Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A protein kinase which phosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia-tolerant gastropod mollusc Busycon canaliculatum. Purification involved four steps: poly(ethylene glycol) fractionation, affinity chromatography on Blue agarose, ion-exchange chromatography on phosphocellulose and preparative isoelectric focusing (pI = 5.5). The activity was monitored by following changes in pyruvate kinase I50 values for L-alanine which have previously been linked to changes in the degree of enzyme phosphorylation. The correlation between enzyme phosphorylation and changes in the L-alanine inhibition constant was also directly demonstrated in the present paper by radioactively labelling PK with [tau-32P]ATP. The final purified protein kinase solution gave a single band on SDS-gel electrophoresis with a molecular weight of 37,000 +/- 2000. Kinetic analysis of the purified protein kinase (PK-kinase) showed a pH optimum of 7.0, an absolute requirement for magnesium ions (Km = 1.29 mM), a relatively high affinity for MgATP (Km = 57 microM), and inhibition by increasing salt concentrations (I50 = 55 mM KCl). The protein kinase activity was not affected by either spermine, heparin, cAMP, cGMP or concentrations of CaCl2 less than 10 mM. The enzyme did not phosphorylate either phosphofructokinase or glycogen phosphorylase, two enzymes that are also phosphorylated during anoxia in whelks. The purified enzyme is different from the catalytic subunit of cAMP-dependent protein kinase as shown by the inability of cAMP to stimulate the protein kinase at all stages of the preparation; cAMP did not activate either crude enzyme, the 7% poly(ethylene glycol) supernatant, or any of the column eluant peak fractions when measured by changes in pyruvate kinase kinetic parameters.
...
PMID:The role of protein kinases in anoxia tolerance in facultative anaerobes: purification and characterization of a protein kinase that phosphorylates pyruvate kinase. 200 78

The endogenous phosphorylation pattern of the extract prepared from rice young panicle has been examined. The extract was phosphorylated in vitro by incubating with [gamma-32p] ATP, and then analyzed by SDS-PAGE and autoradiography. At least eight major phosphoproteins with apparent molecular weights of 70, 60, 52, 40, 33, 30, 20, 16 and 15 kd were detected in the crude extract of rice young panicle. The pp70 which represents the major phosphoprotein in the crude extract of young panicle of rice is a cytosolic protein. Photoaffinity labeling with 8-azido-ATP revealed that a major protein with molecular weight 42,000 dalton but not pp70 can be specifically bound ATP. This suggests that pp70 is not a protein kinase itself, but a substrate of protein kinase. The in vitro phosphorylation of the pp70 occurs at a serine or threonine residue and is time and temperature dependent. The phosphorylation of pp70 does not depend by exogenous Ca++ or cAMP, suggesting that pp70 is a major substrate of an Ca++ or cAMP independent protein kinase in rice young panicle.
...
PMID:Characterization of the pp70 protein phosphorylation in the extract of rice young panicle. 201 95

Modification of cellular proteins via phosphorylation is known to be a major regulatory mechanism whereby external stimuli control intracellular events. We demonstrated that bacterial LPS induced a distinct set of phosphorylated protein (pp) in murine peritoneal macrophages, and that the LPS-induced pp were specifically located in cytosol and/or membrane fractions. One of the most heavily phosphorylated substrate proteins with a molecular mass of 65 kDa (pp65) was purified to homogeneity via SDS-PAGE analysis and autoradiography by sequential chromatography on Sephacryl S-200, HPLC anion exchange, and hydroxyapatite HPLC. Our pp65 is apparently the first purified LPS-induced pp, and is thought to be a novel protein. Serine residues on pp65 were found to be exclusively phosphorylated, indicating a contribution by LPS-inducible serine kinase. Interestingly, LPS-induced phosphorylation of pp65 was not observed in macrophages from a LPS-nonresponsive C3H/HeJ strain of mice, although their macrophages had about the same amounts of unphosphorylated p65 as normal macrophages when detected under Western blot analysis by using polyclonal anti-pp65 antibodies. This suggests that the functional defect of C3H/HeJ macrophages exists somewhere in the process before the pp65 phosphorylation. Moreover, the degree of the pp65 phosphorylation in macrophages stimulated with LPS or lipd A correlated well to that of cellular responses such as IL-1 production in the same macrophages. Considering these observations, the pp65 seems to play a crucial role in macrophage activation, and the studies on the structure and function of the pp65 should lead to progress in our understanding of the mechanisms of macrophage activation by LPS.
...
PMID:Purification and characterization of the 65-kDa protein phosphorylated in murine macrophages by stimulation with bacterial lipopolysaccharide. 202 84

cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with the molecular masses determined for the subunits of the native enzyme. The recombinant alpha subunit exhibited protein kinase activity which was greatest in the absence of monovalent ions. With increasing amounts of salt, alpha subunit kinase activity declined rapidly. Addition of the beta subunit led to maximum stimulation at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 microM) was comparable to the mammalian enzyme (40-60 microM), whereas the alpha subunit alone had a Km of 240 microM. After sucrose density gradient analysis, the reconstituted holoenzyme sedimented at the same position as the mammalian CKII holoenzyme.
...
PMID:Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria. 204 Feb 87

