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1.
1. Human platelets contain a calmodulin-dependent phosphatase (calcineurin) that has many properties similar to those of bovine brain calmodulin-dependent phosphatase. 2. The activity of calcineurin phosphatase accounts for a small fraction of the total phosphatase activity in human platelets. 3. Labeling of human platelets with 32P yielded many phosphoproteins. 4. Incubation of a lysate of the 32P-labeled platelets with bovine brain calmodulin-dependent phosphatase led to preferential dephosphorylation of a 28 kDa protein (P28), a minor component of platelet proteins. 5. P28 is one of several proteins that were rapidly labeled upon stimulation of platelets with thrombin. 6. Even though the enzyme is known to catalyze the dephosphorylation of many substrates in vitro, its apparent preference for P28 suggests that its activity is highly selective.  相似文献   

2.
Identification of an autoinhibitory domain in calcineurin   总被引:16,自引:0,他引:16  
The hypothesis that calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, contains an autoinhibitory domain was tested using synthetic peptides corresponding to regions of the carboxyl-terminus of calcineurin. Of the several peptides analyzed, one, containing residues I-T-S-F-E-E-A-K-G-L-D-R-I-N-E-R-M-P-P-R-R-D-A-M-P, gave complete inhibition of its protein phosphatase activity. Using [32P]myosin light chain as substrate an IC50 of about 10 microM was obtained with either native calcineurin, assayed in the presence of Ca2+/calmodulin, or with calcineurin subjected to partial proteolysis which converts it to a fully active phosphatase when assayed in the presence of [ethylenebis (oxyethylenenitrilo)]tetraacetic acid. With 50 mM p-nitrophenylphosphate as substrate an IC50 of about 40 microM was observed. Studies with overlapping peptides suggested that the sequence P-P-R-R-D-A-M-P was essential but not sufficient for the observed inhibition. Kinetic analysis indicated that the inhibition of phosphatase activity was not competitive with respect to [32P]myosin light chain. This peptide did not show significant inhibition of the catalytic subunits of protein phosphatases type I or type IIA or of Ca2+/calmodulin-dependent protein kinase II. These results indicate that amino acids within this sequence of calcineurin constitute a unique autoinhibitory domain which interacts with the active site and is responsible for the low basal phosphatase activity in the absence of Ca2+/calmodulin.  相似文献   

3.
4.
To elucidate Ca(2+)-mediated regulation of aflatoxin production, the status of Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation was investigated employing toxigenic and non-toxigenic strains of Aspergillus parasiticus. Incubation of cytoplasmic extracts with [gamma-(32)P]ATP followed by SDS-PAGE and autoradiography revealed total absence of protein phosphorylation during periods corresponding to aflatoxin production in the toxigenic strain (NRRL 2999). In contrast, protein phosphorylation was unaffected in the non-toxigenic strain (SRRC 255). Aflatoxin production in the toxigenic strain was also accompanied by enhanced (26-fold) activity of calcineurin (calmodulin-dependent protein phosphatase 2B) concomitant with a lowered (6-fold) activity of calmodulin-dependent protein kinase. In addition, the in vitro activity of Ca(2+)/calmodulin-dependent protein kinase was susceptible to dose-dependent inhibition by aflatoxin. Since calcineurin remains active in the absence of phosphorylation by calmodulin-dependent protein kinase, it is suggested that calcineurin-mediated dephosphorylation of regulatory enzymes ensures continued production of aflatoxins.  相似文献   

