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

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The regulation of kinase activity associated with insulin receptor by phosphorylation and dephosphorylation has been examined using partially purified receptor immobilized on insulin-agarose. The immobilized receptor preparation exhibits predominately tyrosine but also serine and threonine kinase activities toward insulin receptor beta subunit and exogenous histone. Phosphorylation of the insulin receptor preparation with increasing concentrations of unlabeled ATP, followed by washing to remove the unreacted ATP, results in a progressive activation of the receptor kinase activity when assayed in the presence of histone and [gamma-32P]ATP. A maximal 4-fold activation is achieved by prior incubation of receptor with concentrations of ATP approaching 1 mM. High pressure liquid chromatographic analysis of tryptic hydrolysates of the 32P-labeled insulin receptor beta subunit reveals three domains of phosphorylation (designated peaks 1, 2, and 3). Phosphotyrosine and phosphoserine residues are present in these three domains while peak 2 contains phosphothreonine as well. Thus, at least seven sites are available for phosphorylation on the beta subunit of the insulin receptor. Incubation of the phosphorylated insulin receptor with alkaline phosphatase at 15 degrees C results in the selective dephosphorylation of the phosphotyrosine residues on the beta subunit of the receptor while the phosphoserine and phosphothreonine contents are not affected. The dephosphorylation of the receptor is accompanied by a marked 65% inhibition of the receptor kinase activity. Almost 90% of the decrease in [32P]phosphate content of the receptor after alkaline phosphatase treatment is accounted for by a decrease in phosphotyrosine content in peak 2, while very small decreases are observed in peaks 1 and 3, respectively. These results demonstrate that the extent of phosphorylation of tyrosine residues in receptor domain 2 closely parallels the receptor kinase activity state, suggesting phosphorylation of this domain may play a key role in regulating the insulin receptor tyrosine kinase.
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PMID:Tyrosine phosphorylation of the insulin receptor beta subunit activates the receptor-associated tyrosine kinase activity. 653 76

Partially purified liver insulin receptors from full-term pregnant rats show decreased autophosphorylation rates if compared with receptors from virgins. We studied the molecular mechanism of this phenomenon, looking at possible structural and functional changes of several domains. The ATP-binding domain seems to be unaltered in receptors from pregnant rats since Km for ATP was similar to that observed in virgins. In contrast, the Vmax. is decreased some 45%, suggesting changes in the kinase domain. Truncation of a fragment of 10 kDa from the C-terminal tail does not normalize the kinase activity in receptors from pregnant rats, suggesting that this domain is not involved in the inhibitory regulation. Treatment with alkaline phosphatase increases the [32P]Pi incorporation into receptors from pregnant rats; however, the autophosphorylation remains lower than that observed in virgin rats. Tryptic phosphopeptide maps of phosphorylated receptors show that the same phosphopeptides are present in receptors from virgin and pregnant rats. However, the progression through the autoactivation cascade in the kinase domain is impaired in receptors from pregnant rats. Differences in the cleavage by trypsin at the two alternative sites in the kinase domain were observed, indicating possible structural changes in receptors from pregnant rats that could be related to the impairment of the autoactivation cascade. Integrity of the alpha- and beta-subunits, as well as differential expression of the two receptor isotypes, were shown to be unaltered. We conclude that (1) the decreased autophosphorylation rate of the liver insulin receptor from pregnant rats is associated with the impairment of its autoactivation cascade, probably as a consequence of the basal Ser/Thr phosphorylation; and (2) the inhibition of the autoactivation cascade does not account for the overall inhibition of autophosphorylation observed in receptors from pregnant rats.
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PMID:Impairment of the liver insulin receptor autoactivation cascade at full-term pregnancy in the rat. 748 90

