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At least 1,189 records · Page 66Linked to original sources
PubMed1986

Phosphorylation of rabbit muscle glycogen synthase by casein/glycogen synthase kinase-1 (CK-1). Stoichiometry and distribution of the phosphorylation sites on the glycogen synthase subunit.

The stoichiometry of the phosphorylation of rabbit muscle glycogen synthase by casein/glycogen synthase kinase-1 (CK-1) depended on the concentration of protein kinase in the assay and reached values of 7-8 mol/mol subunit at high concentrations. Phosphorylation by CK-1 above 4 mol/mol subunit promoted a further decrease of glycogen synthase activity when determined by the low glucose-6-phosphate/high glucose-6-phosphate activity ratio assay. Analysis by limited proteolysis with trypsin and chymotrypsin showed that all of the regions in glycogen synthase phosphorylated by casein/glycogen synthase kinase-2 (CK-2), the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase), FA/glycogen synthase kinase-3 (FA/GSK-3) and phosphorylase b kinase were also phosphorylated by CK-1. Digestion with CNBr of glycogen synthase phosphorylated by CK-1 revealed the presence of the two phosphopeptides also labeled by the other protein kinases, the largest phosphopeptide (CB2) containing more phosphorylation sites for CK-1 than the smallest one (CB1). Three phosphopeptides (CB2-c, CB2-d and CB2-e) were obtained by trypsinization of CB2 phosphorylated by CK-1. None of them coincided with those labeled by A-kinase, a fact that was confirmed by the additivity of the effect of both protein kinases. In contrast, CB2-d comigrated with the peptide phosphorylated by FA/GSK-3, and CB2-e with that labeled by CK-2, whereas CB2-c would correspond to a new phosphopeptide.

Animals↗
PubMed1991

Fetal lung development in rats with a glycogen storage disorder.

A New Zealand strain of rats (NZR/Mh) is unable to mobilize liver glycogen due to a deficiency of phosphorylase b kinase. Affected homozygous rats (gsd/gsd) were used to assess the developmental relationship between lung glycogen loss and surfactant phospholipid and protein biosynthesis. Phosphorylase a and phosphorylase b kinase activities were negligible in gsd/gsd fetal lungs compared with controls from gestational day (D18) until postnatal day 1 (D + 1). At D20, tissue glycogen content was 158 +/- 5 and 181 +/- 6 mumol/g lung for control and gsd/gsd, respectively. Control rats mobilized 84% of their lung glycogen by D + 1, whereas the gsd/gsd strain retained 70-80% of D19-20 levels. This apparent fall in gsd/gsd glycogen per gram lung was due to an increase in cellular protein and size. Thus, in controls, total glycogen per lung decreased 65% from D20 to D + 1, whereas DNA doubled. In contrast, gsd/gsd lung growth resulted in a doubling of total lung glycogen, whereas the glycogen-to-DNA ratio remained constant. A lack of cellular glycogenolysis was confirmed by electron microscopy where gsd/gsd type II cells remained large and glycogen-rich over the entire perinatal interval. The potential for glycogen breakdown by a lysosomal alpha-amyloglucosidase in gsd/gsd lungs was estimated in tissue homogenates, whereas rates of hydrolysis of glycogen or p-nitrophenylglucoside were significant and equal to controls at all ages tested. Incorporation of [14C]choline into phosphatidylcholine (PC) of incubated lung slices increased 1.7-fold in control lungs from D20-D21. Over the same interval, PC synthesis in gsd/gsd lungs was 40% lower and did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1992

The mechanism of activation of protein kinase FA (the activator of type-1 protein phosphatase) in brain synaptosomes.

The ATP.Mg-dependent type-1 protein phosphatase and its activating factor (protein kinase FA) were identified to exist in brain synaptosome. The inactive protein phosphatase was found to exist in the synaptosomal cytosol whereas its activating factor (protein kinase FA) was present in the synaptosomal membrane, indicating that the inactive protein phosphatase and its activating factor FA are localized in two separate subcellular compartments. The membrane-bound FA was found to exist in two forms; approximately 75% of FA is inactive and trypsin-resistant, whereas 25% of FA is active and trypsin-labile. When membranes were incubated with exogenous phospholipase C, the inactive/trypsin-resistant FA could be activated and sequestered to become the active/trypsin-labile FA in a time- and dose-dependent manner. Taken together, the results provide initial evidence that the activation-sequestration of membrane-bound protein kinase FA may represent one mode of control modulating the activity of protein kinase FA and thereby to activate protein phosphatase in brain synaptosome, representing an efficient regulatory mechanism for regulating neurotransmission in the central nervous system.

Adenosine Triphosphate↗
PubMed1982

The influences of glucagon, epinephrine and adrenergic agents on glycogen phosphorylase a and pyruvate kinase activities in hepatocytes from juvenile and adult rabbits.