5.
We have investigated regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and calcineurin (Ca2+/calmodulin-dependent protein phosphatase) after transient forebrain ischemia. Immunoreactivity and enzyme activity of CaM kinase II decreased in regions CA1 and CA3, and in the dentate gyrus, of the hippocampus early (6-12 h) after ischemia, but the decrease in immunoreactivity gradually recovered over time, except in the CA1 region. Furthermore, the increase in Ca2+/calmodulin-independent activity was detected up to 3 days after ischemia in all regions tested, suggesting that the concentration of intracellular Ca2+ increased. In contrast to CaM kinase II, as immunohistochemistry and regional immunoblot analysis revealed, calcineurin was preserved in the CA1 region until 1.5 days and then lost with the increase in morphological degeneration of neurons. Immunoblot analysis confirmed the findings of the immunohistochemistry. These results suggest that there is a difference between CaM kinase II and calcineurin in regional and temporal loss after ischemia and that imbalance of Ca2+/calmodulin-dependent protein phosphorylation-dephosphorylation may occur.  相似文献   

6.
A calmodulin-binding protein from sea urchin eggs consisting of two subunits (55 and 17K-daltons) was identified as a Ca2+-dependent phosphoprotein phosphatase similar to calcineurin in mammalian brain and to phosphatase 2B in skeletal muscle. Peptide mappings showed that the 55K subunit was different from 61K subunit of calcineurin, whereas the 17K subunit was similar to 19K subunit of calcineurin but different from calmodulin. The 55K + 17K protein of sea urchin eggs dephosphorylated 32P-inhibitor-1 in a Ca2+- and calmodulin-dependent manner. Vmax and Km for inhibitor-1 in the presence of Ca2+ and calmodulin were 2,100 pmol Pi/min/mg and 2.7 microM. Ca2+-dependent phosphatase activity for inhibitor-1 was detected in homogenates of both unfertilized and fertilized eggs, but was not detected in isolated cortices and mitotic apparatus.  相似文献   

7.
The regulatory effect of regucalcin on Ca2+/calmodulin-dependent phosphatase activity and the binding of regucalcin to calmodulin was investigated. Phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine in rat liver cytosol was significantly increased by the addition of Ca2+ (100 μM) and calmodulin (0.30 μM). Thess increases were clearly inhibited by the addition of regucalcin (0.50–1.0 μM) into the enzyme reaction mixture. The cytosolic phosphoamino acid phosphatase activity was significantly elevated by the presence of anti-regucalcin monoclonal antibody (0.2 μg/ml), suggesting that endogenous regucalcin in the cytosol has an inhibitory effect on the enzyme activity. This elevation was prevented by the addition of regucalcin (0.50 μM). Purified calcineurin phosphatase activity was significantly increased by the addition of calmodulin (0.12 μM) in the presence of Ca2+ (1 and 10 μM). This increase was completely inhibited by the presence of regucalcin (0.12 μM). The inhibitory effect of regucalcin was reversed by the addition of calmodulin with the higher concentration (0.36 μM). Regucalcin has been demonstrated to bind on calmodulin-agarose beads by analysis with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The present study demonstrates that regucalcin inhibits Ca2+/calmodulin-dependent protein phosphatase activity in rat liver cytosol, and that regucalcin can bind to calmodulin. J. Cell. Biochem. 71:140–148, 1998. © 1998 Wiley-Liss, Inc.  相似文献   

8.
A 20-residue peptide analogue (IASGRTGRRNAIHDILVSSA) of the 8000-dalton heat-stable cAMP-dependent protein kinase inhibitor undergoes efficient calcium-dependent binding by calmodulin, with Kd approximately 70 nM when calcium is present. It is a potent inhibitor of smooth muscle myosin light chain kinase and of the calmodulin-dependent phosphatase activity of calcineurin. At concentrations above 3 microM, the peptide stimulates the basal activity of calcineurin. The native protein kinase inhibitor has no effect on the catalytic activity of myosin light chain kinase and is moderately inhibitory to both the calmodulin-dependent and -independent phosphatase activity of calcineurin. Competition experiments using excess concentrations of calcineurin and calmodulin suggest that the primary interaction of the native heat-stable inhibitor is with the catalytic subunit of protein kinase. Dansylcalmodulin exhibits only a weak interaction with the inhibitor. Observations on deletion peptides of the 20-residue analogue help to delineate the overlapping peptide binding specificities of the cAMP-dependent protein kinase [Scott, J. D., Glaccum, M. B., Fischer, E. H., & Krebs, E. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1613-1616] and calmodulin. In both cases, the most effectively bound peptides contain the RTGRR sequence.  相似文献   