Insulin stimulation of differentiated 3T3-L1 adipocytes or Chinese hamster ovary cells expressing high levels of the insulin receptor resulted in a time-dependent decrease in the electrophoretic mobility of SOS on sodium dodecyl sulfate-polyacrylamide gels. The reduction in SOS mobility was completely reversed by alkaline phosphatase treatment, and the in vitro phosphorylation of SOS by mitogen-activated protein kinase resulted in a decrease of electrophoretic mobility identical to that following in vivo insulin stimulation. Immunoprecipitation of Grb2 followed by SOS immunoblotting demonstrated a disassociation of the SOS-Grb2 complex that paralleled the decrease in SOS electrophoretic mobility. Similarly, SOS immunoprecipitation followed by Grb2 immunoblotting also indicated an uncoupling of the SOS-Grb2 complex. Further, incubation of whole-cell extracts with glutathione-S-transferase-Grb2 fusion proteins demonstrated that insulin stimulation resulted in a decreased affinity of SOS for Grb2. In contrast, the dissociation of SOS from Grb2 did not affect the interactions between Grb2 and tyrosine-phosphorylated Shc. In addition to insulin, several other agents which activate the mitogen-activated protein kinase pathway (platelet-derived growth factor, serum, and phorbol ester) also resulted in the uncoupling of the SOS-Grb2 complex. Consistent with these results, expression of v-ras and v-raf resulted in a constitutive decrease in the association between SOS and Grb2. Together, these data suggest a molecular mechanism accounting for the transient activation of ras due to the uncoupling of the SOS-Grb2 complex following SOS phosphorylation.
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PMID:Insulin-stimulated disassociation of the SOS-Grb2 complex. 773 60

A synthetic tris-sulfotyrosyl dodecapeptide (TRDIY(S)ETDY(S)Y(S)RK-amide), whose primary sequence is identical to the 1142-1153 sequence of the insulin proreceptor, inhibited insulin receptor dephosphorylation in solubilized membranes, and digitonin-permeabilized cells derived from Chinese hamster ovary (CHO) cells expressing high levels of human insulin receptors (CHO/HIRc). It also inhibited the dephosphorylation of a synthetic tyrosine phosphorylated substrate by recombinant PTP-1B, a protein tyrosine phosphatase (PTPase), indicating that it acted via interaction with PTPase(s). A N-stearyl derivative of the peptide caused an approximately 4.5-fold increase in insulin-stimulated receptor autophosphorylaction in intact CHO/HIRc cells. The peptide displayed specificity toward tyrosine-class phosphatases only, as it had no effect on the activities of the serine/threonine phosphatases PP-1 and PP-2A, or alkaline phosphatase. The tyrosine sulfate ester bonds of the peptide were stable when incubated with PTP-1B (1 h, 30 degrees C). These data suggest that the sulfotyrosyl peptide functions as a nonhydrolyzable phosphotyrosyl peptide analogue capable of direct interaction with PTPase catalytic domain.
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PMID:A synthetic tris-sulfotyrosyl dodecapeptide analogue of the insulin receptor 1146-kinase domain inhibits tyrosine dephosphorylation of the insulin receptor in situ. 808 98

Glycosyl-phosphatidylinositol-anchored membrane proteins (GPI-proteins) are normally identified either by cleavage of the lipid anchor using (glycosyl)phosphatidylinositol-specific phospholipases C or D (GPI-PLs) or by metabolic labeling of the lipid moiety with specific building blocks. Therefore, methods for discrimination between transmembrane proteins and GPI-proteins on the basis of their physicochemical properties are desirable. Here we are presenting a selective extraction method for typical well-characterized mammalian GPI-proteins, e.g., acetylcholine esterase, alkaline phosphatase, 5'-nucleotidase, and lipoprotein lipase, using a derivative of taurocholate. The results were compared to those obtained with well-characterized transmembrane proteins, e.g., insulin receptor and hydroxymethyl glutaryl coenzyme A-reductase, glucose transporters, or aminopeptidase M and several commercially available detergents. With regard to total membrane proteins, it was possible to selectively enrich GPI-proteins up to 8- to 14-fold by using concentrations between 0.1 and 0.3% of 4'-NH2-amino-7 beta-benzamido-taurocholic acid (BATC). In addition, the cleavage specificity and efficiency of (G)PI-PLs were increased in the presence of identical concentrations of BATC compared to commonly used detergents, e.g., Nonidet P-40. Therefore, the present study shows that the use of BATC facilitates the identification of glycosyl-phosphatidylinositol-anchored membrane proteins.
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PMID:4'-Amino-benzamido-taurocholic acid selectively solubilizes glycosyl-phosphatidylinositol-anchored membrane proteins and improves lipolytic cleavage of their membrane anchors by specific phospholipases. 813 45

Insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor and differentiated 3T3L1 adipocytes resulted in a time-dependent reduction in the SDS-polyacrylamide gel electrophoretic mobility of STAT3. The decreased STAT3 mobility initially occurred by 2 min and was quantitative by 5 min. In addition, the change in STAT3 mobility was concentration-dependent and was detectable at 0.3 nm insulin with maximal effect between 1 and 3 nm. Although both these cell types also express the STAT1 alpha, STAT1 beta, STAT5, and STAT6 isoforms, only STAT3 was observed to undergo an insulin-dependent reduction in mobility. Immuno-precipitation of STAT1 and STAT3 from 32P-labeled cells demonstrated that only STAT3 was phosphorylated in response to insulin whereas phosphoamino acid analysis indicated that this phosphorylation event occurred exclusively on serine residues. Furthermore, treatment of cell extracts with alkaline phosphatase reversed the insulin-stimulated decrease in STAT3 mobility. Together, these data demonstrate that insulin is a specific activator of STAT3 serine phosphorylation without affecting the other STAT isoforms.
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PMID:Insulin stimulates the serine phosphorylation of the signal transducer and activator of transcription (STAT3) isoform. 864

Insulin is known to be an important osteotropic hormone. To date, no study has specifically addressed the possibility that insulin receptor expression may be regulated by differentiation in bone. We report a heterogeneous distribution of insulin receptor (IR) within neonatal rat calvaria using a specific monoclonal antibody to the beta-subunit of the rat insulin receptor (CT-1). Specific binding of CT-1 to mature osteoblasts was demonstrated, with little binding over periosteal tissues or osteocytes. Using enzymatically derived subpopulations of calvarial cells, we showed a correlation between alkaline phosphatase activity and insulin-stimulated 2-deoxyglucose (2-DOG) uptake and increased 125I-insulin binding. Since primary calvarial cultures contain many cell types, we compared 125I-insulin binding, insulin-stimulated 2-DOG uptake, and Northern blot analysis of IR mRNA in the clonal preosteoblast-like cell line UMR 201-10B and the mature osteoblast cell line UMR 106-01. It is shown that UMR 106-01 cells possess higher levels of IR mRNA, insulin binding, and insulin-stimulated glucose uptake, and that insulin up-regulated expression of mRNA of the glucose transporter GLUT1 by 3-fold. In contrast, insulin binding was negligible in UMR 201-10B cells, which expressed much lower levels of IR mRNA. UMR 201-10B cells did not possess an insulin-sensitive glucose uptake system, although they express GLUT1 mRNA. These data are consistent with the hypothesis that, as in muscle and fat, insulin receptor expression correlates with the stage of osteoblast differentiation in vivo and in vitro.
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PMID:Insulin receptor expression in bone. 886 6

