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)

To study phosphorylation of D. melanogaster nuclear lamins in vivo, we used Kc tissue culture cells. Kc cells contain products of both lamin genes, the lamin Dm0 gene encoding constitutive polypeptides expressed in almost all cell types and the developmentally regulated lamin C gene. We grew Kc cells in low phosphate medium and labelled them with (32P(H3PO4. To obtain mitotic cells we used vinblastine to arrest cells in metaphase. Cells were collected, washed, lysed and resultant extracts fractionated in the presence of protein phosphatase inhibitors. D. melanogaster proteins were then denatured by boiling in SDS plus DTT, followed by immunoaffinity chromatography and SDS-PAGE purification. As anticipated, we found that a CNBr fragment derived from the N-terminal part of lamin Dm0-derivatives (amino acid residues 2-158; fragment A) was phosphorylated during both interphase and mitosis. Interphase but not mitotic phosphorylation was found on an internal CNBr fragment (derived from the end of the central rod domain and the first part of the C-terminal lamin tail; amino acid residues 385-548; fragment D). Interphase only phosphorylation was also detected on another CNBr fragment derived from the extreme C-terminal portion of lamin Dm0-derivatives (amino acid residues 549-622; fragment E). To supplement these data, we used 2-D tryptic peptide mapping followed by phosphorImager analysis. We routinely detected at least seven 'spots' derived from interphase lamins but only a single mitotic lamin phosphopeptide.
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PMID:In vivo phosphorylation of Drosophila melanogaster nuclear lamins during both interphase and mitosis. 1237 69

Recombinant calcineurin heterodimer with the full length delta-isoform of the catalytic subunit (CaN(500)) was expressed in insect cells using the baculovirus system and compared to native bovine brain enzyme in its response to divalent metal ions, redox reagents, and enzymatic modification of arginine residues. The response to various metal ions showed essentially the same profile as bovine brain calcineurin, although Co2+ and Zn2+ did not support recombinant activity as well. Kinetic analysis showed that metal ion and substrate binding were not independent, as found for the bovine brain calcineurin. Incubation with DTT or ascorbate alone caused similar effects on the activity of both enzymes, but different responses were observed when incubated with both DTT and ascorbate; only the recombinant enzyme showed activation. Arginine deimination of recombinant calcineurin by peptidylarginine deiminase resulted in the loss of 60-80% of its phosphatase activity with protection observed if calmodulin was present. Recombinant calcineurin was reactivated by treatment with the protease clostripain, suggesting that deimination of an arginine in the carboxyl terminal domain may be responsible for the loss of phosphatase activity and decreased calmodulin binding [Arch. Biochem. Biophys. 318 (1995) 370]. Supporting this conclusion, a truncated variant of the catalytic subunit lacking the carboxyl terminus showed no loss of phosphatase activity compared to full length calcineurin subunit and contained lower amounts of citrulline than the full length subunit after deimination. These different responses of recombinant calcineurin are consistent with conformational differences compared to bovine brain calcineurin and raise questions about its utility for studying the mechanism of calcineurin.
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PMID:Response of recombinant calcineurin to metal ions, reduction-oxidation agents, and enzymatic modification. 1240 72

Protein kinase-A (PKA) is activated during beta-adrenergic stimulation of the heart and is known to phosphorylate several sarcomeric proteins including the giant polypeptide titin. A PKA phosphorylation site on titin is located within the N2B-unique sequence, which is present in the elastic segment of the two major isoforms of cardiac titin, N2B and N2BA, but not in the skeletal-muscle isoforms of the N2A-type. In bovine and rat cardiomyocytes, PKA-mediated phosphorylation decreases passive tension (PT), an effect ascribed to titin phosphorylation. Whether titin is phosphorylated by PKA upon beta-adrenergic stimulation in human heart has not been shown to date. Here we report that PKA induces phosphorylation of N2B and N2BA titin isoforms, as well as a characteristic proteolytic fragment of titin, T2, in human donor hearts. The PKA-induced phosphorylation signals were stronger when myofilaments were first de-phosphorylated by protein phosphatase-1, suggesting inherent phosphorylation of titin in human heart. Titin phosphorylation was associated with a reduction in PT of skinned human cardiac strips; the relative decrease was higher at shorter than at longer physiological sarcomere lengths. The PKA-dependent PT drop was substantially larger when fibers were pre-treated with protein phosphatase-1, indicating that inherent phosphorylation of titin is important for the basal myocardial PT level. Mechanical measurements on isolated myofibrils from rat heart confirmed the PKA effect on passive stiffness and also showed a more pronounced effect in the presence of reducing agent, DTT. In contrast, PKA did not alter the PT of single skinned rat diaphragm muscle fibers; however, the kinase was still able to phosphorylate the skeletal N2A-titin isoform, which lacks the N2B-unique sequence. Thus, an additional phosphorylation site in titin may exist outside the cardiac N2B-unique sequence. We conclude that PKA mediates phosphorylation of titin in normal human myocardium. Titin phosphorylation lowers titin-based passive stiffness in heart but not in skeletal muscle.
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PMID:Protein kinase-A phosphorylates titin in human heart muscle and reduces myofibrillar passive tension. 1689 74

Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.
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PMID:Reversible oxidation of phosphatase and tensin homolog (PTEN) alters its interactions with signaling and regulatory proteins. 2656 76