Genome-wide screening for genes associated with FK506 sensitivity in fission yeast

We have been studying calcineurin signal transduction pathway in fission yeast Schizosaccharomyces pombe (S. pombe) by developing a genetic screen for mutants that show hypersensitivity to the immunosuppressive calcineurin inhibitor FK506 (tacrolimus). In the present study, to identify nonessential genes that are functionally related to the calcineurin signaling pathway, we performed a genome-wide screen of 3004 haploid deletion strains and confirmed 72 deletion strains to be FK506 sensitive. These 72 genes are classified into nine functional groups to include membrane trafficking (16 genes), signal transduction (10 genes), ubiquitination (8 genes), chromatin remodeling (6 genes), cytokinesis (4 genes), ribosomal protein (3 genes), RNA binding protein (3 genes), and a variety of other known functions (17 genes) or still unknown functions (5 genes) in the biological system. In our previous screening of FK506-sensitive mutants we isolated several membrane-trafficking mutants showing defective cell wall integrity. Here, we further examined the vacuolar fusion, the v-SNARE synaptobrevin Syb1 localization, and the sensitivity to the β-glucan synthase inhibitor micafungin in these 72 FK506-sensitive strains. Results showed that 25 deletion strains exhibited abnormal vacuole fusion, 19 deletion strains exhibited Syb1 mislocalization, and 14 deletion strains exhibited both abnormal vacuole fusion and Syb1 mislocalization, while 42 deletion strains showed both normal vacuole fusion and Syb1 localization. Likewise, 16 deletion strains showed sensitivity to micafungin. Altogether, our present study indicates that calcineurin mediates a plethora of physiological processes in fission yeast, and that calcineurin is extensively involved in cross-talk between signaling pathways.     

Substrate selectivity and sensitivity to inhibition by FK506 and cyclosporin A of calcineurin heterodimers composed of the alpha or beta catalytic subunit.

The calcineurin (CaN) alpha and beta catalytic subunit isoforms are coexpressed within almost all cell types. The enzymatic properties of CaN heterodimers comprised of the regulatory B subunit (CnB) with either the alpha or beta catalytic subunit were compared using in vitro phosphatase assays. CaN containing the alpha isoform (CnA alpha) has lower K(m) and higher V(max) values than CaN containing the beta isoform (CnA beta) toward the PO4-RII, PO4-DARPP-32(20-38) peptides, and p-nitrophenylphosphate (pNPP). CaN heterodimers containing the alpha or beta catalytic subunit isoform displayed identical calmodulin dissociation rates. Similar inhibition curves for each CaN heterodimer were obtained with the CaN autoinhibitory peptide (CaP) and cyclophilin A/cyclosporin A (CyPA/CsA) using each peptide substrate at K(m) concentrations, except for a five- to ninefold higher IC50 value measured for CaN containing the beta isoform with p-nitrophenylphosphate as substrate. No difference in stimulation of phosphatase activity toward p-nitrophenylphosphate by FKBP12/FK506 was observed. At low concentrations of FKBP12/FK506, CaN containing the alpha isoform is more sensitive to inhibition than CaN containing the beta isoform using the phosphopeptide substrates. Higher concentrations of FKBP12/FK506 are required for maximal inhibition of beta CaN using PO4-DARPP-32(20-38) as substrate. The functional differences conferred upon CaN by the alpha or beta catalytic subunit isoforms suggest that the alpha:beta and CaN:substrate ratios may determine the levels of CaN phosphatase activity toward specific substrates within tissues and specific cell types. These findings also indicate that the alpha and beta catalytic subunit isoforms give rise to substrate-dependent differences in sensitivity toward FKBP12/FK506.     

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.     

