Effect of mouse calcineurin on induction and growth of rice callus transformed by the calcineurin gene

Calcineurin is a Ca²⁺/calmodulin dependent serine/threonine protein phosphatase, and has multiple functions in animal cells. In this work, mouse calcineurin was introduced into wild-type rice and the expression of calcineurin inhibited the induction and growth of rice calli. Inhibitor analysis showed that untransformed and CNAtr transgenic callus cultures had different sensitivity to cyclosporin A (CsA), a specific inhibitor of protein phosphatase calcineurin. When callus cultures were subject to 1 μM of CsA, the growth of calli induced from untransformed wild-type rice was inhibited. Interestingly, the growth inhibition of CNAtr transgenic calli was not detected in presence of 1 μM of CsA. Our findings showed that the heterologous calcineurin might be involved in the regulation of cell growth in plant cells.     

Effects of cyclosporin A, FK506 and rapamycin on calcineurin phosphatase activity in mouse brain

Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase expressed at high levels in brain. The immunosuppressive drugs cyclosporin A and FK506, but not rapamycin are specific inhibitors of calcineurin, the inhibitory effects of which have been elucidated in the immune system. Here by using these compounds as inhibitors, we assayed the enzyme in mouse brain after injection of 12.5 nmol cyclosporin A, FK506, or rapamycin into the left lateral ventricle of mouse brain. Data from calcineurin activity assay suggest that infusion of cyclosporin A or FK506, rather than rapamycin inhibited calcineurin activity in brain and in a substrate noncompetitive manner, which is revealed by the in vitro enzyme kinetic analysis. Cyclosporin A or FK506 injected into brains also affected the inhibitory effects of cyclosporin A or FK506 added to brain extracts on calcineurin activity. The results may be ascribed to the decreased free immunophilin in brain after infusion of corresponding immunosuppressant, or the fact that two immunophilin-immunosuppressant complexes have not completely identical interaction sites on calcineurin.     

Calcineurin dephosphorylates glycogen synthase kinase-3 beta at serine-9 in neuroblast-derived cells

This study examined the role of calcineurin, a major calcium-dependent protein phosphatase, in dephosphorylating Ser-9 and activating glycogen synthase kinase-3β (GSK-3β). Treatment with calcineurin inhibitors increased phosphorylation of GSK-3β at Ser-9 in SH-SY5Y human neuroblastoma cells. The over-expression of a constitutively active calcineurin mutant, calcineurin A beta (1–401), led to a significant decrease in phosphorylation at Ser-9, an increase in the activity of GSK-3β, and an increase in the phosphorylation of tau. K_m of calcineurin for a GSK-3β phosphopeptide was 469.3 μM, and specific activity of calcineurin was 15.2 nmol/min/mg. In addition, calcineurin and GSK-3β were co-immunoprecipitated in neuron-derived cells and brain tissues, and calcineurin formed a complex only with dephosphorylated GSK-3β. We conclude that in vitro, calcineurin can dephosphorylate GSK-3β at Ser-9 and form a stable complex with GSK-3β, suggesting the possibility that calcineurin regulates the dephosphorylation and activation of GSK-3β in vivo .     

Reversible inhibition of calcineurin by the polyphenolic aldehyde gossypol

The reversible inhibition of calcineurin (CaN), which is the only Ca(2+)/calmodulin-dependent protein Ser/Thr phosphatase, is thought to be a key functional event for most cyclosporin A (CsA)- and tacrolimus (FK506)-mediated biological effects. In addition to CaN inhibition, however, CsA and FK506 have multiple biochemical effects because of their action in a gain-of-function model that requires prior binding to immunophilic proteins. We screened a small molecule library for direct inhibitors of CaN using CaN-mediated dephosphorylation of (33)P-labeled 19-residue phosphopeptide substrate (RII phosphopeptide) as an assay and found the polyphenolic aldehyde gossypol to be a novel CaN inhibitor. Unlike CsA and FK506, gossypol does not require a matchmaker protein for reversible CaN inhibition with an IC(50) value of 15 microm. Gossypolone, a gossypol analog, showed improved inhibition of both RII phosphopeptide and p-nitrophenyl phosphate dephosphorylation with an IC(50) of 9 and 6 microm, respectively. In contrast, apogossypol hexaacetate was inactive. Gossypol acts noncompetitively, interfering with the binding site for the cyclophilin 18.CsA complex in CaN. In contrast to CsA and FK506, gossypol does not inactivate the peptidyl-prolyl-cis/trans-isomerase activity of immunophilins. Similar to CsA and FK506, T cell receptor signaling induced by phorbol 12-myristate 13-acetate/ionomycin is inhibited by gossypol in a dose-dependent manner, demonstrated by the inhibition of nuclear factor of activated T cell (NFAT) c1 translocation from the cytosol into the nucleus and suppression of NFAT-luciferase reporter gene activity.     