1. Glucagon, epinephrine, norepinephrine, isoproterenol and phenylephrine each increases significantly glucose appearance and glycogen disappearance from hepatocytes of both juvenile and adult fed rabbits. Such increases caused by catecholamines and adrenergic agonists are suppressed significantly by the beta-adrenergic antagonist propranolol but are unchanged by the alpha-antagonist phentolamine. 2. Glucagon, epinephrine, norepinephrine, isoproterenol and phenylephrine each increases significantly glycogen phosphorylase a activity and decreases significantly the pyruvate kinase activity ratio (assayed with 0.8 mM phosphoenolpyruvate +/- 200 microM fructose 1,6-bisphosphate) in hepatocytes from both juvenile and adult rabbits. Changes induced by catecholamines and adrenergic agonists in the activities of both enzymes are significantly diminished by propranolol but unaltered by phentolamine. 3. These observations suggest that regulation of glycogenolysis and gluconeogenesis in rabbits by glucagon and catecholamines is at least partially due to activation of glycogen phosphorylase and inhibition of pyruvate kinase. Contrary to the age-related changes observed in the adrenergic nature of catecholamines' regulation of these two processes in rats, such regulation of both processes by catecholamines is beta-adrenergic in rabbits regardless of age.

Aging↗
PubMed1981

Diabetes alters the myocardial cAMP-protein kinase cascade system.

An isolated perfused working rat heart preparation was used to assess the effect of alloxan-induced diabetes on the cAMP cascade system. Diabetes did not alter basal cAMP, cGMP content, or protein kinase or phosphorylase activities, but depressed (50%) isoproterenol-induced changes in cAMP content and protein kinase activity ratios. In contrast, phosphorylase activation and increased left ventricular pressure (LVP) were unaltered by diabetes. The relationship between cAMP and protein kinase activation is linear in hearts from both normal and diabetic rats. Diabetes did not alter this relationship. The relationship between protein kinase and phosphorylase activation or increases in LVP is also linear. An increase in the slopes obtained with diabetic hearts (P less than 0.05) was observed, suggesting an increased gain in the amplification cascade to protein kinase. The in vivo administration of insulin diminished this response. Thus, diabetes alters the ability of heart to accumulate cAMP and alters the gain of the amplification cascade system subsequent to protein kinase activation. This second effect may indicate an unmasking of a parallel regulatory pathway and in the beta-adrenergic regulation of phosphorylase activity and LVP.

Alloxan↗
PubMed1984

Multiple phosphorylation of rat-liver glycogen synthase by protein kinases.

The phosphorylation sites in liver synthase were studied using gel filtration and high performance liquid chromatography of 32P-labeled tryptic peptides. Phosphorylase b kinase, calmodulin-dependent glycogen synthase kinase and glycogen synthase kinase 4 from liver phosphorylated the same low Mr tryptic peptide. cAMP-dependent protein kinase mainly phosphorylated the low Mr tryptic peptide, but also incorporated phosphate into two other peptides. Glycogen synthase kinase 5 phosphorylated a single tryptic peptide, whereas glycogen synthase kinase 3 phosphorylated several tryptic peptides. Calcium-phospholipid-dependent protein kinase phosphorylated two tryptic peptides, the major one of which had the same chromatographic properties as the low Mr peptide described above. These findings confirm that liver glycogen synthase undergoes multi-site phosphorylation and suggest that the topography of the sites is generally similar to that in muscle glycogen synthase.

Animals↗
PubMed1992

Cyclic inhibition-potentiation of the crosslinking of synapsin I with brain microtubules by protein kinase FA (an activator of ATP.Mg-dependent protein phosphatase).

The ATP.Mg-dependent type-1 protein phosphatase activating factor (FA) was identified as a protein kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton and is believed to be involved in the modulation of neurotransmission. More importantly, more than 90% of the phosphates in 32P-synapsin I phosphorylated by FA could be removed by the activated ATP.Mg-dependent type-1 protein phosphatase and the synapsin I phosphatase activity was found to be strictly FA-dependent. Functional study further revealed that as a synapsin I kinase, factor FA could phosphorylate synapsin I and thereby inhibits crosslinking of synapsin I with tubulin, while as a synapsin I phosphatase activator, FA could promote the crosslinking copolymerization of synapsin I with tubulin. Taken together, the results provide initial evidence that a cyclic modulation of the crosslinking copolymerization of synapsin I with brain microtubules can be controlled by factor FA, representing an efficient cyclic cascade control mechanism for the regulation of axonal transport process during neurotransmission.

Animals↗
PubMed1980

The role of Ca2+ and cyclic AMP in the phosphorylation of rat-liver soluble proteins by endogenous protein kinases.

