UNIPROT:P67775 - FACTA Search

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Query: UNIPROT:P67775 (alpha isoform)
797 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have shown previously (Nishimura, M., Fedorov, S., and Uyeda, K. (1994) (J. Biol. Chem. 269, 26100-26106) that the administration of high concentrations of glucose stimulates dephosphorylation of Fru-6-P,2-kinase: Fru-2,6-bisphosphatase in perfused liver, and xylulose (Xu) 5-P activates the dephosphorylation reaction. To characterize the protein phosphatase, we have purified the Xu 5-P-activated protein phosphatase to homogeneity from livers of rats injected with high glucose. Several protein phosphatases in the livers were separated by DEAE-cellulose chromatography, but only one peak of the enzyme was activated by Xu 5-P. The protein phosphatase was inhibited by okadaic acid (IC50 = 1-3 nM) and did not require Mg2+ or Ca2+, suggesting that the enzyme was type 2A. The enzyme was a heterotrimer (M(r) = 150,000) and consisted of structural (A, 65 kDa) catalytic (C, 36 kDa), and regulatory (B, 52 kDa) subunits. Amino acid sequences of five tryptic peptides derived from the B subunit showed similarity with those of the B alpha isoform of rat protein phosphatase 2A, but five out of 73 residues were different. The protein phosphatase catalyzed dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase, phosphorylase alpha, and pyruvate kinase, and the Km values were 0.8 microM, 3.7 microM, and 2.2 microM, respectively. Among these substrates dephosphorylation of only the bifunctional enzyme was activated by Xu 5-P, and the K alpha value for Xu 5-P was 20 microM. Xu 5-P was the only sugar phosphate which activated the PP2A among all the sugar phosphates examined. These results demonstrated the existence and isolation of a unique heterotrimeric protein phosphatase 2A in rat liver which catalyzed the dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase and was activated specifically by Xu 5-P. The Xu 5-P-activated protein phosphatase 2A explains the increased Fru 2,6-P2 level in liver after high glucose administration.
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PMID:Purification and characterization of a novel xylulose 5-phosphate-activated protein phosphatase catalyzing dephosphorylation of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. 759 45

The effects of granulocyte/macrophage-colony-stimulating factor (GM-CSF) are mediated by interaction with its composite receptor (GMR), which consists of a unique alpha subunit (GMR alpha) and a beta subunit (GMR beta) that is common to the receptors for GM-CSF, interleukin 3, and interleukin 5. GMR beta is required for high-affinity binding, cell proliferation, and protein phosphorylation but has no intrinsic GM-CSF-binding activity. GMR alpha in isolation binds to GM-CSF with low affinity and can signal for increased glucose uptake. In addition to the membrane-bound receptor (mGMR alpha), there is a naturally occurring soluble isoform (sGMR alpha) that is released free into the pericellular milieu. Analysis of genomic sequences reveals that the soluble GMR alpha isoform comes about by alternative mRNA splicing. To examine GMR alpha expression, we developed a quantitative reverse transcription-polymerase chain reaction assay based on serial dilutions of in vitro transcribed GMR alpha RNA. This assay provides a strict log-log measure of GMR alpha RNA expression, distinguishes transcripts related to the soluble and membrane-associated isoforms, and quantitatively detects 0.1 fg of GMR alpha-related mRNA. There was little or no GMR alpha expression in two human lymphoid cell lines and in the erythroblastic leukemia cell line K562, but all myeloid cell lines tested expressed both the membrane-associated and soluble isoforms of GMR alpha. Baseline level of expression of both isoforms varied > 20-fold among the myeloid cell lines studied. Differentiation of HL-60 cells to neutrophils with dimethyl sulfoxide led to a 2-fold downregulation of sGMR alpha and a 20-fold upregulation of mGMR alpha. These differentiation-induced transcriptional changes were unrelated to changes in mRNA stability. These findings indicate that sGMR alpha is differentially expressed from mGMR alpha in human hematopoietic cells and that programmed downregulation of sGMR alpha may be important in myeloid maturation.
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PMID:Membrane-associated and soluble granulocyte/macrophage-colony-stimulating factor receptor alpha subunits are independently regulated in HL-60 cells. 789 72