Substitution in position 3 of cyclosporin A abolishes the cyclophilin-mediated gain-of-function mechanism but not immunosuppression

Binary complex formation between the immunosuppressive drug cyclosporin A (CsA) and cyclophilin 18 is the prerequisite for the ability of CsA to inhibit the protein phosphatase activity of calcineurin, a central mediator of antigen-receptor signaling. We show here that several CsA derivatives substituted in position 3 can inhibit calcineurin without prior formation of a complex with cyclophilin 18. [Methylsarcosine(3)]CsA was shown to inhibit calcineurin, either in its free form with an IC(50) value of 10 microm, or in its complex form with cyclophilin 18 with an IC(50) of 500 nm. [Dimethylaminoethylthiosarcosine(3)]CsA ([Dat-Sar(3)]CsA) was found to inhibit calcineurin on its own, with an IC(50) value of 1.0 microm, but was not able to inhibit calcineurin after forming the [Dat-Sar(3)]CsA-cyclophilin 18 binary complex. Despite their different inhibitory properties, both CsA and [Dat-Sar(3)]CsA suppressed T cell proliferation and cytokine production mainly through blocking NFAT activation and interleukin-2 gene expression. Furthermore, to demonstrate that [Dat-Sar(3)]CsA can inhibit calcineurin in a cyclophilin-independent manner in vivo, we tested its effect in a Saccharomyces cerevisiae strain (Delta12), in which all the 12 cyclophilins and FKBPs were deleted. [Dat-Sar(3)]CsA, but not CsA, bypassed the requirement for cellular cyclophilins and caused growth inhibition in the salt-stressed Delta12 strain.     

Targeting calcineurin activation as a therapeutic strategy for T-cell acute lymphoblastic leukemia

Calcineurin is a calcium-activated serine/threonine phosphatase critical to a number of developmental processes in the cardiovascular, nervous and immune systems. In the T-cell lineage, calcineurin activation is important for pre-T-cell receptor (TCR) signaling, TCR-mediated positive selection of thymocytes into mature T cells, and many aspects of the immune response. The critical role of calcineurin in the immune response is underscored by the fact that calcineurin inhibitors, such as cyclosporin A (CsA) and FK506, are powerful immunosuppressants in wide clinical use. We observed sustained calcineurin activation in human B- and T-cell lymphomas and in all mouse models of lymphoid malignancies analyzed. In intracellular NOTCH1 (ICN1)- and TEL-JAK2-induced T-cell lymphoblastic leukemia, two mouse models relevant to human malignancies, in vivo inhibition of calcineurin activity by CsA or FK506 induced apoptosis of leukemic cells and rapid tumor clearance, and substantially prolonged mouse survival. In contrast, ectopic expression of a constitutively activated mutant of calcineurin favored leukemia progression. Moreover, CsA treatment induced apoptosis in human lymphoma and leukemia cell lines. Thus, calcineurin activation is critical for the maintenance of the leukemic phenotype in vivo, identifying this pathway as a relevant therapeutic target in lymphoid malignancies.     

