Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.16 (calcineurin)
 17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lowe syndrome, also known as oculocerebrorenal syndrome, is caused by mutations in the X chromosome-encoded OCRL gene. The OCRL protein is 51% identical to inositol polyphosphate 5-phosphatase II (5-phosphatase II) from human platelets over a span of 744 aa, suggesting that OCRL may be a similar enzyme. We engineered a construct of the OCRL cDNA that encodes amino acids homologous to the platelet 5-phosphatase for expression in baculovirus-infected Sf9 insect cells. This cDNA encodes aa 264-968 of the OCRL protein. The recombinant protein was found to catalyze the reactions also carried out by platelet 5-phosphatase II. Thus OCRL converts inositol 1,4,5-trisphosphate to inositol 1,4-bisphosphate, and it converts inositol 1,3,4,5-tetrakisphosphate to inositol 1,3,4-trisphosphate. Most important, the enzyme converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The relative ability of OCRL to catalyze the three reactions is different from that of 5-phosphatase II and from that of another 5-phosphatase isoenzyme from platelets, 5-phosphatase I. The recombinant OCRL protein hydrolyzes the phospholipid substrate 10- to 30-fold better than 5-phosphatase II, and 5-phosphatase I does not cleave the lipid at all. We also show that OCRL functions as a phosphatidylinositol 4,5-bisphosphate 5-phosphatase in OCRL-expressing Sf9 cells. These results suggest that OCRL is mainly a lipid phosphatase that may control cellular levels of a critical metabolite, phosphatidylinositol 4,5-bisphosphate. Deficiency of this enzyme apparently causes the protean manifestations of Lowe syndrome.
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PMID:The protein deficient in Lowe syndrome is a phosphatidylinositol-4,5-bisphosphate 5-phosphatase. 776 12

Two short amino acid motifs, WXGDXNXR and PXWCDRXL, define a large family of inositol polyphosphate 5-phosphatases. We tested the importance of seven of these conserved amino acids to substrate binding and catalysis by mutating each to alanine in the platelet 75 kDa inositol polyphosphate 5-phosphatase II (5-phosphatase II). Native and mutant forms of 5-phosphatase II were expressed in baculovirus-infected Sf9 cells, and the recombinant proteins were purified by Mono Q chromatography and studied for enzyme activity. Mutants D476A, N478A, D553A, and R554A had no detectable activity using all four known substrates for this enzyme. Mutants R480A, W551A, and I555A showed greatly reduced hydrolysis of Ins(1,4,5)P3 when compared to native enzyme [Km = 75 microM, Vm = 8300 nmol of Ins(1,4,5)P3 hydrolyzed min-1 (mg of protein)-1]. Mutants W551A and I555A had a Km for Ins(1,4,5)P3 hydrolysis similar to that of the native enzyme (35 microM and 81 microM, respectively), suggesting that these amino acids do not play a role in binding substrate. By contrast, mutant R480A had both increased Km (634 microM) and decreased Vm [855 nmol of Ins(1,4,5)P3 hydrolyzed min-1 (mg of protein)-1]. As judged by measurement of Km, mutant R480A retained normal binding of Ins(1,3,4,5)P4, suggesting that the arginine in motif 2 has a greater role in Ins(1,4,5)P3 binding than in Ins(1,3,4,5)P4 binding. Mutant I555A bound Ins(1,3,4,5)P4 with 8-fold reduced affinity. These mutations markedly reduced 5-phosphatase II hydrolysis of the three other substrates, Ins(1,3,4,5)P4, PtdIns(4,5)P2, and PtdIns(3,4,5)P3. We also tested a mutation comparable to D553A, D460A, in the 110 kDa form of the signaling inositol polyphosphate 5-phosphatase (5SIP110). 5SIP110 D460A had no detectable enzyme activity but retained the ability to bind GRB2. These results are consistent with a role for these conserved amino acids in substrate binding and catalysis.
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PMID:Mutation of the conserved domains of two inositol polyphosphate 5-phosphatases. 867 90

