ABSTRACT
The catalytic activity of the C subunit of serine/threonine phosphatase 2A is regulated by the association with A (PR65) and B subunits. It has been reported that the alpha4 protein, a yeast homolog of the Tap42 protein, binds the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases such as protein phosphatase 4 and protein phosphatase 6. In the present study, we showed that alpha4 binds these three phosphatases and the association of alpha4 reduces the activities of these phosphatases in vitro. In contrast, PR65 binds to the C subunit of serine/threonine phosphatase 2A but not to protein phosphatase 4 and protein phosphatase 6. These results suggest that the alpha4 protein is a common regulator of the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases.
Subject(s)
Fungal Proteins/genetics , Phosphoprotein Phosphatases/genetics , Saccharomyces cerevisiae Proteins , Adaptor Proteins, Signal Transducing , Animals , Cell Line , Fungal Proteins/metabolism , Gene Expression Regulation, Enzymologic , Humans , Mice , Molecular Sequence Data , Phosphoprotein Phosphatases/metabolism , Protein Phosphatase 2ABSTRACT
Amino acid deprivation of Chinese hamster ovary cells overexpressing human insulin receptors results in deactivation of p70 S6 kinase (p70) and dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), which become unresponsive to insulin; readdition of amino acids restores these responses in a rapamycin-sensitive manner, suggesting that amino acids and mammalian target of rapamycin signal through common effectors. Contrarily, withdrawal of medium amino acids from the hepatoma cell line H4IIE does not abolish the ability of insulin to stimulate p70 and 4E-BP1. The addition of 3-methyladenine (3MA) to H4IIE cells deprived of amino acids inhibited the increment in protein degradation caused by amino acid withdrawal nearly completely at 10 mM and also strongly inhibited the ability of insulin to stimulate p70 and 4E-BP1 at 10 mM. Treatment of H4IIE cells with 3MA did not alter the ability of insulin to activate tyrosine phosphorylation, phosphoinositide 3-kinase, or mitogen-activated protein kinase. In conclusion, the ability of H4IIE cells to maintain the insulin responsiveness of the mammalian target of rapamycin-dependent signaling pathways impinging on p70 and 4E-BP1 without exogenous amino acids reflects the generation of amino acids endogenously through a 3MA-sensitive process, presumably autophagy, a major mechanism of facultative protein degradation in liver.
Subject(s)
Amino Acids/metabolism , Autophagy , Protein Biosynthesis/drug effects , Signal Transduction , Sirolimus/metabolism , Animals , CHO Cells , Cell Line , Cricetinae , Humans , Insulin/pharmacology , Phosphorylation , Rats , Receptor, Insulin/drug effects , Tumor Cells, CulturedABSTRACT
Recent studies have revealed that the alpha4 protein, a mammalian homolog of yeast Tap42, is associated with the protein phosphatase 2A catalytic subunit (PP2A-C), however, effects of the association of alpha4 with PP2A-C on its phosphatase activity have not been examined, especially using physiologically relevant substrates in the signaling pathway of mTOR (the mammalian target of rapamycin) protein. Here, we report how this association affects the enzymatic activity of PP2A-C using the recombinant eIF-4E binding protein (4E-BP1) phosphorylated by immunoprecipitated mTOR as a substrate. PP2A-C dephosphorylated 4E-BP1 in vitro. The association of alpha4 and Tap42 with PP2A-C inhibited the phosphatase activity toward 4E-BP1. Rapamycin treatment, however, neither induced restoration of the phosphatase activity of PP2A-C nor caused dissociation of alpha4 and Tap42 from PP2A-C. Our study is the first report to reveal a potential regulatory role of alpha4 and Tap42 to inibit the phosphatase activity of PP2A-C toward the physiologically relevant substrate in the mTOR signaling.