Estradiol-17 beta (E2) predetermined protein phosphorylation systems have been identified recently in midpregnant rat corpus luteum. Major type protein kinase activities in these systems were explored here using as probes protein kinase inhibitors. Luteal nuclear, mitochondrial, microsomal and cytosolic fractions were obtained from rats hysterectomized and hypophysectomized on day 12 of pregnancy and then treated for 72 h with E2. In vitro phosphate transfer from [gamma-32P]ATP was monitored by SDS-PAGE followed by autoradiography. Polymyxin B (PMB), 1-200 microM, a PKC inhibitor, completely blocked, in a dose dependent manner, the Ca2+ phospholipid (PL) stimulated radiolabeling of nuclear fraction Mr 79,000 substrate(s) as expected. Similarly, the calmodulin (CaM) antagonist compound 48/80, 1-20 micrograms/ml, inhibited the Ca2+/CaM-dependent phosphorylation of the microsomal fraction Mr 60,000 and Mr 56,000 proteins. The Ca2+ PL-enhanced labeling of mitochondrial fraction Mr 76,000 substrate(s) was only partially susceptible to inhibition by PMB or compound 48/80. Studies of microsomal fraction phosphoprotein bands not stimulated by added cofactors indicated that the radiolabeling of Mr 75,000 protein(s) was partially blocked by compound 48/80 but not by PMB. Phosphate transfer to Mr 41,000 protein(s) was inhibited by the cAMP-dependent kinase protein inhibitor (PKI), while the phosphorylation of Mr 31,000 protein(s) was refractory to all inhibitors employed here. Surprisingly, regardless of hormonal pretreatment, PMB and compound 48/80 activated in every subcellular fraction the cofactor independent appearance of at least one phosphoprotein band, between Mr 87,000-99,000. This novel observation should be instrumental in understanding the actions of these compounds towards living cells.
...
PMID:Inhibition and stimulation of rat luteal protein phosphorylation by protein kinase effectors. 204 6

A calcium-sensitive, phospholipid-dependent protein kinase (protein kinase C) and its three isozymes were purified from rat heart cytosolic fractions utilizing a rapid purification method. The purified protein kinase C enzyme showed a single polypeptide band of 80 KDa on SDS-polyacrylamide gel electrophoresis, and was totally dependent on the presence of Ca2+ and phospholipid for activity. Diacylglycerol was also found to stimulate enzymatic activity. Autophosphorylation of the purified PKC showed an 80 KDa polypeptide. The identity of the purified protein was also verified with monoclonal antibodies specific for PKC. Further fractionation of the purified PKC on a hydroxylapatite column yielded three distinct peaks of enzyme activity, corresponding to type I, II and III based on similar chromatographic behaviour as the rat brain enzyme. All three forms were entirely Ca2+ and phosphatidylserine dependent. Type II was found to be the most abundant. Type I was found to be highly unstable. PKC activity studies demonstrate that types II and III isozymic forms are different with respect to their sensitivity to Ca2+.
...
PMID:Purification and characterization of protein kinase C isozymes from rat heart. 207 92

As a first step in understanding the function of the 68-kDa Alz-50 antigen (A68) in the pathophysiology of Alzheimer's disease (AD), we have reexamined preliminary observations in our laboratory (Wolozin and Davies, 1986) of a protein kinase activity associated with crude preparations of the protein. This study was undertaken to determine whether the kinase activity is an inherent property of the Alz-50 antigen, or is a property of an associated protein. Phosphorylation was therefore examined by incubating A68-enriched preparations with radiolabelled ATP. This resulted in the appearance of a labelled 68-kDa phosphoprotein, comigrating with the Alz-50 reactive A68 protein. The labelling of this 68-kDa protein occurred in the presence of 2% SDS, suggesting that it is more likely to represent an autophosphorylation than a transfer of phosphate mediated by another kinase. Upon further inspection, it was found that the autophosphorylated 68-kDa protein was not localized to regions of AD brain where A68 was detectable, but displayed a more ubiquitous distribution. In addition, this phosphoprotein was also observed to be present in similar preparations from normal brain, which lacked the Alz-50 antigen (Wolozin et al, 1986). These findings indicate that the auto-kinase activity at 68 kDa is not closely associated with the A68 protein, but with a comigrating contaminant in the preparation. Other experiments in this study indicate that A68 is not a substrate for in vitro phosphorylation. Following incubation of A68 preparations with radiolabelled ATP, immunoprecipitation of the antigen did not reveal any phosphate transfer to the protein. These results were unaffected by a prior incubation with alkaline phosphatase, even when the subsequent phosphorylation reactions were conducted in the presence of protein kinase activators. Incubation with alkaline phosphatase did not produce any alterations in electrophoretic mobility of A68, nor did it affect the binding of antibodies directed against phosphatase-sensitive epitopes with A68. Thus, despite the suggestion that A68 is a modified form of tau, the antigen exhibits remarkable differences from tau with regard to its sensitivity to kinases and to alkaline phosphatase.
...
PMID:Phosphorylation characteristics of the A68 protein in Alzheimer's disease. 212 70