FK506 and the role of immunophilins in nerve regeneration

FK506 is a new FDA-approved immunosuppressant used for prevention of allograft rejection in, for example, liver and kidney transplantations. FK506 is inactive by itself and requires binding to an FK506 binding protein-12 (FKBP-12), or immunophilin, for activation. In this regard, FK506 is analogous to cyclosporin A, which must bind to its immunophilin (cyclophilin A) to display activity. This FK506-FKBP complex inhibits the activity of the serine/threonine protein phosphatase 2B (calcineurin), the basis for the immunosuppressant action of FK506. The discovery that immunophilins are also present in the nervous system introduces a new level of complexity in the regulation of neuronal function. Two important calcineurin targets in brain are the growth-associated protein GAP-43 and nitric oxide (NO) synthase (NOS). This review focuses on studies showing that systemic administration of FK506 dose-dependently speeds nerve regeneration and functional recovery in rats following a sciatic-nerve crush injury. The effect appears to result from an increased rate of axonal regeneration. The nerve regenerative property of this class of agents is separate from their immunosuppressant action because FK506-related compounds that bind to FKBP-12 but do not inhibit calcineurin are also able to increase nerve regeneration. Thus, FK506's ability to increase nerve regeneration arises via a calcineurin-independent mechanism (i.e., one not involving an increase in GAP-43 phosphorylation). Possible mechanisms of action are discussed in relation to known actions of FKBPs: the interaction of FKBP-12 with two Ca²⁺ release-channels (the ryanodine and inositol 1,4,5-triphosphate receptors) which is disrupted by FK506, thereby increasing Ca²⁺ flux; the type 1 receptor for the transforming growth factor-β (TGF-β1), which stimulates nerve growth factor (NGF) synthesis by glial cells, and is a natural ligand for FKBP-12; and the immunophilin FKBP-52/FKBP-59, which has also been identified as a heat-shock protein (HSP-56) and is a component of the nontransformed glucocorticoid receptor. Taken together, studies of FK506 indicate broad functional roles for the immunophilins in the nervous system. Both calcineurin-dependent (e.g., neuroprotection via reduced NO formation) and calcineurin-independent mechanisms (i.e., nerve regeneration) need to be invoked to explain the many different neuronal effects of FK506. This suggests that multiple immunophilins mediate FK506's neuronal effects. Novel, nonimmunosuppressant ligands for FKBPs may represent important new drugs for the treatment of a variety of neurological disorders.     

The regulatory domains of CNA have different effects on the inhibition of CN activity by FK506 and CsA

Calcineurin (CN) is the common receptor for two immunophilin-immunosuppressant complexes, Cyp-CsA and FKBP-FK506. Calcineurin is composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). CNA contains the catalytic domain and three regulatory domains: a CNB-binding domain (BBH, 350-370), a calmodulin- binding domain (CBD, 389-413), and an autoinhibitory domain (AID, 457-482). To investigate the effects of these three regulatory domains on the inhibition of CN by the two drugs we constructed three C-terminal deletion mutants: CNAabc (1-456), CNAab (1-388) and CNAa (1-347). Inhibition of CNA and its derivatives by the two drugs was examined and compared with inhibition by peptides (AID [457-482] and LCBD [389-456], CBD and the extension of the AID were included). Our results show that the BBH is critical for inhibition of CN by Cyp-CsA and FKBP-FK506. The LCBD has no effect and the AID reduces the inhibition of CN by two complexes. In addition, LCBD and AID as autoinhibitors may inhibit enzyme activity via different sites.     

Role of the Rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.     

Mechanism for the paradoxical inhibition and stimulation of calcineurin by the immunosuppresive drug tacrolimus (FK506)

We examined the paradoxical inhibition and stimulation of calcineurin, the calcium-activated protein phosphatase, using the drug FK506 (tacrolimus) which acts as a complex together with its binding protein; the complex is designated here as FKC. We reproduced FKC inhibition with RIIp, a phosphorylated peptide substrate, and FKC stimulation with p-nitrophenylphosphate (pNPP) as substrate. The presence of RIIp in the pNPP assay caused inhibition. Yet, under these conditions, FKC still stimulated pNPP dephosphorylation to the same extent. The effects of Mn2+ were strikingly different for the two substrates when calcineurin was measured under otherwise identical conditions: Mn2+ stimulated pNPP dephosphorylation several fold, but only stimulated RIIp dephosphorylation by about 50%. When Pi was used as product inhibitor, FKC stimulation, but not calmodulin stimulation, was attenuated. We conclude that FKC enhances substrate binding to the enzyme. This would lead to inhibition with RIIp, known to bind calcineurin tightly, but stimulation with pNPP, known to bind calcineurin weakly. The result not only resolves the paradox but also elucidates the mechanism of action for this class of immunosuppressive drugs.     

Rapid screen of human genes for relevance to cancer using fission yeast

A total of 437 human full-length cDNAs isolated by microarray analysis of liver and/or gastric cancer tissues were evaluated for their relevance to cancer using the fission yeast Schizosaccharomyces pombe. Overexpression of 161 human cDNAs in S. pombe caused growth inhibition and/or morphological changes, which can be considered as cancer-related phenotypes of S. pombe. Sixteen genes causing growth defects and morphological changes at the same time were chosen to validate their ostensible oncogenic properties. They were highly expressed in liver and/or gastric cancer cell lines. Also, when the mouse embryonic fibroblast cell type NIH3T3 was transfected with these genes, the proliferation rates of cells were increased by 32% to 120%. This study demonstrates that fission yeast can be used as an advantageous and powerful tool for the rapid screening of human genes relevant to cancer. Furthermore, the human genes screened can be tested further as diagnostic markers and potential therapeutic targets for liver and stomach cancers. They also can be studied further for the elucidation of mechanisms involved in carcinogenesis.     