Relative resistance of Cdk5-phosphorylated CRMP2 to dephosphorylation

Collapsin response mediator protein 2 (CRMP2) binds to microtubules and regulates axon outgrowth in neurons. This action is regulated by sequential phosphorylation by the kinases cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) at sites that are hyperphosphorylated in Alzheimer disease. The increased phosphorylation in Alzheimer disease could be due to increases in Cdk5 and/or GSK3 activity or, alternatively, through decreased activity of a CRMP phosphatase. Here we establish that dephosphorylation of CRMP2 at the residues targeted by GSK3 (Ser-518/Thr-514/Thr-509) is carried out by a protein phosphatase 1 family member in vitro, in neuroblastoma cells, and primary cortical neurons. Inhibition of GSK3 activity using insulin-like growth factor-1 or the highly selective inhibitor CT99021 causes rapid dephosphorylation of CRMP2 at these sites. In contrast, pharmacological inhibition of Cdk5 using purvalanol results in only a gradual and incomplete dephosphorylation of CRMP2 at the site targeted by Cdk5 (Ser-522), suggesting a distinct phosphatase targets this residue. A direct comparison of dephosphorylation at the Cdk5 versus GSK3 sites in vitro shows that the Cdk5 site is comparatively resistant to phosphatase treatment. The presence of the peptidyl-prolyl isomerase enzyme, Pin1, does not affect dephosphorylation of Ser-522 in vitro, in cells, or in Pin1 transgenic mice. Instead, the relatively high resistance of this site to phosphatase treatment is at least in part due to the presence of basic residues located nearby. Similar sequences in Tau are also highly resistant to phosphatase treatment. We propose that relative resistance to phosphatases might be a common feature of Cdk5 substrates and could contribute to the hyperphosphorylation of CRMP2 and Tau observed in Alzheimer disease.     

Phosphorylation of tau is regulated by PKN

For the phosphorylation state of microtubule-associated protein, tau plays a pivotal role in regulating microtubule networks in neurons. Tau promotes the assembly and stabilization of microtubules. The potential for tau to bind to microtubules is down-regulated after local phosphorylation. When we investigated the effects of PKN activation on tau phosphorylation, we found that PKN triggers disruption of the microtubule array both in vitro and in vivo and predominantly phosphorylates tau in microtubule binding domains (MBDs). PKN has a catalytic domain highly homologous to protein kinase C (PKC), a kinase that phosphorylates Ser-313 (= Ser-324, the number used in this study) in MBDs. Thus, we identified the phosphorylation sites of PKN and PKC subtypes (PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, -zeta, and -lambda) in MBDs. PKN phosphorylates Ser-258, Ser-320, and Ser-352, although all PKC subtypes phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352. There is a PKN-specific phosphorylation site, Ser-320, in MBDs. HIA3, a novel phosphorylation-dependent antibody recognizing phosphorylated tau at Ser-320, showed immunoreactivity in Chinese hamster ovary cells expressing tau and the active form of PKN, but not in Chinese hamster ovary cells expressing tau and the inactive form of PKN. The immunoreactivity for phosphorylated tau at Ser-320 increased in the presence of a phosphatase inhibitor, FK506 treatment, which means that calcineurin (protein phosphatase 2B) may be involved in dephosphorylating tau at Ser-320 site. We also noted that PKN reduces the phosphorylation recognized by the phosphorylation-dependent antibodies AT8, AT180, and AT270 in vivo. Thus PKN serves as a regulator of microtubules by specific phosphorylation of tau, which leads to disruption of tubulin assembly.     

Calcineurin enhances MEF2 DNA binding activity in calcium-dependent survival of cerebellar granule neurons.

Myocyte enhancer factor 2 (MEF2) has been shown recently to be necessary for mediating activity-dependent neuronal survival. In this study, we show that calcium signals regulate MEF2 activity through a serine/threonine phosphatase calcineurin. In cultured primary cerebellar granule neurons, the electrophoretic mobility of MEF2A protein was sensitive to the level of extracellular potassium chloride (KCl) and depolarizing concentrations of KCl led to hypophosphorylation of the protein. The specific inhibitors of calcineurin cyclosporin A (CsA) and FK506 could overcome KCl-dependent MEF2A hypophosphorylation. The effects of CsA and FK506 were KCl specific as they had little effect on MEF2A phosphorylation when granule neurons were cultured in the presence of full media. Hyperphosphorylation of MEF2A led to the loss of its DNA binding activity as determined by DNA mobility shift assay. Consistent with this, CsA/FK506 also inhibited MEF2-dependent reporter gene expression. These findings demonstrate that regulation of MEF2A by calcium signals requires the action of protein phosphatase calcineurin. By maintaining MEF2A in a hypophosphorylated state, calcineurin enhances the DNA binding activity of MEF2A and therefore maximizes its transactivation capability. The identification of MEF2 as a novel target of calcineurin may provide in part a biochemical explanation for the therapeutic and toxic effects of immunosuppressants CsA and FK506.     