We investigated cell cycle-dependent regulation of protein kinase activity encoded by the viral mos gene by using a normal rat kidney cell line (NRK-6m2) chronically infected with a temperature-sensitive mutant (ts110) of Moloney murine sarcoma virus, which produces the P85gag-mos transforming protein. In elutriation experiments, in which cells in various phases of the cell cycle are separated based upon size, a twofold increase in the specific activity of the P85gag-mos protein kinase was observed as cells progressed from G0/G1 through S and G2/M. A three- to fourfold increase in gas-mos protein kinase specific activity relative to unsynchronized cells was observed in mitotic NRK-6m2 cells synchronized by treatment with thymidine followed by colcemid or with nocodazole alone. Interestingly, the gag-mos protein was structurally altered in mitotic cells generating a protein species moving slower than P85gag-mos in SDS-polyacrylamide gels. Our findings indicate that viral mos protein kinase activity is regulated during the cell cycle via phosphorylation. We propose that the mos transforming protein functions in a pleiotropic manner, affecting both cytoplasmic and nuclear targets.
...
PMID:Cell cycle-mediated structural and functional alteration of P85gag-mos protein kinase activity. 213 25

The entire coding sequence of wild-type mouse p53 was expressed in Escherichia coli under control of the PL promoter of bacteriophage lambda. The bacterial p53 protein had identical mobility to p53 from SV3T3 cells on SDS polyacrylamide gels and was recognized in bacterial lysates by three p53-specific monoclonal antibodies, including PAb246 which is specific for wild-type mouse p53. Immunoprecipitates of the bacterial p53 were phosphorylated by a highly purified preparation of rat casein kinase II; the stoichiometry of incorporation was approximately 1 mol of phosphate per mol of p53. The phosphorylated residue was identified by phosphopeptide mapping as serine 389, which is a major site of p53 phosphorylation in vivo. p53 (serine 389) kinase activity was detected on lysates of SV3T3 cells; this activity co-purified with casein kinase II on phosphocellulose and Mono Q columns and was inhibited by heparin. Immunoprecipitates of the p53-T antigen complex from SV3T3 cells also had associated serine 389 kinase activity. Phosphorylation of serine 389 by this kinase was potently inhibited by heparin and quenched by excess unlabelled GTP. The data indicate that p53 is a physiological substrate of casein kinase II, which is stimulated in response to mitogens, phosphorylates nuclear oncoproteins, and may play a role in the transduction of extracellular signals to the nucleus.
...
PMID:The p53 tumour suppressor protein is phosphorylated at serine 389 by casein kinase II. 214 48

Incubation of intact rat fat cells with maximally effective concentrations of insulin (1 nM, 12 min) or isoprenaline (300 nM, 3 min) increased particulate cGMP- and cilostamide-inhibited, low-Km cAMP phosphodiesterase (cAMP-PDE) activity by about 50% and 100%, respectively. In 32P-labeled cells, these agents induced serine 32P-phosphorylation of a 135-kDa particulate protein and, to a variable and lesser extent, a 44-kDa protein, which were selectively immunoprecipitated by anti-cAMP-PDE, as analyzed by SDS/PAGE and autoradiography. In the absence of hormonal stimulation, little phosphorylation was detected (less than 10% of that with the hormones). The two phosphoproteins were identified as cAMP-PDE or a closely related molecule (in the case of the 44-kDa species, perhaps a proteolytic fragment) since (i) amounts of 32P in the immunoprecipitated 135-kDa protein paralleled enzyme inactivation, (ii) prior incubation of the anti-cAMP-PDE with the pure rat or bovine enzyme selectively blocked the immunoprecipitation of the phosphoproteins, (iii) 135- and 44-kDa proteins reacted with the anti-cAMP-PDE on Western immunoblots, and (iv) the two phosphoproteins copurified with cAMP-PDE activity through DEAE-Sephacel chromatography and were isolated by highly selective affinity chromatography on cilostamide-agarose. Thus, in fat cells, catecholamine- and insulin-induced activation of the cAMP-PDE may be mediated via phosphorylation by cAMP-dependent protein kinase and an insulin-activated serine protein kinase, respectively.
...
PMID:Evidence that insulin and isoprenaline activate the cGMP-inhibited low-Km cAMP phosphodiesterase in rat fat cells by phosphorylation. 215 56


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---