Cyclic ADP-ribose (cADPR) is generated in pancreatic islets by glucose stimulation, serving as a second messenger for Ca2+ mobilization from the endoplasmic reticulum for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). In the present study, we observed that the addition of calmodulin (CaM) to rat islet microsomes sensitized and activated the cADPR-mediated Ca2+ release. Inhibitors for CaM-dependent protein kinase II (CaM kinase II) completely abolished the glucose-induced insulin secretion as well as the cADPR-mediated and CaM-activated Ca2+ mobilization. Western blot analysis revealed that the microsomes contain the alpha isoform of CaM kinase II but do not contain CaM. When the active 30-kDa chymotryptic fragment of CaM kinase II was added to the microsomes, fully activated cADPR-mediated Ca2+ release was observed in the absence of CaM. These results along with available evidence strongly suggest that CaM kinase II is required to phosphorylate and activate the ryanodine-like receptor, a Ca2+ channel for cADPR as an endogenous activator, for the cADPR-mediated Ca2+ release.
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PMID:Requirement of calmodulin-dependent protein kinase II in cyclic ADP-ribose-mediated intracellular Ca2+ mobilization. 853 Apr 41

We report the carboxylmethylation of a 36-kDa protein in intact normal rat islets and clonal beta (INS-1) cells. This protein was predominantly cytosolic. Its carboxylmethylation, as assessed by vapor phase equilibration assay, was resistant to inhibition by N-acetyl-S-trans, trans-farnesyl-L-cysteine, a competitive substrate for cysteine methyl transferases. These data suggest that the methylated C-terminal amino acid is not cysteine. The methylated protein was identified as the catalytic subunit of protein phosphatase 2A (PP2Ac) by immunoblotting. The carboxylmethylation of the PP2Ac increased its catalytic activity, suggesting a key role in the functional regulation of PP2A. Therefore, we studied okadaic acid, a selective inhibitor of PP2A that acts by an unknown mechanism. Okadaic acid (but not 1-nor-okadaone, its inactive analog) inhibited (Ki = 10 nM) the carboxylmethylation of PP2Ac and phosphatase activity in the cytosolic fraction (from normal rat islets and clonal beta-cells) as well as in intact rat islets. Furthermore, methylated PP2Ac underwent rapid demethylation (t 1/2 = 40 min) catalyzed by a methyl esterase localized in islet homogenates. Ebelactone, a purported inhibitor of methyl esterases, significantly delayed (> 200 min) the demethylation of PP2Ac. Furthermore, ebelactone reversibly inhibited glucose- and ketoisocaproate-induced insulin secretion from normal rat islets. These data identify, for the first time, a methylation-demethylation cycle for PP2Ac in the beta-cell and suggest a key functional relationship between PP2A activity and the carboxylmethylation of its catalytic subunit. These findings thus suggest a negative modulatory role for PP2A in nutrient-induced insulin exocytosis.
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PMID:Carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting cells: evidence for functional consequences on enzyme activity and insulin secretion. 864 Nov 81

Induction of protein kinase C (PKC) pathway in the vascular tissues by hyperglycemia has been associated with many of the cellular changes observed in the complications of diabetes. Recently, we have reported that the use of a novel, orally effective specific inhibitor of PKC beta isoform (LY333531) normalized many of the early retinal and renal hemodynamics in rat models of diabetes. In the present study, we have characterized a spectrum of biochemical and molecular abnormalities associated with chronic changes induced by glucose or diabetes in the cultured mesangial cells and renal glomeruli that can be prevented by LY333531. Hyperglycemia increased diacylglycerol (DAG) level in cultured mesangial cells exposed to high concentrations of glucose and activated PKC alpha and beta1 isoforms in the renal glomeruli of diabetic rats. The addition of PKC beta selective inhibitor (LY333531) to cultured mesangial cells inhibited activated PKC activities by high glucose without lowering DAG levels and LY333531 given orally in diabetic rats specifically inhibited the activation of PKC beta1 isoform without decreasing PKC alpha isoform activation. Glucose-induced increases in arachidonic acid release, prostaglandin E2 production, and inhibition of Na+-K+ ATPase activities in the cultured mesangial cells were completely prevented by the addition of LY333531. Oral feeding of LY333531 prevented the increased mRNA expression of TGF-beta1 and extracellular matrix components such as fibronectin and alpha1(IV) collagen in the glomeruli of diabetic rats in parallel with inhibition of glomerular PKC activity. These results suggest that the activation of PKC, predominately the beta isoform by hyperglycemia in the mesangial cells and glomeruli can partly contribute to early renal dysfunctions by alteration of prostaglandin production and Na+-K+ ATPase activity as well as the chronic pathological changes by the overexpression of TGF-beta1 and extracellular matrix components genes.
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PMID:Characterization of protein kinase C beta isoform activation on the gene expression of transforming growth factor-beta, extracellular matrix components, and prostanoids in the glomeruli of diabetic rats. 920 63