Neuroprotectant FK506 inhibits glutamate-induced apoptosis of astrocytes in vitro and in vivo

Neuron-astrocyte interactions are critical for signalling, energy metabolism, extracellular ion and glutamate homeostasis, volume regulation and neuroprotection in the CNS. Glutamate uptake by astrocytes may prevent excitotoxic glutamate elevation and determine neuronal survival. However, an excess of glutamate can cause the death of astrocytes. FK506, an inhibitor of calcineurin, and an immunosuppressive drug, is neuroprotective in animal models of neurologic diseases, including focal and global ischaemia. In the present work, we demonstrate that a single injection of FK506 60 min after a transient middle cerebral artery occlusion (MCAo) significantly decreases the number of terminal deoxynucleotidyl transferase nick-end labelling (TUNEL)-positive cells in the ischaemic cortex and striatum. Using 3-D confocal microscopy we found that, 24 h after MCAo, many TUNEL-positive cells in the ischaemic striatum and cortex are astrocytes. Furthermore, we demonstrate that exposure of cultured cortical astrocytes to 50-100 mM Glu for 24 h induces apoptotic alterations in nuclear morphology, DNA fragmentation, dissipation of mitochondrial transmembrane potential (DeltaPsi) and caspase activation. FK506 (1 muM) efficiently inhibits Glu-induced apoptosis of cultured astrocytes, DNA fragmentation and changes in mitochondrial DeltaPsi. Our findings suggest that modulation of glutamate-induced astrocyte death early after reperfusion may be a novel mechanism of FK506-mediated neuroprotection in ischaemia.     

Dissecting the fission yeast regulatory network reveals phase-specific control elements of its cell cycle

Background  

Fission yeast Schizosaccharomyces pombe and budding yeast Saccharomyces cerevisiae are among the original model organisms in the study of the cell-division cycle. Unlike budding yeast, no large-scale regulatory network has been constructed for fission yeast. It has only been partially characterized. As a result, important regulatory cascades in budding yeast have no known or complete counterpart in fission yeast.     

Role of fission yeast primase catalytic subunit in the replication checkpoint

To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe, spp1(+) and spp2(+) (S. pombe primase 1 and 2). spp1(+) encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polalpha to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1(+) gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1(+) exhibit an allele-specific abnormal mitotic phenotype. Mutations of spp1(+) reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polalpha-primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Polalpha has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polalpha. Together, these have led us to propose that suboptimal cellular levels of polalpha-primase complex due to the allele-specific mutations of Spp1 might not allow Polalpha to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.     

Mutations in the regulatory subunit of yeast acetohydroxyacid synthase affect its activation by MgATP

Isoleucine, leucine and valine are synthesized via a common pathway in which the first reaction is catalysed by AHAS (acetohydroxyacid synthase; EC 2.2.1.6). This heterotetrameric enzyme is composed of a larger subunit that contains the catalytic machinery and a smaller subunit that plays a regulatory role. The RSU (regulatory subunit) enhances the activity of the CSU (catalytic subunit) and mediates end-product inhibition by one or more of the branched-chain amino acids, usually valine. Fungal AHAS differs from that in other organisms in that the inhibition by valine is reversed by MgATP. The fungal AHAS RSU also differs from that in other organisms in that it contains a sequence insert. We suggest that this insert may form the MgATP-binding site and we have tested this hypothesis by mutating ten highly conserved amino acid residues of the yeast AHAS RSU. The modified subunits were tested for their ability to activate the yeast AHAS CSU, to confer sensitivity to valine inhibition and to mediate reversal of the inhibition by MgATP. All but one of the mutations resulted in substantial changes in the properties of the RSU. Unexpectedly, four of them gave a protein that required MgATP in order for strong stimulation of the CSU and valine inhibition to be observed. A model to explain this result is proposed. Five of the mutations abolished MgATP activation and are suggested to constitute the binding site for this modulator.     

The biosynthetic pathway of FK506 and its engineering: from past achievements to future prospects

Journal of Industrial Microbiology & Biotechnology - FK506, a 23-membered macrolide produced by several Streptomyces species, is an immunosuppressant widely used to prevent the rejection of...     

Protein phosphatase type 2B (calcineurin)-mediated, FK506-sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions.

To assess the physiological function of Ca(2+)-dependent protein phosphatase (PP2B) in the yeast Saccharomyces cerevisiae, the phenotypes of PP2B-deficient mutants were investigated. Although PP2B was dispensable for growth under normal conditions, the mutations did, however, cause growth inhibition under certain stress circumstances. The growth of the mutants was inhibited by NaCl and LiCl, but not by KCl, CaCl2, MgCl2 or nonspecific osmotic stresses. Upon shift to high NaCl medium, intracellular Na+ levels of both wild type yeast and the mutants initially increased at a comparable rate. However, internal Na+ in wild type cells started to decline more rapidly than the mutant cells during cultivation in high NaCl medium, indicating that PP2B is important in maintaining a gradient across the membrane. The protection against salt stress was achieved, at least in part, by the stimulation of Na+ export. The maintenance of a high level of internal K+ in high NaCl medium was also PP2B-dependent. In the presence of the immunosuppressant FK506, the growth behaviour and intracellular Na+ and K+ of wild type cells in high NaCl medium became very similar to those of the PP2B-deficient mutant in a manner dependent on the presence of the FK506 binding protein.     