Regulation of tumor necrosis factor cytotoxicity by calcineurin

Cyclosporin (CsA) inhibits mitochondrial death signaling and opposes tumor necrosis factor (TNF)-induced apoptosis in vitro. However, CsA is also a potent inhibitor of calcineurin, a phosphatase that may participate in cell death. Therefore, we tested the hypothesis that calcineurin regulates TNF cytotoxicity in rat hepatoma cells (FTO2B). TNF-treated FTO2B cells appeared apoptotic by DNA fragmentation, nuclear condensation, annexin V binding, and caspase activation. We studied two calcineurin inhibitors, CsA and FK506, and found that each potently inhibited TNF cytotoxicity. Western blot demonstrated calcineurin in FTO2B homogenates. In a model of mitochondrial permeability transition (MPT), we found that CsA prevented MPT and cytochrome c release, while FK506 inhibited neither. In summary, we present evidence that calcineurin participates in an apoptotic death pathway activated by TNF. CsA may oppose programmed cell death by inhibiting calcineurin activity and/or inhibiting mitochondrial signaling.     

Tau becomes a more favorable substrate for GSK-3 when it is prephosphorylated by PKA in rat brain

Microtubule-associated protein tau is abnormally hyperphosphorylated in Alzheimer's disease (AD) and other tauopathies and is believed to lead to neurodegeneration in this family of diseases. Here we show that infusion of forskolin, a specific cAMP-dependent protein kinase A (PKA) activator, into the lateral ventricle of brain in adult rats induced activation of PKA by severalfold and concurrently enhanced the phosphorylation of tau at Ser-214, Ser-198, Ser-199, and or Ser-202 (Tau-1 site) and Ser-396 and or Ser-404 (PHF-1 site), which are among the major abnormally hyperphosphorylated sites seen in AD. PKA activation positively correlated to the extent of tau phosphorylation at these sites. Infusion of forskolin together with PKA inhibitor or glycogen synthase kinase-3 (GSK-3) inhibitor revealed that the phosphorylation of tau at Ser-214 was catalyzed by PKA and that the phosphorylation at both the Tau-1 and the PHF-1 sites is induced by basal level of GSK-3, because forskolin activated PKA and not GSK-3 and inhibition of the latter inhibited the phosphorylation at Tau-1 and PHF-1 sites. Inhibition of cdc2, cdk5, or MAPK had no significant effect on the forskolin-induced hyperphosphorylation of tau. Forskolin inhibited spatial memory in a dose-dependent manner in the absence but not in the presence of R(p)-adenosine 3',5'-cyclic monophosphorothioate triethyl ammonium salt, a PKA inhibitor. These results demonstrate for the first time that phosphorylation of tau by PKA primes it for phosphorylation by GSK-3 at the Tau-1 and the PHF-1 sites and that an associated loss in spatial memory is inhibited by inhibition of the hyperphosphorylation of tau. These data provide a novel mechanism of the hyperphosphorylation of tau and identify both PKA and GSK-3 as promising therapeutic targets for AD and other tauopathies.     

Synthesis, maturation, and trafficking of human Na+-dicarboxylate cotransporter NaDC1 requires the chaperone activity of cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na⁺-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.     

Delineation of the calcineurin-interacting region of cyclophilin B

The immunosuppressant drug cyclosporin A (CsA) inhibits T-cell function by blocking the phosphatase activity of calcineurin. This effect is mediated by formation of a complex between the drug and cyclophilin (CyP), which creates a composite surface able to make high-affinity contacts with calcineurin. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineurin than the CyPA/CsA and CyPC/CsA complexes, pointing to fine structural differences in the calcineurin-binding region. To delineate the calcineurin-binding region of CyPB, we mutated several amino acids, located in two loops corresponding to CyPA regions known to be involved, as follows: R76A, G77H, D155R, and D158R. Compared to wild-type CyPB, the G77H, D155R, and D158R mutants had intact isomerase and CsA-binding activities, indicating that no major conformational changes had taken place. When complexed to CsA, they all displayed only reduced affinity for calcineurin and much decreased inhibition of calcineurin phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of CyPB/CsA with calcineurin, in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in CyPA and might therefore account for the higher affinity of the CyPB/CsA complex for calcineurin.     