9.
Calcineurin, which is a Ca2+/calmodulin-dependent protein phosphatase, is a key mediator in calcium signaling in diverse biological processes and of clinical importance as the target of the immunosuppressant FK506. To identify a mutant(s) in which calcineurin is activated, inhibiting cellular growth as a result, we screened for a mutant(s) whose temperature sensitivity would be suppressed by FK506 from the budding yeast non-essential gene deletion library. We found that the temperature sensitivity of cells in which the conserved Verprolin VRP1 gene had been deleted, which gene is required for actin organization and endocytosis, was suppressed by either FK506 or by cnb1 deletion. Indeed, the calcineurin activity increased significantly in the ?vrp1 cells. Finally, we demonstrated that the ?vrp1 strain to be useful as an indicator in a positive screening for bioactive compounds inhibiting calcineurin.  相似文献   

10.
Yu DY  Luo J  Bu F  Song GJ  Zhang LQ  Wei Q 《Biological chemistry》2006,387(7):977-983
Calcineurin is a Ca2+/calmodulin-dependent phosphatase that dephosphorylates numerous substrates in different neuronal compartments. Genetic and pharmacological studies have provided insight into its involvement in the brain. Cyclosporin A (CsA) is used as a specific calcineurin inhibitor in many pharmacological experiments. However, the calcineurin activity of CsA-treated brain has not been reported. To examine the relationship between calcineurin activity and brain function, we injected CsA into the left lateral ventricle of the mouse brain and assayed calcineurin activity. CsA reduced calcineurin activity in a dose-dependent manner, without affecting the amount of calcineurin protein. Assays of the effect of protein phosphatase inhibitors on CsA-injected mouse brain extracts and kinetic analysis revealed that CsA inhibited calcineurin activity in a non-competitive manner in vivo, in agreement with in vitro results. Injection of CsA led to enhanced phosphorylation of tau at Ser-262 (12E8 site), Ser-198, Ser-199, and/or Ser-202 (Tau-1 site) and Ser-396 and/or Ser-404 (PHF-1 site), as well as to impaired spatial memory, which are two characteristic features of Alzheimer's disease. We propose that inhibition of calcineurin may play an important role in Alzheimer's disease.  相似文献   

11.
Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.  相似文献   

12.
Summary Calcineurin was dicovered as an inhibitor of calmodulin stimulated cyclic AMP phosphodiesterase and its ability to act as a calmodulin binding protein largely explains its inhibitory action on calmodulin regulated enzymes. Recent studies establish calcineurin as the enzyme protein phosphatase whose activity is regulated by calmodulin and a variety of divalent metals. In this work, we have investigated the effects of several agents including sulfhydryl agents, trifluoperazine (a calmodulin antagonist), PPi, NaF and orthovanadate and of tryptic proteolysis on the calcineurin inhibition of cyclic AMP phosphodiesterase (called inhibitory activity) and on protein phosphatase activity. Inhibitors for sulfhydryl groups (pHMB, NEM) inhibited phosphatase activity without any effect on the inhibitory activity. Dithioerythritol completely reversed the inhibition by pHMB. Limited proteolysis of calcineurin caused an activation of basal phosphatase activity with a complete loss of inhibitory activity. Phosphatase activity of the proteolyzed calcineurin was not stimulated by calmodulin. The presence of calmodulin along with calcineurin during tryptic digestion appeared to preserve the stimulation of phosphatase by Ca2+-calmodulin. [3H]-Trifluoperazine (TFP) was found to be incorporated irreversibly into calcineurin in the presence of ultraviolet light. This incorporation was evident into the A and B subunits of calcineurin. TFP-caused a decrease in the phosphatase activity and an increase in its inhibitory activity. [3H]-TFP incorporation into the A subunit was drastically decreased in the proteolyzed calcineurin. This was also true when the [3H]-TFP incorporated calcineurin was subjected to tryptic proteolysis. The incorporation into the B unit was essentially unaffected in the trypsinized calcineurin. Phosphatase activity was inhibited by orthovanadate, NaF, PPi, and EDTA. Inhibitions by these compounds were more pronounced when the phosphatase was determined in the presence of Ca2+-cahnodulin than in their absence.  相似文献   