Molecular mechanisms of TGF beta receptor-triggered signaling cascades rapidly induced by the calcineurin inhibitors cyclosporin A and FK506

The calcineurin inhibitor (CNI)-induced renal fibrosis is attributed to an exaggerated deposition of extracellular matrix, which is mainly due to an increased expression of TGFbeta. Herein we demonstrate that the CNI cyclosporin A and tacrolimus (FK506), independent of TGFbeta synthesis, rapidly activate TGFbeta/Smad signaling in cultured mesangial cells and in whole kidney samples from CNI-treated rats. By EMSA, we demonstrate increased DNA binding of Smad-2, -3, and -4 to a cognate Smad-binding promoter element (SBE) accompanied by CNI-triggered activation of Smad-dependent expression of tissue inhibitor of metalloprotease-1 (TIMP-1) and connective tissue growth factor. Using an activin receptor-like kinase-5 (ALK-5) inhibitor and by small interfering RNA we depict a critical involvement of both types of TGFbeta receptors in CNI-triggered Smad signaling and fibrogenic gene expression, respectively. Mechanistically, CNI cause a rapid activation of latent TGFbeta, which is prevented in the presence of the antioxidant N-acetyl cysteine. A convergent activation of p38 MAPK is indicated by the partial blockade of CNI-induced Smad-2 activation by SB203580; conversely, both TGFbeta-RII and TGFbeta are critically involved in p38 MAPK activation by CNI. Activation of both signaling pathways is similarly triggered by reactive oxygen species. Finally, we show that neutralization of TGFbeta markedly reduced the CNI-dependent Smad activation in vitro and in vivo. Collectively, this study demonstrates that CNI via reactive oxygen species generation activate latent TGFbeta and thereby initiate the canonical Smad pathway by simultaneously activating p38 MAPK, which both synergistically induce Smad-driven gene expression.     

Immunolocalization of calcineurin and FKBP12, the FK506-binding protein, in Hassall's corpuscles of human thymus and epidermis

Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, plays an important role in various physiological functions including T cell activation. This enzyme is a target molecule for the immunosuppressants, cyclosporin A and FK506. In the present study, we investigated immunohistochemical localization of calcineurin and FKBP12, an FK506-binding protein, in human thymus and epidermis. The catalytic subunit (calcineurin A) of calcineurin was abundantly expressed in Hassall's corpuscles which were localized in the thymic medulla and represented the terminal stages of thymic medullary epithelium. The regulatory subunit (calcineurin B) of calcineurin was also expressed in high amounts in Hassall's corpuscles. In the epidermis, which shows similarities to Hassall's corpuscles, both subunits were also abundantly expressed, and their expression increased with the differentiation of keratinocytes. FKBP12 was observed to be expressed abundantly, both in Hassall's corpuscles and the entire epidermis. These findings suggest that the differentiated forms of the two cell types, which are the thymic medullary epithelial cell and the keratinocyte, are the target for pharmacological actions of FK506.     

Localization and activity of the calcineurin catalytic and regulatory subunit complex at the septum is essential for hyphal elongation and proper septation in Aspergillus fumigatus

Calcineurin, a heterodimer composed of the catalytic (CnaA) and regulatory (CnaB) subunits, plays key roles in growth, virulence and stress responses of fungi. To investigate the contribution of CnaA and CnaB to hyphal growth and septation, ΔcnaB and ΔcnaAΔcnaB strains of Aspergillus fumigatus were constructed. CnaA colocalizes to the contractile actin ring early during septation and remains at the centre of the mature septum. While CnaB's septal localization is CnaA-dependent, CnaA's septal localization is CnaB-independent, but CnaB is required for CnaA's function at the septum. Catalytic null mutations in CnaA caused stunted growth despite septal localization of the calcineurin complex, indicating the requirement of calcineurin activity at the septum. Compared to the ΔcnaA and ΔcnaB strains, the ΔcnaAΔcnaB strain displayed more defective growth and aberrant septation. While three Ca(2+) -binding motifs in CnaB were sufficient for its association with CnaA at the septum, the amino-terminal arginine-rich domains (16-RRRR-19 and 44-RLRKR-48) are dispensable for septal localization, yet required for complete functionality. Mutation of the 51-KLDK-54 motif in CnaB causes its mislocalization from the septum to the nucleus, suggesting it is a nuclear export signal sequence. These findings confirm a cooperative role for the calcineurin complex in regulating hyphal growth and septation.