Anisomycin过度激活丝裂原活化蛋白激酶使tau发生过度磷酸化

阿尔茨海默病(Alzheimer's disease,AD)的病理特征之一是神经元内存在神经原纤维缠结(neurofibrillary tangles,NFTs),后者是由过度磷酸化的微管相关蛋白tau形成的双股螺旋细丝(paired helical filaments,PHFs)构成.为了探讨丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)在微管相关蛋白tau磷酸化中的作用及机制,本实验用0.1 μg/mL、0.2 μg/mL和0.4μg/mL三种不同浓度的MAPK激动剂anisomycin处理小鼠成神经瘤细胞株(mouse neuroblastoma cells,N2a),检测MAPK活性的变化及其与tau蛋白多个AD相关位点过度磷酸化的关系,并检测糖原合酶激酶-3(glycogen synthase kinase-3,GSK-3)和蛋白激酶A(protein kinase A,PKA)的活性变化.结果显示,anisomycin以剂量依赖的方式激活MAPK活性,但免疫印迹结果显示tau蛋白的Ser-198/199/202位点和Ser-396/404位点的过度磷酸化只在anisomycin浓度为0.4 μg/mL时出现,三种浓度的anisomycin均未引起tau蛋白Ser-214位点磷酸化的改变;同时,GSK-3活性在anisomycin为0.1 μg/mL时没有明显变化,当anisomycin浓度升高到0.2 μg/mL和0.4 μg/mL时出现明显增高,而PKA的活性没有明显的改变.使用GSK-3的特异性抑制剂氯化锂(LiCl)则完全阻断MAPK被过度激活导致的tau蛋白磷酸化水平的增高,而同时MAPK活性不受影响.以上结果提示:过度激活MAPK可以导致tau蛋白Ser-198/199/202和Ser-396/404位点过度磷酸化,其机制可能涉及MAPK激活GSK-3的间接作用.     

A Magnaporthe grisea cyclophilin acts as a virulence determinant during plant infection

Cyclophilins are peptidyl prolyl cis-trans isomerases that are highly conserved throughout eukaryotes and that are best known for being the cellular target of the immunosuppressive drug cyclosporin A (CsA). The activity of CsA is caused by the drug forming a complex with cyclophilin A and inhibiting the calmodulin-dependent phosphoprotein phosphatase calcineurin. We have investigated the role of CYP1, a cyclophilin-encoding gene in the phytopathogenic fungus Magnaporthe grisea, which is the causal agent of rice blast disease. CYP1 putatively encodes a mitochondrial and cytosolic form of cyclophilin, and targeted gene replacement has shown that CYP1 acts as a virulence determinant in rice blast. Cyp1 mutants show reduced virulence and are impaired in associated functions, such as penetration peg formation and appressorium turgor generation. CYP1 cyclophilin also is the cellular target for CsA in Magnaporthe, and CsA was found to inhibit appressorium development and hyphal growth in a CYP1-dependent manner. These data implicate cyclophilins as virulence factors in phytopathogenic fungi and also provide evidence that calcineurin signaling is required for infection structure formation by Magnaporthe.     

Ser-59 is the major phosphorylation site in alphaB-crystallin accumulated in the brains of patients with Alexander's disease

The phosphorylation state of alphaB-crystallin accumulated in the brains of two patients with Alexander's disease (one infantile and one juvenile type) was determined by means of SDS-PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific antibodies against alphaB-crystallin and each of three phosphorylated serine residues. The level of mammalian small heat shock protein of 25-28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of alphaB-crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser-59 in both cases. A significant level of phosphorylation at Ser-45 but not Ser-19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. alphaB-crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. AlphaB-crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with Alzheimer's disease and aged controls. The phosphorylation site was mostly at Ser-59 in all cases. Immunohistochemically, alphaB-crystallin was stained in Rosenthal fibers in brains of patients with Alexander's disease and their peripheral portions were immunostained with antibodies recognizing phosphorylated Ser-59. These results indicate that the major phosphorylation site in alphaB-crystallin in brains of patients with Alexander's disease or Alzheimer's disease as well as in aged controls is Ser-59.