and bases was investigated by incubating isolated rat liver mitochondria at 37°C under non-phosphorylating conditions in the presence of oligomycin and carboxyatractyloside. Within 30 min the adenine nucleotides were degraded by about 25 per cent. The main products formed were adenosine and inosine the contents of which increased five- to sevenfold.Compartmentation studies revealed that about 50 to 60 per cent of the adenosine formed remained inside the organelles whereas inosine was almost completely released into the surrounding medium. Outside the mitochondria only very small amounts of adenine nucleotides were detected. Similar incubations in the presence of [14C]-adenosine yielded no [14C]-inosine ruling out extramitochondrial adenosine deamination.It is concluded that endogenous adenine nucleotides can be degraded in mitochondria via AMP dephosphorylation and subsequent adenosine deamination. A purine nucleoside transport system mediating at least the efflux of inosine from the mitochondria is suggested. 相似文献
20.
Protein kinases and phosphatases are responsible for several cellular events mediated by protein phosphorylation and dephosphorylation. Among these events are cell growth and differentiation and cellular metabolism. Casein kinase I (CKI) and casein kinase II (CKII) are involved in the phosphorylation of several substrates. Endogenous protein phosphorylation and casein kinase activity were investigated in the megagametophyte of the native Brazilian conifer Araucaria angustifolia, during seed development. It was observed that a number of different polypeptides are phosphorylated in vitro in the three megagametophyte stages of development tested (from globular, cotyledonary and mature embryos, respectively) and the phosphate was incorporated mainly in serine residues. The use of okadaic acid and vanadate in the phosphorylation reactions increased phosphate incorporation in several polypeptides suggesting the presence of serine/threonine as well as tyrosine phosphatases in the megagametophyte. Also, the results obtained in experiments with CKII inhibitor, GTP as phosphate donor, RNA hybridizations, and in-gel kinase assays indicate the presence of CKII in the A. angustifolia megagametophyte. 相似文献