Subject(s)
Fungal Proteins/metabolism , Phosphoprotein Phosphatases/metabolism , Phosphoproteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Adaptor Proteins, Signal Transducing , Animals , Catalysis , Cell Line , Eukaryotic Initiation Factor-4E , Humans , Intracellular Signaling Peptides and Proteins , Kinetics , Mammals , Molecular Chaperones , Peptide Initiation Factors/metabolism , Phosphorylation , Protein Phosphatase 2 , Recombinant Fusion Proteins/metabolism , Signal Transduction , Substrate Specificity , TransfectionABSTRACT
Phorbol ester treatment of Chinese hamster ovary cells stably overexpressing the delta isoform of protein kinase C induced the association of the isoform with the particulate fraction and the tyrosine phosphorylation of a small portion of the delta isoform. The delta isoform without tyrosine phosphorylation was recovered as an enzyme dependent on phospholipid and diacylglycerol, whereas the tyrosine-phosphorylated delta isoform was recovered in two fractions, one dependent on, and the other independent of, phospholipid and diacylglycerol. The tyrosine-phosphorylated delta isoform independent of lipid activators might be associated with phorbol ester and phospholipids. Immunoblot analysis revealed that the delta isoform is a doublet protein of 76 and 78 kDa, and that the delta isoform fraction without tyrosine phosphorylation contained 76- and 78-kDa proteins, whereas the tyrosine-phosphorylated delta isoform contained the 78-kDa protein but not the 76-kDa protein. In vitro analysis showed that the 78-kDa protein of the delta isoform without tyrosine phosphorylation is an efficient substrate of tyrosine kinase only when phosphatidylserine and either diacylglycerol or phorbol ester are present; however, the 76-kDa protein can not be tyrosine-phosphorylated even in the presence of these lipid activators. The phospholipid and diacylglycerol-dependent form of the tyrosine-phosphorylated enzyme isolated from the cell line required lower concentrations of phosphatidylserine and phorbol ester for its activity in vitro as compared with the enzyme without tyrosine phosphorylation. These results suggest that the tyrosine-phosphorylated enzyme generated upon stimulation of the cells may associate with membranes and exert its full activity even with the lower concentrations of the lipid activators.
Subject(s)
Isoenzymes/isolation & purification , Protein Kinase C/isolation & purification , Tyrosine/metabolism , Animals , CHO Cells , Cricetinae , Phorbol Esters/pharmacology , Phosphorylation , Protein Kinase C-deltaABSTRACT
The main cause of death in chronic hemodialysis patients is heart failure. We studied cardiac function prospectively in diabetic and non-diabetic patients who had recently been started on hemodialysis. We analyzed the mechanocardiograms and echocardiograms in addition to routine blood chemistry tests, chest X-rays, and electrocardiograms of ten hemodialysis patients at our hospital from 1989 to 1991. Ejection fraction (EF) and fractional shortening (FS) were not significantly different between diabetic patients and non-diabetic patients at the start of hemodialysis. After starting hemodialysis, EF and FS declined in diabetic patients. In contrast, non-diabetic patients did not show any changes in EF and FS during hemodialysis. The results demonstrated a poor prognosis in cardiac systolic function in hemodialysis patients with diabetes mellitus.
Subject(s)
Diabetes Mellitus/physiopathology , Renal Dialysis , Stroke Volume , Adult , Aged , Echocardiography , Female , Heart Rate , Humans , Male , Middle Aged , Prospective Studies , Renal Dialysis/adverse effectsABSTRACT
The presence of immunoreactive xanthine oxidase protein was proven in a xanthinuric patient, using a polyclonal antibody against xanthine oxidase. The antibody was raised against purified human liver xanthine oxidase in a rabbit. Double immunodiffusion method demonstrated the existence of an immunologically reactive xanthine oxidase which did not possess xanthine oxidase activity. In addition, urinary excretion of oxypurines in the patient and her family was investigated. The results indicated that a brother and a sister had xanthinuria.
Subject(s)
Duodenum/enzymology , Intestinal Mucosa/enzymology , Purine-Pyrimidine Metabolism, Inborn Errors/diagnosis , Xanthine Oxidase/analysis , Xanthine Oxidase/deficiency , Xanthines/urine , Adult , Antibodies , Family , Female , Humans , Male , Purine-Pyrimidine Metabolism, Inborn Errors/urine , Xanthine Oxidase/urineABSTRACT
The antibody was raised against purified human liver xanthine oxidase in a rabbit. In a xanthinuric patient, the double immunodiffusion method demonstrated the existence of an immunologically reactive duodenal mucosa xanthine oxidase which did not possess xanthine oxidase activity. These results indicated that xanthine oxidase protein is abnormal in structure and/or amino acid sequence.
Subject(s)
Liver/enzymology , Purine-Pyrimidine Metabolism, Inborn Errors/enzymology , Xanthine Oxidase/metabolism , Xanthines/urine , Female , Humans , Hypoxanthine , Hypoxanthines/blood , Hypoxanthines/urine , Immunodiffusion , Male , Purine-Pyrimidine Metabolism, Inborn Errors/genetics , Purine-Pyrimidine Metabolism, Inborn Errors/metabolism , Reference Values , Xanthine , Xanthine Oxidase/analysis , Xanthine Oxidase/deficiency , Xanthines/bloodABSTRACT
The metabolism of pyrazinamide and allopurinol was studied in three xanthinuric patients from two families with hereditary xanthinuria to determine whether both substrates were oxidized only by xanthine oxidase or by other oxidases as well. One xanthinuric patient could neither metabolize pyrazinamide into 5-hydroxypyrazinamide nor allopurinol into oxypurinol. Two xanthinuric patients could metabolize both pyrazinamide into 5-hydroxypyrazinamide and allopurinol into oxypurinol but could not oxidize pyrazinoic acid to 5-hydroxypyrazinoic acid. These findings suggest that xanthinuria comprises at least two subgroups.