Pleckstrin is the major substrate phosphorylated on serine and threonine in response to stimulation of human platelets by thrombin (Abrams, C. S., Zhao, W., Belmonte, E., and Brass, L. F. (1995) J. Biol. Chem. 270, 23317-23321). We now show that pleckstrin in platelets is in a complex with inositol polyphosphate 5-phosphatase I (5-phosphatase I). This enzyme hydrolyzes the 5-phosphate from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thus serves as a calcium signal-terminating enzyme, since the substrates but not the products mobilize intracellular calcium. Pleckstrin co-immunoprecipitates with 5-phosphatase I in homogenates of platelets. Platelet homogenates fractionated by anion exchange chromatography show co-elution of pleckstrin and 5-phosphatase I. Fractions containing phosphorylated pleckstrin have 7-fold greater 5-phosphatase activity than those containing unphosphorylated pleckstrin. Mixing experiments with recombinant 5-phosphatase I and pleckstrin in vitro show that they form a stoichiometric complex. A mutant form of pleckstrin, in which the serine and threonine residues that are phosphorylated by protein kinase C are substituted with glutamic acid (pseudophosphorylated pleckstrin), activates recombinant 5-phosphatase I 2-3-fold while native unphosphorylated pleckstrin does not stimulate the enzyme. Thus pleckstrin functions to terminate calcium signaling in platelets when it is phosphorylated by binding to and activating 5-phosphatase I.
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PMID:Phosphorylation of platelet pleckstrin activates inositol polyphosphate 5-phosphatase I. 899 61

The 75-kDa inositol polyphosphate 5-phosphatase (5-phosphatase II) hydrolyzes various signaling molecules including the following: inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidylinositol 3,4, 5-trisphosphate. Although studied extensively, a demonstrably full-length cDNA encoding 5-phosphatase II has yet to be isolated. In this study we used a human partial 2.3-kilobase pair (kb) cDNA to screen mouse brain and kidney cDNA libraries, resulting in the isolation of a 3.7-kb cDNA (M5), which by multiple criteria represents a full-length cDNA encoding a 115-kDa 5-phosphatase II. We also isolated a smaller cDNA (M22) with a unique N terminus that encodes a 104-kDa polypeptide. Analysis of these cDNAs suggests a further 87-kDa isoform may arise from differential splicing resulting in translation at methionine 234 in M5. RNA analysis of tissues demonstrates expression of two mRNA species of approximately 4.0 or 3.0 kb, respectively. Probes unique to the 5' end of M5 or M22 hybridized to the 4.0- or 3.0-kb transcripts, respectively. RNA analysis using probes derived from sequence 3' to the potential splice site in M5 and M22 hybridized to both transcripts. Expression of the recombinant 115-kDa protein, or a smaller recombinant protein lacking the N terminus transiently in COS-7 cells, showed localization of enzyme activity to the membrane. Removal of the C-terminal CAAX motif resulted in a significant translocation of the protein lacking the N terminus but not the 115-kDa 5-phosphatase to the cytosol. Western blot analysis of membrane and cytosolic fractions of multiple mouse tissues confirmed the 115-kDa 5-phosphatase II was located in the membrane, whereas the 104- and 87-kDa isoforms were prominent in the cytosol. Collectively these studies demonstrate the widespread expression of at least three isoforms of 5-phosphatase II derived from RNA splicing events. This allows differential distribution of the 5-phosphatase II activity between the membrane and cytosol of the cell and thereby may regulate enzyme access to phosphoinositide-derived signaling molecules.
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PMID:Distinct membrane and cytosolic forms of inositol polyphosphate 5-phosphatase II. Efficient membrane localization requires two discrete domains. 952 32

Fc(epsilon)RI-induced Ca2+ signaling in mast cells is initiated by activation of cytosolic tyrosine kinases. Here, in vitro phospholipase assays establish that the phosphatidylinositol 3-kinase (PI 3-kinase) lipid product, phosphatidylinositol 3,4,5-triphosphate, further stimulates phospholipase Cgamma2 that has been activated by conformational changes associated with tyrosine phosphorylation or low pH. A microinjection approach is used to directly assess the consequences of inhibiting class IA PI 3-kinases on Ca2+ responses after Fc(epsilon)RI cross-linking in RBL-2H3 cells. Injection of antibodies to the p110beta or p110delta catalytic isoforms of PI 3-kinase, but not antibodies to p110alpha, lengthens the lag time to release of Ca2+ stores and blunts the sustained phase of the calcium response. Ca2+ responses are also inhibited in cells microinjected with recombinant inositol polyphosphate 5-phosphatase I, which degrades inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), or heparin, a competitive inhibitor of the Ins(1,4,5)P3 receptor. This indicates a requirement for Ins(1,4,5)P3 to initiate and sustain Ca2+ responses even when PI 3-kinase is fully active. Antigen-induced cell ruffling, a calcium-independent event, is blocked by injection of p110beta and p110delta antibodies, but not by injection of 5-phosphatase I, heparin, or anti-p110alpha antibodies. These results suggest that the p110beta and p110delta isoforms of PI 3-kinase support Fc(epsilon)RI-induced calcium signaling by modulating Ins(1,4,5)P3 production, not by directly regulating the Ca2+ influx channel.
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PMID:p110beta and p110delta phosphatidylinositol 3-kinases up-regulate Fc(epsilon)RI-activated Ca2+ influx by enhancing inositol 1,4,5-trisphosphate production. 1127 65