13.
We have isolated a cDNA clone encoding a homolog of mammalian calcineurin B (the regulatory subunit of calmodulin-dependent protein phosphatase) by screening a cDNA expression library of Saccharomyces cerevisiae with antiserum against bovine calcineurin B. The yeast calcineurin B homolog (YCNB) is composed of 175 amino acids with a calculated molecular mass of 19,639 daltons and contains four putative Ca(2+)-binding domains. The amino-acid alignment of YCNB with human calcineurin B demonstrates 53% sequence identity and 82% homology. Southern blot analysis indicates that the gene for YCNB is a single-copy gene. Thus, yeast calmodulin-dependent protein phosphatase apparently has a heterodimeric structure similar to that of the enzyme in mammalians.  相似文献   

14.
Abstract: Neurotransmission requires rapid docking, fusion, and recycling of neurotransmitter vesicles. Several of the proteins involved in this complex Ca2+-regulated mechanism have been identified as substrates for protein kinases and phosphatases, e.g., the synapsins, synaptotagmin, rabphilin3A, synaptobrevin, munc18, MARCKS, dynamin I, and B-50/GAP-43. So far most attention has focused on the role of kinases in the release processes, but recent evidence indicates that phosphatases may be as important. Therefore, we investigated the role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in exocytosis and subsequent vesicle recycling. Calcineurin-neutralizing antibodies, which blocked dynamin I dephosphorylation by endogenous synaptosomal calcineurin activity, but had no effect on the activity of protein phosphatases 1 or 2A, were introduced into rat permeabilized nerve terminals and inhibited Ca2+-induced release of [3H]noradrenaline and neuropeptide cholecystokinin-8 in a specific and concentration-dependent manner. Our data show that the Ca2+/calmodulin-dependent phosphatase calcineurin plays an essential role in exocytosis and/or vesicle recycling of noradrenaline and cholecystokinin-8, transmitters stored in large dense-cored vesicles.  相似文献   

15.
C. elegans coelomocytes are macrophage-like scavenger cells that provide an excellent in vivo system for the study of clathrin-mediated endocytosis. Using this in vivo system, several genes involved in coelomocyte endocytosis have been identified previously. However, the detailed mechanism of endocytic pathway is still unknown. Here, we report a new function of calcineurin, an evolutionarily conserved Ca2+/calmodulin-dependent Ser/Thr protein phosphatase, in coelomocyte endocytosis. We found that calcineurin mutants show defective coelomocyte endocytosis. Genetic analysis suggests that calcineurin and a GTPase, dynamin (DYN-1), may function upstream of an orphan receptor, CUP-4, to regulate endocytosis. Therefore, we propose a model in which calcineurin may regulate coelomocyte endocytosis via DYN-1 and CUP-4 in C. elegans.  相似文献   

16.
The Dictyostelium genome harbors single copy genes for both the catalytic and regulatory subunits of the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Since molecular genetic approaches to reduce the expression of these genes have failed so far, we attempted to pharmacologically target calcineurin activity in vivo by using the recently described calcineurin inhibitor, gossypol. Up-regulation of expression of the gene for the Ca2+-ATPase PAT1 in conditions of Ca2+ stress was reduced by gossypol. Dictyostelium wild-type cells treated with 12.5-100 microM gossypol showed reduced growth rates and impaired development. In addition, cell signalling was affected. A cell line that overproduces the catalytic subunit of calcineurin was more resistant to gossypol.  相似文献   