Recently, we demonstrated that the 36 kDa catalytic subunit of protein phosphatase 2A (PP2Ac) undergoes methylation at its C-terminal leucine in normal rat islets, human islets and isolated beta cells; this modification increases the catalytic activity of PP2A [Kowluru et al. Endocrinology. 137:2315-2323, 1996]. Previous studies have suggested that adenine and guanine nucleotides or glycolytic intermediates [which are critical mediators in beta cell function] also modulate phosphatase activity in the pancreatic beta cell. Therefore, we examined whether these phosphorylated molecules specifically regulate the carboxyl methylation and the catalytic activity of PP2A in beta cells. Micromolar concentrations of ATP, ADP, GTP or GDP each inhibited the carboxyl methylation of PP2Ac and, to a lesser degree, the catalytic activity of PP2A. Likewise, the carboxyl methylation of PP2Ac and its catalytic activity were inhibited by [mono- or di-] phosphates of glucose or fructose. Additionally, however, the carboxyl methylation of PP2Ac was significantly stimulated by divalent metal ions (Mn2+ > Mg2+ > Ca2+ > control). The nucleotide or sugar phosphate-mediated inhibition of carboxyl methylation of PP2Ac and the catalytic activity of PP2A were completely prevented by Mn2+ or Mg2+. These data indicate that divalent metal ions protect against the inhibition by purine nucleotides or sugar phosphates of the carboxyl methylation of PP2Ac perhaps permitting PP2A to function under physiologic conditions. Therefore, these data warrant caution in interpretation of extant data on the regulation of phosphatase function by purine nucleotides.
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PMID:Purine nucleotide- and sugar phosphate-induced inhibition of the carboxyl methylation and catalysis of protein phosphatase-2A in insulin-secreting cells: protection by divalent cations. 987 31

Activation of protein kinase C (PKC) by hyperglycemia is implicated in the pathogenesis of long-term diabetic complications. Monocyte activation and transformation into macrophages is a key step in the atherosclerotic process. Therefore, in this study, we sought to determine 1) the effect of hyperglycemia on monocyte PKC activity and on the distribution of Ca2+-dependent and diacylglycerol-sensitive PKC isoforms; and 2) whether the effects on these parameters are determined by hyperglycemia per se, independent of the diabetic state. The studies were performed in 19 type 2 diabetic patients and 14 control subjects. Plasma glucose concentration was higher and insulin sensitivity lower (both P < 0.01) in diabetic patients than in control subjects. Monocytes from diabetic patients showed similar cytosol PKC activity to those from control subjects but higher membrane PKC activity (78+/-6 vs. 50+/-5 pmol x min(-1) x mg(-1) protein; P < 0.01). A direct correlation was observed between fasting plasma glucose and membrane PKC activity (r2 = 0.4008, P = 0.0001). In contrast, a reciprocal correlation was observed between membrane PKC activity and insulin sensitivity index (r2 = 0.28, P < 0.05). Using immunoblotting analysis, we found that membrane beta2, but not alpha, isoform of PKC was more abundant in monocytes from diabetic patients. In diabetic patients, when euglycemia was acutely induced, membrane PKC activity decreased by approximately 42% and beta2 isoform by approximately 15%. In two normal subjects in whom hyperglycemia was induced, membrane PKC increased from 63 and 57 to 92 and 128.6 pmol x min(-1) x mg(-1) protein, respectively. This increase was associated with an increase in the membrane isoform beta2; alpha isoform was unchanged. We conclude that 1) monocytes express the glucose-sensitive beta2 isoform of PKC; 2) the prevailing plasma glucose acutely regulates the activity of the membrane PKC and the content of membrane PKC beta2 isoform; and 3) this effect appears to be a direct effect of glucose per se, since the phenomenon was observed in normal control subjects when hyperglycemia was induced. Monocyte PKC activation may account for the accelerated atherosclerosis of patients with type 2 diabetes.
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PMID:Protein kinase C activity is acutely regulated by plasma glucose concentration in human monocytes in vivo. 1034 22

Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane. Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses. The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class IA Pi3ks (ref. 2). Mice lacking only the p85 alpha isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants. Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites. We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class IA Pi3k catalytic subunits; nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.
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PMID:Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha. 1106 85