In fetal brown adipocyte primary cultures, insulin rapidly (at 5 min) induced tyrosine phosphorylation of the insulin receptor beta-subunit; this effect was maximal at physiological concentrations (1 nM). Insulin also stimulated insulin receptor substrate-1 tyrosine phosphorylation and subsequently activated phosphatidylinositol 3-kinase. Moreover, a 3-fold increase in the Ras.GTP active form and a 6-fold increase in Raf-1 kinase activity were induced after insulin stimulation. An immortalized brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo cotransfection) showed a reduced maximal responsiveness to insulin in the same range of insulin concentrations studied (1-100 nM). Transformed brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo H-ras(lys12) cotransfection) developed insulin resistance upstream from Ras, showing an impairment in the insulin receptor autophosphorylation, and in insulin receptor substrate-1 tyrosine phosphorylation and its association with phosphatidylinositol 3-kinase upon treatment with 1 nM insulin, although insulin receptor number and affinity (Kd) remained unaltered. This lack of effect was ameliorated upon treatment with higher insulin concentrations, in a dose-dependent manner. However, downstream from Ras, events such as formation of the Ras.GTP active form, and Raf-1 kinase and 12-O-tetradecanoylphorbol-13-acetate response element-chloramphenicol transferase (transiently transfected) activities were overstimulated, compared with those in primary and immortalized cells, in an insulin-independent manner. Wheat-germ lectin-purified receptors from H-ras(lys12)-transformed brown adipocytes showed a marked phosphorylation in the basal state, which was suppressed by serine-threonine phosphatase pretreatment. Moreover, alkaline phosphatase pretreatment restored the tyrosine kinase activity of the receptor in response to insulin. We conclude that the decreased tyrosine autophosphorylation rate of the insulin receptor from H-ras(lys12)-transformed brown adipocytes is a consequence of its basal serine/threonine phosphorylation, resulting in severe insulin resistance.
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PMID:Alterations in the insulin signaling pathway induced by immortalization and H-ras transformation of brown adipocytes. 923 68

Burn injury is associated with insulin resistance. The molecular basis of this resistance was investigated by examining insulin receptor signaling in rats after thermal injury. The impaired insulin-stimulated transport of [3H]2-deoxyglucose into soleus muscle strips confirmed the insulin resistance following burns. In vivo insulin-stimulated phosphoinositide 3-kinase activity, pivotal in translocation of GLUT4, was decreased in burns when assessed by its insulin receptor substrate-1 (IRS-1)-associated activity. Insulin-induced tyrosine kinase activity of insulin receptor (IR) and tyrosine phosphorylation of IRS-1 were also attenuated. Immunoprecipitated IR, however, appeared to have normal insulin-responsive kinase activity. Finally, immunoprecipitated IRS-1 was tested for its effect on partially purified recombinant IR and was found to inhibit its kinase activity. This inhibitory effect of IRS-1 was abolished by prior treatment of IRS-1 with alkaline phosphatase, indicating that burn injury-related hyperphosphorylation of IRS-1 is similar to that observed in TNFalpha-induced inhibition of IR signaling. All of these changes were observed in the absence of quantitative changes in IR, IRS-1, and phosphoinositide 3-kinase. Alterations in postreceptor insulin signaling, therefore, may be responsible for the insulin resistance after thermal injury.
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PMID:Analysis of thermal injury-induced insulin resistance in rodents. Implication of postreceptor mechanisms. 931 46

We have synthesized a tris-sulfotyrosyl dodecapeptide (3S-peptide-I) that corresponds to the major autophosphorylation domain within the insulin receptor beta-subunit and showed that it potently inhibited insulin receptor dephosphorylation by protein tyrosine phosphatases (PTPases) in vitro. 3S-peptide-I also inhibited tyrosine dephosphorylation of a synthetic peptide by the recombinant PTPase PTP-1B, indicating that 3S-peptide-I interacts directly with PTPase, causing its inactivation. The peptide had no effect on the activity of serine/threonine phosphatases, PP-1 and PP-2A, or alkaline phosphatase. Furthermore, we found that the introduction of a N-stearyl derivative of 3S-peptide-I in CHO/HIRc cells caused a significant increase in insulin-stimulated phosphorylation of the insulin receptor. In contrast, ligand-stimulated phosphorylation of epidermal growth factor (EGF) receptor in CHO cells overexpressing EGF receptors was not affected by the presence of N-stearyl-3S-peptide-I. These data suggest that by inhibiting dephosphorylation of the insulin receptor in intact cells, 3S-peptide-I may specifically enhance insulin signalling.
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PMID:Specific inhibition of insulin receptor dephosphorylation by a synthetic dodecapeptide containing sulfotyrosyl residues as phosphotyrosyl mimetic. 934 28


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