17.
Protein kinases and phosphatases are organized into complex intracellular signaling networks designed to coordinate their activities in both space and time. In order to better understand the molecular mechanisms underlying the regulation of signal transduction networks, it is important to define the spatiotemporal dynamics of both protein kinases and phosphatases within their endogenous environment. Herein, we report the development of a genetically-encoded protein biosensor designed to specifically probe the activity of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. Our reporter design utilizes a phosphatase activity-dependent molecular switch based on the N-terminal regulatory domain of the nuclear factor of activated T-cells as a specific substrate of calcineurin, sandwiched between cyan fluorescent protein and yellow fluorescent protein. Using this reporter, calcineurin activity can be monitored as dephosphorylation-induced increases in fluorescence resonance energy transfer and can be simultaneously imaged with intracellular calcium dynamics. The successful design of a prototype phosphatase activity sensor lays a foundation for studying targeting and compartmentation of phosphatases.  相似文献   

18.
The Ca2+/calmodulin-stimulated protein phosphatase calcineurin is a critical component of Ca2+ signaling cascades in eukaryotic cells. Myristoylation of the regulatory subunit of calcineurin (CNB) is conserved from yeast to humans. Here, we show that CNB myristoylation antagonizes phosphatase activation in yeast. Disruption of CNB myristoylation by mutation of the myristoylated glycine triggered constitutive expression of a calcineurin-dependent reporter gene and enhanced calcineurin-dependent phenotypes. Basal phosphatase activity was also increased in nmt1–181 yeast with reduced N-myristoyltransferase activity. Our findings are the first demonstration of a functional role for CNB myristoylation and reveal the importance of Nmt1 in modulating cellular calcineurin activation.  相似文献   

19.
A series ofNeurospora crassamutants affected in the ability to regulate entry into conidiation (an asexual developmental program) were isolated by using an insertional mutagenesis procedure followed by a screening protocol. One of the mutants isolated by this approach consisted entirely of cells with an abnormal morphology. The mutant produces chains of swollen septated cells. The developmentally regulatedccg-1gene is constitutively expressed in these cells, suggesting that they have entered the conidial developmental program. The insertionally disrupted genecnb-1was isolated by plasmid rescue and found to encode calcineurin B, the regulatory subunit of the Ca2+and calmodulin-dependent protein phosphatase calcineurin. The data demonstrate that calcineurin B is required for normal vegetative growth inN. crassaand suggest that thecnb-1mutant is unable to repress entry into the asexual developmental program. The results suggest that Ca2+may play an important role in regulating fungal morphology.  相似文献   

20.
Calcineurin is a Ca2+- and calmodulin-dependent serine/threonine phosphatase and has multiple functions in animal cells including regulating ionic homeostasis. We generated transgenic rice plants that not only expressed a truncated form of the catalytic subunit of mouse calcineurin, but also were able to grow and fertilize normally in the field. Notably, the expression of the mouse calcineurin gene in rice resulted in its higher salt stress tolerance than the non-transgenic rice. Physiological studies have indicated that the root growth of transgenic plants was less inhibited than the shoot growth, and that less Na+ was accumulated in the roots of transgenic plants after a prolonged period of salt stress. These findings imply that the heterologous calcineurin plays a significant role in maintaining ionic homeostasis and the integrity of plant roots when exposed to salt. In addition, the calcineurin gene expression in the stems of transgenic plants correlated with the increased expression of the Rab16A gene that encodes a group 2-type late-embryogenesis-abundant (LEA) protein. Altogether our findings provide the first genetic and physiological evidence that expression of the mouse calcineurin protein functionally improves the salt stress tolerance of rice partly by limiting Na+ accumulation in the roots.  相似文献   

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