The activity and allosteric properties of plant phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) are controlled posttranslationally by specific reversible phosphorylation of a strictly conserved serine residue near the N-terminus. This up/down-regulation of PEPC is catalyzed by a dedicated and highly regulated serine/threonine (Ser/Thr) kinase (PEPC-kinase) and an opposing type-2A Ser/Thr phosphatase (PP2A). In marked contrast to PEPC-kinase, the PP2A holoenzyme from photosynthetic tissue has been virtually unstudied to date. In the present investigation, we have partially purified and characterized the native form of this PP2A from illuminated leaves of maize (Zea mays L.), a C4 plant, using maize [32P]PEPC as substrate. Various conventional chromatographic matrices, together with thiophosphorylated C4 PEPC-peptide and microcystin-LR affinity-supports, were exploited for the enrichment of this PP2A from soluble leaf extracts. Biochemical and immunological results indicate that the C4-leaf holoenzyme is analogous to other eukaryotic PP2As in being a approximately 170-kDa heteromer comprised of a core PP2Ac-A heterodimer (approximately 38- and approximately 65-kDa subunits, respectively) complexed with a putative, approximately 74-kDa B-type regulatory/targeting subunit. This heterotrimer lacks any strict substrate specificity in that it dephosphorylates C4 PEPC, mammalian phosphorylase a, and casein in vitro. This activity is independent of free Me2+, insensitive to levamisole and the Inhibitor-2 protein that targets PP1, activated by several polycations such as protamine and poly-L-lysine, and highly sensitive to inhibition by microcystin-LR and okadaic acid (IC50 approximately 30 pM), all of which are diagnostic features of yeast and mammalian PP2As. In addition, this C4-leaf PP2A holoenzyme (i) is inhibited in vitro by physiological concentrations of certain C4 PEPC-related metabolites (L-malate, PEP, glucose 6-phosphate, but not the activator glycine) when either 32P-labeled maize PEPC or rabbit muscle phosphorylase a is used as substrate, suggesting a direct effect on this Ser/Thr phosphatase; and (ii) displays, at best, only modest light/dark effects in vivo on its apparent molecular mass, component core subunits and activity against C4 PEPC, in marked contrast to the opposing activity of PEPC-kinase in C4 and Crassulacean acid metabolism leaves. This report represents one of the few studies of a heteromeric PP2A holoenzyme from photosynthetic tissue that dephosphorylates a known target enzyme in plants, such as PEPC, sucrose-phosphate synthase or nitrate reductase.
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PMID:Partial purification and biochemical characterization of a heteromeric protein phosphatase 2A holoenzyme from maize (Zea mays L.) leaves that dephosphorylates C4 phosophoenolpyruvate carboxylase. 1150 60

The heterotrimeric G protein G(s) couples hormone receptors (as well as other receptors) to the effector enzyme adenylyl cyclase and is therefore required for hormone-stimulated intracellular cAMP generation. Receptors activate G(s) by promoting exchange of GTP for GDP on the G(s) alpha-subunit (G(s)alpha) while an intrinsic GTPase activity of G(s)alpha that hydrolyzes bound GTP to GDP leads to deactivation. Mutations of specific G(s)alpha residues (Arg(201) or Gln(227)) that are critical for the GTPase reaction lead to constitutive activation of G(s)-coupled signaling pathways, and such somatic mutations are found in endocrine tumors, fibrous dysplasia of bone, and the McCune-Albright syndrome. Conversely, heterozygous loss-of-function mutations may lead to Albright hereditary osteodystrophy (AHO), a disease characterized by short stature, obesity, brachydactyly, sc ossifications, and mental deficits. Similar mutations are also associated with progressive osseous heteroplasia. Interestingly, paternal transmission of GNAS1 mutations leads to the AHO phenotype alone (pseudopseudohypoparathyroidism), while maternal transmission leads to AHO plus resistance to several hormones (e.g., PTH, TSH) that activate G(s) in their target tissues (pseudohypoparathyroidism type IA). Studies in G(s)alpha knockout mice demonstrate that G(s)alpha is imprinted in a tissue-specific manner, being expressed primarily from the maternal allele in some tissues (e.g., renal proximal tubule, the major site of renal PTH action), while being biallelically expressed in most other tissues. Disrupting mutations in the maternal allele lead to loss of G(s)alpha expression in proximal tubules and therefore loss of PTH action in the kidney, while mutations in the paternal allele have little effect on G(s)alpha expression or PTH action. G(s)alpha has recently been shown to be also imprinted in human pituitary glands. The G(s)alpha gene GNAS1 (as well as its murine ortholog Gnas) has at least four alternative promoters and first exons, leading to the production of alternative gene products including G(s)alpha, XLalphas (a novel G(s)alpha isoform that is expressed only from the paternal allele), and NESP55 (a chromogranin-like protein that is expressed only from the maternal allele). A fourth alternative promoter and first exon (exon 1A) located approximately 2.5 kb upstream of the G(s)alpha promoter is normally methylated on the maternal allele and transcriptionally active on the paternal allele. In patients with isolated renal resistance to PTH (pseudohypoparathyroidism type IB), the exon 1A promoter region has a paternal-specific imprinting pattern on both alleles (unmethylated, transcriptionally active), suggesting that this region is critical for the tissue-specific imprinting of G(s)alpha. The GNAS1 imprinting defect in pseudohypoparathyroidism type IB is predicted to decrease G(s)alpha expression in renal proximal tubules. Studies in G(s)alpha knockout mice also demonstrate that this gene is critical in the regulation of lipid and glucose metabolism.
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PMID:Endocrine manifestations of stimulatory G protein alpha-subunit mutations and the role of genomic imprinting. 1158 48

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