Rat liver soluble proteins were phosphorylated by endogenous protein kinase with [gamma-32P]ATP. Proteins were separated in dodecyl sulphate slab gels and detected with the aid of autoradiography. The relative role of cAMP-dependent, cAMP-independent and Ca2+-activated protein kinases in the phosphorylation of soluble proteins was investigated. Heat-stable inhibitor of cAMP-dependent protein kinase inhibits nearly completed the phosphorylation of seven proteins, including L-type pyruvate kinase. The phosphorylation of eight proteins is not influenced by protein kinase inhibitor. The phosphorylation of six proteins, including phosphorylase, is partially inhibited by protein kinase inhibitor. These results indicate that phosphoproteins of rat liver can be subdivided into three groups: phosphoproteins that are phosphorylated by (a) cAMP-dependent protein kinase or (b) cAMP-independent protein kinase; (c) phosphoproteins in which both cAMP-dependent and cAMP-independent protein kinase play a role in the phosphorylation. The relative phosphorylation rate of substrates for cAMP-dependent protein kinase is about 15-fold the phosphorylation rate of substrates for cAMP-independent protein kinase. The Km for ATP of cAMP-dependent protein kinase and phosphorylase kinase is 8 microM and 38 microM, respectively. Ca2+ in the micromolare range stimulates the phosphorylation of (a) phosphorylase, (b) a protein with molecular weight of 130 000 and (c) a protein with molecular weight of 15 000. The phosphate incorporation into a protein with molecular weight of 115 000 is inhibited by Ca2+. Phosphorylation of phosphorylase and the 15 000-Mr protein in the presence of 100 microM Ca2+ could be completely inhibited by trifluoperazine. It can be concluded that calmodulin is involved in the phosphorylation of at least two soluble proteins. No evidence for Ca2+-stimulated phosphorylation of subunits of glycolytic or gluconeogenic enzymes, including pyruvate kinase, was found. This indicates that it is unlikely that direct phosphorylation by Ca2+-dependent protein kinases is involved in the stimulation of gluconeogenesis by hormones that act through a cAMP-independent, Ca2+-dependent mechanism.

Adenosine Triphosphate↗
PubMed2005

Site-specific phosphorylation of L-form starch phosphorylase by the protein kinase activity from sweet potato roots.

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.

Amino Acid Sequence↗
PubMed1984

Guanosine cyclic 3',5'-phosphate dependent protein kinase, a chimeric protein homologous with two separate protein families.

The amino acid sequence of bovine lung cGMP-dependent protein kinase has been determined by degradation and alignment of two primary overlapping sets of peptides generated by cleavage at methionyl or arginyl residues. The protein contains 670 residues in a single N alpha-acetylated chain corresponding to a molecular weight of 76 331. The function of the molecule is considered in six segments of sequence which may correspond to four folding domains. From the amino terminus, the first segment is related to the dimerizing property of the protein. The second and third segments appear to have evolved from an ancestral tandem internal gene duplication, generating twin cGMP-binding domains which are homologous to twin domains in the regulatory subunits of cAMP-dependent protein kinase and to the cAMP-binding domain of the catabolite gene activator of Escherichia coli. The fourth and fifth segments may comprise one domain which is homologous to the catalytic subunits of cAMP-dependent protein kinase, of calcium-dependent phosphorylase b kinase, and of certain oncogenic viral protein tyrosine kinases. The regulatory, amino-terminal half of cGMP-dependent protein kinase appears to be related to a family of smaller proteins that bind cAMP for diverse purposes, whereas the catalytic, carboxyl-terminal half is related to a family of protein kinases of varying specificity and varying sensitivity to regulators. These data suggest that ancestral gene splicing events may have been involved in the fusion of two families of proteins to generate the allosteric character of this chimeric enzyme.

Amino Acid Sequence↗
PubMed2006

Cloning and characterization of the HPr kinase/phosphorylase gene from Bacillus stearothermophilus No. 236.

The Bacillus stearothermophilus no. 236 gene encoding the bifunctional enzyme HprK/P, the key regulator of carbon catabolite repression/activation (CCR/CCA) in most Gram-positive bacteria, was cloned and the (His)(6)-tagged gene product was characterized in detail. The nucleotide sequence of the hprK/P gene corresponded to an open reading frame of 951 bp that encoded a polypeptide of 316 amino acid residues with a calculated molecular mass of 35,458 Da. The deduced amino acid sequence of the B. stearothermophilus no. 236 HprK/P showed 64.5% identity with the B. subtilis enzyme, allowing us to identify two highly conserved motifs, the nucleotide binding P-loop (Walker motif A) and the HprK/P family signature sequence in the C-terminal half of the protein. Furthermore, complementation experiments showed that the cloned hprK/P gene product was functionally active in the B. subtilis cells. The purified (His)(6)-tagged B. stearothermophilus no. 236 HprK/P migrated on SDS-PAGE gel as a single species with a molecular mass of about 36 kDa, and behaved in gel filtration like a hexameric protein. The recombinant protein catalyzes the pyrophosphate (PPi)-dependent (highest activity at pH 7.0 and 40 degrees C) as well as the ATP-dependent phosphorylation of Ser46 in HPr (maximum activity at pH 8.0 and 45 degrees C). It also catalyzes the inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr, optimally at pH 6.5 and 40 degrees C. BIAcore surface resonance analysis confirmed that a divalent cation, preferentially Mg(2+), was an indispensable cofactor for the three activities of the HprK/P. Fructose-1,6-bisphosphate (FBP) was observed to stimulate ATP-dependent kinase activity, while inorganic phosophate (Pi) inhibited ATP-dependent kinase activity. Mutations in the Walker motif A simultaneously abolished both types of kinase and phosphorylase activities. On the other hand, the conserved signature residues were confirmed to be involved in the PPi-dependent kinase and phosphorylase reactions.

Amino Acid Sequence↗